Tesis - Universidad de Chile

UNIVERSIDAD DE CHILE
FACULTAD DE CIENCIAS QUIMICAS Y FARMACEUTICAS
“THEORETICAL INSIGHT INTO THE CATALYTIC CYCLE OF
COBALAMIN-GLUTAMATE MUTASE COMPLEX”
Tesis presentada a la Universidad de Chile
para optar al grado de Doctor en Química por:
SEBASTIÁN ESTEBAN MIRANDA ROJAS
Directores de Tesis
Dr. Fernando Mendizábal
Dr. Gerald Zapata
SANTIAGO- CHILE
2012
UNIVERSIDAD DE CHILE
FACULTAD DE CIENCIAS QUÍMICAS Y FARMACÉUTICAS
INFORME DE APROBACIÓN
TESIS DE DOCTORADO
Se informa a la Dirección de Postgrado de la Facultad de Ciencias Químicas y
Farmacéuticas que la Tesis de Doctorado presentada por el candidato:
SEBASTIÁN ESTEBAN MIRANDA ROJAS
Ha sido aprobada por la Comisión Informante de Tesis como requisito para optar
al grado de Doctor en Química, en el examen de defensa de Tesis rendido el día
26 de enero del 2012.
Directores de Tesis:
Dr. Fernando Mendizábal
___________________________
Dr. Gerald Zapata
___________________________
Comisión Informante de Tesis:
Dr. Marcelo Kogan (Presidente)
___________________________
Dra. Bárbara Loeb
___________________________
Dra. Patricia Pérez
___________________________
Dr. Eduardo Chamorro
___________________________
“There is a driving force more powerful than the steam,
the electricity and the atomic energy: the will”
Albert Einstein
“Science cannot solve the ultimate mystery of nature.
And that is because, in the last analysis,
we ourselves are a part of the mystery that we are trying to solve”
Max Planck
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ACKNOWLEDGMENT
I want to thanks to my family for their unconditional support, love, patience and
understanding during this long process. Thanks to my mother Erica for her unconditional love
and care. You taught me to be disciplined, dedicated and perfectionist, qualities that aided me in
this complicated pathway called science.
Thanks to my father Miguel whom taught me to never surrender and always finish what I have
started. When I am tired and I want to quit, I always think about you and how you always
continue regardless of everything, you represent my strength to carry on. Thanks to my brother
Miguel for always supporting me and loving me no matter what. I know I can always count on
you. Thanks to my brother Gustavo for giving me the purpose of being a better person and a
better scientist, and helping me when I need it, even for those little details that you may believe I
forget.
I would like to express my gratitude to Dr. Fernando Mendizábal for his support, for the
countless scientific discussions and wise advices. I believe that I am a better scientist than when
I started this thesis thanks to you, my eternal gratitude for you. Special thanks for introducing me
into the amazing world of bioinorganic chemistry
Dr. Gerald Zapata, you has been a friend when I need it, we have shared lots of good
moments and I truly hope to share lots more. Also, you have provided me of computer resources
without which I would never have achieved my scientific goals together with fruitful
discussions, so I will never be able to thank you enough.
Finally, I would like to thank you both for being the supervisors of this thesis work and for
trusting me and supporting me in every hard decision I had to make during this time.
Thanks to the reviewers of my thesis: Dr. Marcelo Kogan, Dr. Bárbara Loeb, Dr. Patricia
Pérez and Dr. Eduardo Chamorro. Your valuable comments certainly helped to increase the
value of this thesis. Special thanks to Dr. Marcelo Kogan whom personally has supported me in
every stage of the evaluation process.
Thanks to Dr. Irma Crivelli for the insightful discussions about inorganic chemistry and
spectroscopy.
iv
Thanks to Professor Ramiro Arratia for his financial support and for giving me the chance of
being part of his amazing research group. Thanks to Dr. Alvaro Muñoz and Dr. Desmond
Mc-Leod.
Thanks to Professor Pekka Pyykkö and Dr. Ville Kayla for receiving me on the University of
Helsinki during my research stay on Finland.
Thanks to Raúl Mera for sharing his knowledge and friendship with me along this pathway.
Thanks to my colleges Darwin Burgos and Daniela Donoso.
Thanks to my friends. Felipe Cartagena, Abigail Tapia, Yair Riquelme, Jorge y Pamela
Hoffmann. Each one of you has made a contribution in my life leading me to accomplish my
dream of being a scientist. I honestly hope that you are as proud of me as I am of you all. I
would like to thank to Jorge and Lyzethly for their support and friendship during the last stages
of this process.
I want to express my deepest gratitude to Paulina Cañete, without you and your love I never
would have been able to finish this thesis. You gave me the strength to continue making every
effort worthy. Thank you for believing in me, I am sure that together we are able to accomplish
even greater things. Thanks to Paulina’s father Arnaldo and to her siblings Rodrigo and
Macarena. I really appreciate your love and support.
Thanks to the secretaría de Postgrado y Postítulo, especially to Helen Gallegos.
Thanks to the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) for
their financial support though the following sholarships: “Beca para Estudios de Postgrado”,
“Beca de Apoyo de Tesis Doctoral” and “Beca para Congresos y Estadías Cortas en el
Extranjero”.
Thanks to the Vicerrectoria de Asuntos Académicos of the Universidad de Chile for the
“short research stays abroad” scholarship which was carried out in the University of HelsinkiFinland.
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LIST OF PAPERS
Fernando Mendizabal, Claudio Olea-Azar, Sebastián Miranda; Theoretical study of the
interaction d(10)-d(8) between Pt(0) and M(I) on the [Pt(PH3)-MPH3](+) complexes (M = Cu,
Ag, Au); International Journal of Quantum Chemistry; p. 1454-1458; 107 (2007)
Carolina Jullian, Sebastián Miranda, Gerald Zapata-Torres, Claudio Olea-Azar, Fernando
Mendizabal; Studies of inclusion complexes of natural and modified cyclodextrin with
(+)catechin by NMR and molecular modeling; Bioorganic & Medicinal Chemistry ; p. 32173224; 15 (2007)
Carolina Jullian, Constanza Cifuentes, Muriel Alfaro, Sebastián Miranda, Germán Barriga,
Claudio Olea-Azar; Spectroscopic characterization of the inclusión complexes of luteolin with
native and derivatized beta-cyclodextrin; Bioorganic & Medicinal Chemistry ; p. 5025-5031; 18
(2010)
Raúl Mera-Adasme, Fernando Mendizábal, Claudio Olea-Azar, Sebastián Miranda-Rojas, and
Patricio Fuentealba; A Computationally Efficient and Reliable Bond Order Measure; The
Journal of Physical Chemistry A; p 4397-4405; 115 (2011)
Ingrid Ponce, J. Francisco Silva, Ruben Oñate, Sebastian Miranda-Rojas, Alvaro MuñozCastro, Ramiro Arratia-Pérez, Fernando Mendizabal, and José H. Zagal; Theoretical and
Experimental Study of Bonding and Optical Properties of Self-Assembly
Metallophthalocyanines Complexes on a Gold Surface. A Survey of the Substrate–Surface
Interaction; The Journal of Physical Chemistry C; p 23512-23518; 115 (2011)
Mera-Adasme, Raul; Sundholm, Dage; Olea-Azar, C.; Mendizabal, Fernando; Miranda-Rojas,
Sebastián; Gonzalez, Mauricio. Computational studies of the Metal-binding Site of the Wildtype and the H46R Mutant of the Copper,zinc Superoxide Dismutase. Inorganic Chemistry;
Submitted Manuscript
Sebastián Miranda-Rojas, Gerald Zapata-Torres, Raúl Mera-Adasme, and Fernando
Mendizábal; Methyl- and Adenosylcobalamin: Theoretical Relationship between the Co-C bond
Strength and their Molecular and Electronic Structures. Organometallics; Submitted Manuscript
Sebastián Miranda-Rojas, Gerald Zapata-Torres, Raúl Mera-Adasme, and Fernando
Mendizábal; Methyl- and Adenosylcobalamin:
To be submitted
Sebastián Miranda-Rojas, Gerald Zapata-Torres, and Fernando Mendizábal; Theoretical Study
of the Reaction Mechanism for the Isomerization Reaction Catalyzed by Glutamate Mutase.
Manuscript in preparation
Sebastián Miranda-Rojas, Gerald Zapata-Torres, and Fernando Mendizábal; Quantum
Chemical Modeling of Cobalamin-Glutamate Mutase Catalytic Reaction Pathway.
Manuscript in preparation
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CONTENTS
CONTENTS .................................................................................................................... vii
FIGURE INDEX .............................................................................................................. ix
TABLE INDEX................................................................................................................ xi
ABBREVIATIONS......................................................................................................... xii
AMINO ACID ABBREVIATIONS .............................................................................. xiii
MODELS DEFINITION ................................................................................................ xiv
ABSTRACT .................................................................................................................... xv
RESUMEN .................................................................................................................... xvii
1
INTRODUCTION..................................................................................................... 1
Enzymes and Quantum Bioinorganic Chemistry................................................................ 1
Density Functional Theory................................................................................................ 3
Cobalt ............................................................................................................................... 7
Cobalamins....................................................................................................................... 8
Cobalamin-dependent Enzymes ...................................................................................... 11
Glutamate mutase ........................................................................................................... 16
Proposed mechanisms for the Co-C bond weakening....................................................... 21
Thesis work .................................................................................................................... 25
2
1.1
HYPOTHESIS ....................................................................................................... 27
1.2
MAIN GOAL ......................................................................................................... 27
1.3
SPECIFIC GOALS................................................................................................. 27
METHODOLOGY .................................................................................................. 30
2.1
FREE COFACTOR SYSTEMS .............................................................................. 30
2.1.1
Free Cofactor: Method of Calculation ................................................................. 30
2.1.2
Free Cofactor: Basis Sets .................................................................................... 30
2.1.3
Free Cofactor: Geometry Optimizations.............................................................. 30
2.1.4
Folding Angle .................................................................................................... 32
2.1.5
Co-C Bond Dissociation Energy ......................................................................... 32
2.1.6
Chemical Hardness............................................................................................. 32
2.1.7
Free Cofactor: Potential Energy Surface Calculation........................................... 33
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2.2
AdoCbl-GLUTAMATE MUTASE COMPLEX...................................................... 34
2.2.1
GM-AdoCbl complex: Model Design ................................................................. 34
2.2.2
GM-AdoCbl complex: Method of Calculation .................................................... 36
2.2.3
GM-AdoCbl complex: Potential Energy Surface Calculation .............................. 37
2.3
3
ISOMERIZATION REACTION: GLUTAMATE TO METHYLASPARTATE...... 38
2.3.1
Enzyme’s Catalytic Site: Model Design .............................................................. 38
2.3.2
Enzyme’s Catalytic Site: Method of Calculation ................................................. 39
2.3.3
Basis Set............................................................................................................. 40
2.3.4
Transition State and Intermediate of the Fragmentation process .......................... 40
RESULTS AND DISCUSSION ............................................................................. 41
3.1
GROUND STATE MODELS ................................................................................. 41
3.1.1
AdoCbl and MeCbl cofactors: Full Atom Models ............................................... 41
3.1.2
Ado-Corrin side chains intramolecular hydrogen bonds ...................................... 44
3.1.3
Modifications in the Nucleosidic Loop and its effect on the cofactor ................... 45
3.1.4
Effects of α-Axial Base Interchange.................................................................... 46
3.1.5
Sterical Strain between Ado and the Corrin Ring ................................................ 49
3.1.6
Structural Fragments Contributions to the Co-C bond Strength on AdoCbl ......... 49
3.1.7
Electronic Structure and Co-C bond Strength Relationship.................................. 52
3.2
CO-C BOND DISSOCIATION PROCESS............................................................. 58
3.2.1
Potential Energy Surface and Spin Density Analysis........................................... 58
3.2.2
Geometric Distortion .......................................................................................... 63
3.2.3
Electronic Charge Rearrangement due to the Co-C bond Dissociation................. 67
3.3
INSIGHTS INTO THE GM CATALYTIC CYCLE................................................ 72
3.4
CATALYTIC CO-C BOND DISSOCIATION PROCESS ...................................... 75
3.4.1
Role of the Aspartate-14 Residue on the Dissociation Process............................. 75
3.4.2
Role of the Enzymatic Environment on the Co-C Bond Dissociation Process ...... 81
3.4.3
Understanding the Role of the Enzymatic Environment ...................................... 86
3.4.4
The Hydrogen Abstraction Pathway Paradigm .................................................... 88
3.5
ISOMERIZATION REACTION: GLM TO MASP................................................. 91
3.5.1
The carbon Skeleton Rearrangement................................................................... 91
3.5.2
Role of the enzymatic environment on the Isomerization Reaction...................... 95
a)
Role of Glutamate-171........................................................................................ 95
viii
b)
Role of the Arginine Claw................................................................................... 96
c)
Chemical Role of the Binding Site....................................................................... 98
4
CONCLUSIONS ................................................................................................... 100
5
REFERENCES ...................................................................................................... 106
FIGURE INDEX
Figure 1. Free energy profiles comparing enzyme-catalyzed and uncatalyzed reactions. ............ 2
Figure 2. Molecular Structure of Cobalamin active systems. ..................................................... 9
Figure 3. Reactions catalyzed by class I enzymes.................................................................... 13
Figure 4. Generalized accepted mechanism for AdoCbl-dependent isomerization reactions..... 14
Figure 5. Proposed reaction mechanism for the free-radical based 1,2 migrations.................... 15
Figure 6. Reaction mechanism for the isomerization of glutamate to methylasparte catalyzed by
glutamate mutase..................................................................................................................... 16
Figure 7. Molecular Architecture of Glutamate mutase. .......................................................... 17
Figure 8. Molecular representation of the Histidine16 - Aspartate14 Hydrogen Bond. ............. 18
Figure 9. Molecular Representation of the Conformational change from confA to confB......... 20
Figure 10. AdoCbl-Glutamate Mutase complex. ..................................................................... 21
Figure 11. Schematic representation of the Mechano-Chemical Trigger mechanism. ............... 22
Figure 12. Schematic representation of AdoCbl and MeCbl with their corresponding reduced
models. ................................................................................................................................... 31
Figure 13. Glutamate Mutase-substrate (glm) complex............................................................ 38
Figure 14. Schematic representation of the optimized geometries of AdoCbl, MeCbl and their
reduced models. ...................................................................................................................... 51
Figure 15. Representation of the frontier molecular orbitals belonging to the full atom models of
AdoCbl and MeCbl. ................................................................................................................ 54
Figure 16. Molecular orbital (MO) and Co-C bond strength relationship diagram.................... 55
Figure 17. Comparison of the potential energy surfaces for the Co-C bond dissociation process
of the free models.................................................................................................................... 59
Figure 18. Comparison of the spin density profiles along the Co-C dissociation process of the
free models.............................................................................................................................. 60
ix
Figure 19. Comparison of the Co-Nax bond length variations along the Co-C bond dissociation
process of the free models. ...................................................................................................... 64
Figure 20. AdoCbl and MeCbl Spin Density distribution. ....................................................... 65
Figure 21. Energetic contribution of the corrin ring flexibility to the Co-C bond dissociation
process. ................................................................................................................................... 66
Figure 22. Electronic Charge Redistribution on Co atom along the Co-C dissociation process of
the free models. ....................................................................................................................... 69
Figure 23. Electronic Charge Redistribution on C atom along the Co-C dissociation process of
the free models. ....................................................................................................................... 70
Figure 24. Electronic Charge Redistribution on Neqs atom along the Co-C dissociation process
of the free models.................................................................................................................... 71
Figure 25. Schematic representation of the steps of the GM-catalytic cycle modelled in this
study. ...................................................................................................................................... 72
Figure 26. Energy Diagram of the steps of the GM-catalytic cycle modelled in this study. ...... 73
Figure 27. Optimized geometries of the AdoCbl-H16/D14 and AdoCbl-H16/D14 models after the
Co-C bond dissociation process. .............................................................................................. 76
Figure 28. AdoCbl-H16/D14 versus AdoCbl-H16. A) Potential energy surfaces of the Co-C bond
dissociation process B) Spin density profiles along the Co-C dissociation................................ 76
Figure 29. AdoCbl-H16/D14 versus AdoCbl-H16.Comparison of the Co-Nax bond length
variations along the Co-C bond dissociation process. ............................................................... 78
Figure 30. AdoCbl-H16/D14 versus AdoCbl-H16. Comparison of the electronic charge
redistribution along the Co-C dissociation process (A) Nax (B) Co (C) C (D) Neqs .................... 80
Figure 31. Optimized geometries of the AdoCbl-H16/D14 and AdoCbl-GM-H16/D14 models after
the Co-C bond dissociation process. ........................................................................................ 82
Figure 32. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14. A) Potential energy surfaces of the CoC bond dissociation process. B) Spin density profiles along the Co-C dissociation process....... 82
Figure 33. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14.Comparison of the Co-Nax bond length
variations along the Co-C bond dissociation process ................................................................ 84
Figure 34. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14. Comparison of the electronic charge
redistribution along the Co-C dissociation process (A) Nax (B) Co (C) C (D) Neqs .................... 85
Figure 35. Optimized geometries of the AdoCbl-GM-H16 and AdoCbl-GM-H16/D14 models after
the Co-C bond dissociation process ......................................................................................... 86
x
Figure 36. AdoCbl-GM-H16/D14 versus AdoCbl-GM-H16 A) Potential energy surfaces of the CoC bond dissociation process B) Spin density profiles along the Co-C dissociation process........ 87
Figure 37. AdoCbl-H16/D14 versus AdoCbl-GM-H16 A) Potential energy surfaces of the Co-C
bond dissociation. B) Spin density profiles along the Co-C dissociation process. ..................... 88
Figure 38. Molecular Representation of the Step-Wise and the Concerted reaction pathways .. 89
Figure 39. Step-Wise versus Concerted reaction pathway. Comparison of the potential energy
surfaces of the Co-C bond dissociation along the respective reaction pathways. ....................... 90
Figure 40. Schematic representation of the steps associated to the isomerization reaction of
glutamate to methylaspartate ................................................................................................... 91
Figure 41. Optimized geometries of the full atom model at the different steps associated to the
isomerization reaction catalyzed by glutamate mutase.............................................................. 92
Figure 42. Potential energy surface of the substrate carbon-carbon bond dissociated during the
fragmentation stage of the isomerization reaction .................................................................... 93
Figure 43. Calculated energy diagram of the mechanism for the isomerization reaction of
glutamate to methylaspartate. .................................................................................................. 94
Figure 44. Role of the Glu171 residue on the energetic of the GM catalytic mechanism. ......... 96
Figure 45. Role of the Arg claw on the energetic of the GM catalytic mechanism.................... 97
Figure 46. Role of the binding site on the energetic of the GM catalytic mechanism................ 98
TABLE INDEX
Table 1. Selected geometric parameters of the Cobalamin Models presented in this study a .... 41
Table 2. Bond Dissociation Energies (BDE) of the Co-C and Co-Nax axial bondsa .................. 42
Table 3. The Energetic associated to the Co-C bond and its Electronic Structure .................... 53
xi
ABBREVIATIONS
AdoCbl
Desoxyadenosylcobalamin
MeCbl
Methylcobalamin
Cbl(II)
Cobalamin
DMB
5,6-dimethylbenzimidazole
Co-C
cobalt-carbon bond
Co-Nax
cobalt-axial nitrogen bond
Co-Neqs
cobalt-equatorial nitrogens bonds
GM
Glutamate mutase
glm
glutamate (substrate)
masp
methylaspartate (product)
DFT
Density Functional Theory
ECP
Effective Core Potential
BP86
Becke88 –Perdew86 Functional
B3LYP
Becke 3 param-Lee Yang Parr Functional
DZ
Double Zeta
COSMO
Conductor-like Screening Model
PES
Potential Energy Surface
BDE
Bond Dissociation Energy
MO
Molecular Orbital
AO
Atomic Orbital
HOMO
Highest Occupied Molecular Orbital
LUMO
Lowest Unoccupied Molecular Orbital
PDB
Protein Data Bank
F/R
Fragmentation/Recombination Mechanism
Arg Claw
Set of arginines from the catalytic site that interacts with the substrate
xii
AMINO ACID ABBREVIATIONS
A/Ala
Alanine
C/Cys
Cysteine
D/Asp
Aspartate
E/Glu
Glutamate
F/Phe
Phenylalanine
G/Gly
Glycine
H/His
Histidine
I/Ile
Isoleucine
K/Lys
Lysine
L/Leu
Leucine
M/Met
Methionine
N/Asn
Asparagine
P/Pro
Proline
Q/Gln
Glutamine
R/Arg
Arginine
S/Ser
Serine
T/Thr
Threonine
V/Val
Valine
W/Trp
Tryptophan
Y/Tyr
Tyrosine
xiii
MODELS DEFINITION
AdoCbl
Full atom model of the AdoCbl cofactor
AdoCblwoSC
Model of AdoCbl without the a and b side chains of the corrin ring
AdoCblwoloop
Model of AdoCbl without the nucleosidic loop
AdoCblwoloopIMI
Model of AdoCbl without the nucleosidic loop and with the DMB ligand
replaced by an imidazole ring
AdoCblred
Model of AdoCbl without the nucleosidic loop and the side chains of the
corrin ring
AdoCblredIMI
Model of AdoCbl without the side chains and the nucleosidic loop of the
corrin ring, with the DMB ligand replaced by an imidazole ring
MeCbl
Full atom model of the MeCbl cofactor
MeCblwoloop
Model of MeCbl without the nucleosidic loop
MeCblwoloopIMI
Model of MeCbl without the nucleosidic loop and with the DMB ligand
replaced by an imidazole ring
MeCblred
Model of MeCbl without the nucleosidic loop and the side chains of the
corrin ring
MeCblredIMI
Model of MeCbl without the nucleosidic loop and the side chains of the
corrin ring, with the DMB ligand replaced by an imidazole ring
xiv
ABSTRACT
Enzymes accelerate chemical reactions with exceptional selectivity making life itself
possible. Glutamate Mutase (GM) is an adenosylcobalamin (AdoCbl)-dependent enzyme that
catalyzes the reversible isomerization of glutamate (glm) to methylaspartate (masp). The AdoCbl
cofactor consists of an equatorial corrin ring, which has a Co+3 as the metal center axially
coordinated by a deoxyadenosyl moiety and intramolecularly by a 5,6-dimethylbenzimidazole
group. The accepted mechanism for the isomerization reaction begins with substrate binding to
the catalytic site, which induces the homolytic cleavage of the cobalt carbon bond (Co-C) of
AdoCbl, being this the first catalytic event. This generates an adenosyl radical (Ado•) that
subsequently abstracts a hydrogen atom from the substrate, forming a substrate-derived radical
and AdoH. The newly formed radical rearranges to a product-related radical, which eventually
re-abstracts the hydrogen from AdoH, leading into Ado• regeneration. Finally, the catalytic cycle
ends by Co-C bond formation. The generation of highly reactive radical intermediaries makes
this reaction very difficult to occur without side reactions. Thus, the need of proper catalytic
machinery able to control the reaction pathway is mandatory.
An intriguing question in bioinorganic chemistry is how upon substrate binding, the cofactorenzyme complex modifies its electronic structure weakening the Co-C bond strength until its
homolytic dissociation. In addition, the exact reaction pathway that connects the Co-C bond
breaking with the hydrogen abstraction from the substrate remains uncertain. Finally, the exact
role of the enzymatic environment on the isomerization reaction mechanism is far from been
completely understood.
The need of deeper insights about the GM catalytic reaction, especially the control of the CoC bond dissociation process and the mechanism to manage the chemical transformation are
interesting challenges to be solved by computational chemistry approaches.
Here we present a large set of quantum chemical models obtained to answer the questions
above presented. First, we explored the nature of the Co-C bond by the comparative study of
AdoCbl and an analogous cofactor known as methylcobalamin (MeCbl), both in their free forms.
Second, a set of models of AdoCbl in complex with GM were obtained to explore the forces
involved in the catalytic Co-C bond dissociation process and the influence of the substrate on it.
Third, in order to shed light into the driving forces of the catalytic cycle, we performed the study
of models of ground state, key intermediates and transition states of the catalytic cycle using
xv
GM-Ado-glm complexes by means of modern electronic structure calculations. In all the
calculation involving the GM as part of the model, the cluster approach was used.
The studies of the Co-C bond dissociation in complex with the enzyme and the study of the
catalytic cycle were focused on the effect of the nearby enzymatic environment. Our main
results revealed that the Co-C bond is weakened in part by the axial ligand replacement
involving a histidine-aspartate pair, and the electrostatic effect exerted by the lysine-glutamate
pair from the enzyme which stabilizes the Ado• radical. After the formation of the Ado•, the
presence of glm leads to the formation of glm• radical, which is largely more stable than Ado•.
Thus, glm has an important role on the propagation of the catalytic cycle toward the product
formation. Finally, our findings revealed that the near enzymatic environment provides
differential stabilization of the reaction intermediaries and transition state mediated by the
properties of the interactions with the catalytic site. Additionally, it provides of an important
structural support. This understanding would allow in the future the design of bio-mimetic able
to catalyze highly complex reactions.
xvi
RESUMEN
Las enzimas son capaces de acelerar reacciones químicas con una gran selectividad,
permitiendo que la vida sea posible como la conocemos. Glutamato mutasa (GM) es una enzima
dependiente de adenosilcobalamina (AdoCbl), la cual cataliza la isomerización reversible de
glutamato (glm) a metilaspartato (masp). AdoCbl es un cofactor que consiste en un macrociclo
ubicado en el plano ecuatorial denominado corrina, el cual tiene como átomo central un Co+3.
Este metal está axialmente coordinado por un ligando desoxiadenosina e intramolecularmente
por un grupo 5,6-dimetilbenzimidazol. El mecanismo propuesto para la reacción de
isomerización comienza con la unión del sustrato al sitio catalítico, la cual induce la ruptura
homolítica del enlace cobalto-carbono (Co-C) del cofactor. Como producto de esta disociación
homolítica se forma el radical desoxiadenosilo (Ado•) que luego abstrae un hidrógeno desde el
sustrato, formando un radical derivado del sustrato y AdoH. Este radical sufre un
reordenamiento que da origen al radical relacionado con el producto, el cual reabstrae el
hidrógeno desde AdoH, regenerando Ado•. El ciclo catalítico termina con la formación del
enlace Co-C. La generación de intermediarios radicalarios altamente reactivos hace que esta
reacción sea muy difícil de llevar a cabo sin reacciones secundarias. Es así que la necesidad de
una maquinaria catalítica capaz de controlar los caminos de reacción sea indispensable.
Una interesante pregunta en química bioinorgánica es cómo luego de la unión del sustrato, el
complejo cofactor-enzima es capaz de modificar su estructura electrónica debilitando el enlace
Co-C hasta su disociación homolítica. Además, el camino de reacción exacto que conecta el
rompimiento del enlace Co-C con el proceso de abstracción del hidrógeno desde el sustrato
permanece incierto. Finalmente, el papel exacto que cumple el entorno enzimático en el
mecanismo de isomerización está lejos de ser completamente comprendido. La necesidad de una
mayor comprensión del mecanismo catalítico de GM, especialmente el proceso de control de la
ruptura del enlace Co-C y el mecanismo para controlar la transformación química catalizada por
GM son interesantes problemas para ser resueltos por métodos químico computacionales.
En este trabajo presentamos un conjunto de modelos químico-cuánticos obtenidos con el
propósito de responder a las preguntar presentadas anteriormente. Primero, exploramos la
naturaleza del enlace Co-C mediante en estudio comparativo de AdoCbl y un cofactor análogo
conocido como MeCbl, ambos en sus formas libres. Segundo, obtuvimos un conjunto de
modelos del cofactor unido a GM para explorar las fuerzas involucradas en el proceso de ruptura
xvii
catalítica del enlace Co-C, junto con la influencia del sustrato en esta. Tercero, con el fin de
comprender las fuerzas que manejan el ciclo catalítico, llevamos a cabo el estudio del estado
fundamental, intermediarios clave y estados de transición del ciclo catalítico usando modelos del
complejo GM-Ado-glm obtenidos a partir de cálculos de estructura electrónica. Los estudios de
la disociación del enlace Co-C en complejo con la enzima y del ciclo catalítico fueron enfocados
en el efecto del entorno enzimático cercano. La aproximación de cluster fue usando en todos los
cálculos que involucraron a GM como parte del modelo.
Nuestros principales resultados revelaron que el enlace Co-C es debilitado por el reemplazo
de base axial que sufre luego de unirse a la enzima por el par histidina-aspartato. Además existe
un efecto electrostático ejercido por el par lisina-glutamato de la enzima que estabiliza al radical
Ado•. La presencia de glm tiene un papel importante luego de la formación del radical Ado• ya
que permite la formación del radical un radical glm• que es más estable, propagando el ciclo
catalítico hacia la formación del producto. Finalmente, nuestros hallazgos revelaron que el
entorno enzimático conduce la reacción de isomerización mediante una estabilización diferencial
de los intermediarios y estados de transición asociados a la reacción. Además provee de un
importante soporte estructural. Los antecedentes entregados en este trabajo permitirán en el
futuro el diseño de bio-miméticos capaces de catalizar reacciones químicas extremadamente
complejas.
xviii
Introduction
1 INTRODUCTION
Enzymes and Quantum Bioinorganic Chemistry
Nature, as the architect of life, has been able through evolution to design and develop a
particular entity in charge of efficiently carry out chemical transformations that are difficult and
in some cases impossible to perform in a laboratory. The importance of these entities lies on the
fact that without them life would be unsustainable. Nowadays, these are known as enzymes
which are not less than highly evolved molecular structures that could be considered as nature’s
own molecular technology.
Enzymes are capable to selectively catalyze chemical reactions by mechanisms that have
been subject of intense studies, even though there is still much about these intriguing systems
under the shadow of mystery.
Considering that enzymes were created and improved during the course of millions of years
of evolution, its understanding will build the basis for the efficient manipulation of nature
resources, like energy conversion and efficient electron transfer devises, bio-mimetics for
catalysis of highly complex chemical reactions, and even the designing of inhibitors for diseases
on which enzymes malfunction are involved among others.
The mechanism of an enzymatic reaction can be studied through its free energy profile, as
depicted on Figure 1, where the energy profile of an enzymatic reaction is compared with its
≠
) and
uncatalyzed analogous. The difference between the energy barrier of the catalyzed ( ∆Gcat
≠
) corresponds to the catalytic power of the enzyme. In terms of
the uncatalyzed reaction ( ∆Guncat
a more accurate description of enzyme catalysis proficiency, it has been defined as the ratio of
the second-order constant of the enzymatic reaction (kcat/KM) and the rate constant of the same
uncatalyzed reaction in neutral aqueous solution (kuncat) [1].
The comprehension of the mechanisms used by enzymes to carry out the catalysis of
chemical reactions, begins with the understanding of how the inter-molecular interactions
formed between the enzyme and the substrate are able to initiate and propagate the processes of
chemical bond breaking and bond forming on the substrate. Thus, to describe how enzymes
work, it is necessary to do it at a molecular level.
1
Introduction
Figure 1. Free energy profiles comparing enzyme-catalyzed and uncatalyzed reactions.
≠
≠
∆Gcat
and ∆Guncat correspond to the activation energy for the catalyzed and the uncatalyzed
≠
reactions, respectively. ∆∆G is defined as the catalytic power of an enzyme. ∆G0 is the
driving force of the reaction.
Since the mid-twenty century, it is well known that atoms and molecules do not follow a
classical behavior as defined by Newton’s laws. Thus, the need of a theory able to describe the
behavior of molecular electronic structure is mandatory to understand the enzymes mechanisms.
Nowadays, Molecular quantum mechanics, also known as quantum chemistry is available and it
provides of a robust theory to model enzyme mechanisms. Quantum chemical methods are able
to give profound mechanistic insights through the study of diverse reaction pathways, describing
the behavior of the electronic structure along these pathways, and testing their energetic
2
Introduction
feasibility. Additionally, quantum chemical methods allow inferring the role of the residues of
the catalytic site of the enzyme on the reaction mechanism, residues that normally correspond to
a small region of the enzyme. Regarding to the constitution of the catalytic site, there are
enzymes whose catalytic machinery is formed only by amino-acid residues, being the most
common type of enzymes. Also, there are enzymes that depend on the binding of one or more
transition metals into their catalytic site in order to be catalytically active. These are known as
metalloenzymes. Finally, there is a group of enzymes known as coenzymes, which need to be
bonded to an additional molecular system to be activated. This additional structure is known as
cofactor, and some of them have a transition metal on its structure, adding more complexity to
the system. The complexity of the latter type of enzymes has attracted the attention of several
researchers around the world.
The branch of chemistry devoted to the computational study of this family of coenzymes
together with metalloenzymes is commonly known as quantum bioinorganic chemistry. Among
the wide variety of approaches to study these systems, one that has proven to be quite powerful
and efficient in the modeling of enzyme active site and reaction mechanism is the quantum
chemical cluster approach. It consists on the design and study of reduced models of the enzymecofactor complex modeled by computational methods. This approach requires including into the
model as many atoms of the active site as possible to accurately modeling the effects that the
active site has on the substrate. This can become a problem when the number of atoms is too
large, as a consequence of the high computational cost that this type of calculations demands.
Presently, this can be afforded due to the development of the Density Functional Theory (DFT)
[2], which allows to provide good insights into the enzymes problematic.
Density Functional Theory
Traditional ab initio quantum chemical methods are mainly derived from the Hartree-Fock
approach. This method is based on the calculation of the systems wavefunction, which depends
on 3N spatial variables for the definition of electrons location and N for the spin variable, with a
total of 4N variables, being computationally very expensive, limiting the study to a small amount
of atoms. This motivated the development of new methods to study more efficiently the
electronic structure of larger molecular systems.
3
Introduction
In 1964 Hohenberg and Kohn stated that the non-degenerate ground state are determined by its
electron probability density ρ ( r ) , which is only function of the 3 spatial coordinates that
defines it ( r = x, y , z ).Thus, the ground state energy of a molecular system is a functional of the
electron density and it can be written as follows
E0 = E0  ρ ( r ) 
(1)
According to the molecular Hamiltonian
H=
1 n 2 n m Zα
1
∇i − ∑∑
+ ∑∑
∑
2 i =1
i =1 α riα
i i > j rij
(2)
The energy is defined by the kinetic energy of the electrons denoted in the first term, the second
term defined as the attractive potential between the electrons and the nuclei, and the repulsion
between the electrons of the molecule which is the last term of the operator. As stated by
Hohenberg and Kohn the energy and consequently its components depend on the electron
density. Then according to the equation (1) the energy can be written as follows
E0 [ ρ 0 ] = T [ ρ 0 ] + V ne [ ρ 0 ] + V ee [ ρ 0 ]
(3)
The second term of equation (3) at the right-hand side is defined as the external potential
originated by the nuclei of the molecule acting over the electrons. This is determined by the
positions of the nuclei from the molecule. If the nuclei positions are kept fixed, the external
potential is only function of the electron density. Then, using the second term of the Hamiltonian
and considering that the electron density is continuous, the Vne [ ρ 0 ] can be expressed as follows
Vne [ ρ 0 ] = ∫ ρ 0 ( r ) v ( r ) dr
(4)
4
Introduction
n
v ( r ) = −∑
i =1
Zα
riα
(5)
Then, in order to calculate the T [ ρ 0 ] and the V ee [ ρ 0 ] terms remaining from equation (3),
Kohn and Sham considered a system of N non-interacting electrons defined as S model, which
has the same external potential than the real systems, and therefore the same electron density. As
expected, the electron density of the S model can be obtained though a set of molecular orbitals
able to describe the S model known as Kohn-Sham orbitals or θ iKS . This is summarized by the
following expression:
N
ρ 0 = ρ s = ∑ θiKS
2
(4)
i =1
Then, the kinetic energy of the S model can be defined using the θiKS as follows
Ts [ ρ ] = −
1
∑ θiKS ( r ) ∇2 θiKS ( r )
2 i
(5)
being the exact T [ ρ 0 ] defined by
T [ ρ 0 ] = Txc [ ρ ] + Ts [ ρ ]
(6)
where Txc [ ρ ] is the energy difference between the non-interacting system and the real system.
The exact electronic repulsion can be obtained following a similar procedure as for the kinetic
energy using the classical definition of the electronic repulsion.
V ee [ ρ 0 ] = Vxc [ ρ 0 ] +
1 ρ ( r1 ) ρ ( r2 )
dr1dr2
2 ∫∫
r12
(7)
5
Introduction
Finally, the two terms needed to obtain the exact energy, namely Txc [ ρ ] and Vxc [ ρ 0 ] are
grouped into a term known as the exchange-correlation functional:
E xc [ ρ ] = Txc [ ρ ] + Vxc [ ρ ]
(8)
Leading to the final expression for the calculation of the ground state energy as a function of the
electron density
E0 [ ρ 0 ] = −
1
1 ρ ( r1 ) ρ ( r2 )
θiKS ( r ) ∇2 θiKS ( r ) + ∫ ρ 0 ( r ) v ( r ) dr + ∫∫
dr1dr2 + E xc [ ρ ]
∑
r12
2 i
2
(9)
This equation allows calculating the exact ground state energy of the molecular systems. The
problem is that the E xc [ ρ ] functional is unknown and in fact only approximated forms of this
functional are used. Thus, the accuracy of the obtained energy closely depends on the final form
of the chosen E xc [ ρ ] functional together with the type of system under study. A complete
survey of the derivation and the final form of several exchange-correlation functionals can be
found on the “Density-Functional Theory of Atoms and Molecules from Parr and Yang” [2] and
the “A Chemist’s Guide to Density Functional Theory from Koch and Holthausen”[3] books.
DFT has become one of the most important advances in the field of computational chemistry,
permitting to handle models within 100 and 250 atoms at reasonable levels of accuracy and
speed. Also, due to the large number of electrons of transition metals elements, other approaches
have been developed to treat these complex systems reducing the computational cost, but
maintaining the high level of theory without a significant lost of accuracy. The most popular
approach is the pseudopotentials (PPs) [4], which divide the atom shells between a set of outer
electrons (that not necessarily match with the valence electrons), and a second group defined as
the core electrons. The latter set is modeled by effective core potentials (ECPs) carefully
parameterized to reproduce experimental results in combination with an appropriated basis set,
thus reducing the number of electrons of the system explicitly treated. The use of DFT and PPs,
accompanied by the constant advances on the development of more efficient computational
6
Introduction
hardware and software, are responsible for the continuous growing in the knowledge of
enzymatic catalysis, increasing the interest into the field of quantum bioinorganic chemistry.
The availability of this combination of methods through several electronic structure software
packages allows for studying very challenging systems. Nonetheless, because of the
approximations inherent to the DFT approach, it is essential that the results obtained from DFT
calculations to be validated on the basis of experimental data. The Cobalamin-dependent
enzymes, which are the focus of our research, are among the most fascinating systems inside the
scope of quantum bioinorganic chemistry. Firstly, this cofactor has been stated by several
experts on the bioinorganic field as one of the most beautiful cofactors in nature [5] and it was
the first biologically relevant system found in nature with a metal-carbon bond in its structure.
Secondly, as it will be discussed later, the types of chemical reactions that this group of enzymes
is able to catalyze are among the most complex chemical transformations observed in nature.
Next, a brief description of the cobalt as a transition metal is presented, which is the metal
involved in the organometallic bond. This is followed by the description of the cofactor,
continued by a complete discussion of the theoretical and experimental background regarding to
cobalamin-dependent enzymes aiming to understand the intricate nature of these cofactors and to
establish the actual state of knowledge about their catalytic mechanism.
Cobalt
Cobalt is a first-row transition metal with atomic number 27 and electron configuration
[Ar]4s23d7. Like Fe and Ni, the usual oxidation states of cobalt are Co+2 and Co+3.
In the presence of six ligands, Co+3 is normally arranged in octahedral coordination geometry
with a large splitting of d orbitals. These complexes can be strong oxidizing agents. Meanwhile,
Co+2 is mainly found adopting two possible coordination geometries, octahedral and tetrahedral;
but it is also found in square pyramidal configuration as observed in cobalamins during their
catalytic process. This is the most common oxidation state for simple cobalt compounds.
Interestingly, it has been found cobalt complexes with Co+1 oxidation state which correspond to
a superreduced state of cobalt. These complexes turned out to be one of the most powerful
nucleofiles known, and it is believed that they are favored by a square planar configuration.
7
Introduction
Cobalt compounds are widely used as oxidation catalyst along with other chemical reactions.
Among these species, there are several organometallic compounds that include a cobalt-carbon
bond and which are efficient catalyst of several reactions. These organocobalt compounds are
able of catalyzing reactions such as the carbonylation of azobenzene, radical mediated
polymerization and hydroformylation of alkenes are only a few examples of the type of chemical
reactions that these compounds are capable of carry out.
Cobalamins
Cobalamins are involved in several enzymatic reactions [6,7] and are among the most
complex organometallic cofactors.[8] Upon crystallographic determination [9-11], their intricate
structure was revealed. They consists of a tetrapyrrole macrocycle named corrin ring, which in
the ground state bear four equatorial nitrogens coordinating a low spin Co+3.
This transition metal is essential to maintain life and it is mainly found as part of cobalamins, but
in some cases it is directly interacting with the active site of enzymes. The complex between
cobalt and the corrin ring provides of the necessary properties that allow generating a very
especial organometallic bond with σ-character, whose stability can be controlled by the
cobalamin-dependent enzymes. This is the key property that nature uses to trigger very complex
reaction mechanisms as it will be further explained and this is not accomplishable by other
transition metals such as Zn, Fe or Cu.
In particular, the corrin ring has 10 saturated carbons which provide certain flexibility to the
cofactor and a lower electron delocalization making it different from other similar macrocycles
biologically relevant, such as porphyrins. The amount of electron delocalization seems to be
crucial for the stabilization of the organometallic bond, being the corrin ring able to provide the
exact electron delocalization in order to obtain the bond strength within the range needed to
carry out the catalytic role of cobalamins. Neither larger nor lower electron delocalization is able
to supply with the bond strength useful for the type of chemical transformations performed by
cobalamins.
Attached to the corrin ring there are seven amide chains named a- through g- (see Figure 2).
On the lower side of the macrocycle (α-side), the metal center is intramolecularly coordinated by
a 5,6-dimethylbenzimidazole (DMB), group which in turn is linked by a nucleoside loop (f-side
chain) to the corrin ring. Meanwhile, on the β-side (upper), the catalytically active forms of
8
Introduction
cobalamin may be either coordinated by a deoxyadenosyl or a methyl group leading to AdoCbl
or MeCbl cofactors, respectively.
Figure 2. Molecular Structure of Cobalamin active systems.
R = deoxyadenosyl for AdoCbl and R = methyl for MeCbl cofactor. The numbering in
deoxyadenosyl moiety is just for analysis purposes. (A-D) designate the ring order and (a-g)
define the identity of the corrin side chains.
In both cases a cobalt-carbon (Co-C) bond is formed, but despite of their similar structure,
there are slight differences on the nature of AdoCbl and MeCbl Co-C bond that defines their
ability to catalyze different chemical reactions. AdoCbl catalyzes isomerization reactions
through the intramolecular 1,2 shift of a hydrogen atom and a variable group on vicinal carbons,
9
Introduction
ranging from heteroatoms to carbon skeleton fragments [12]; whereas MeCbl catalyzes methyl
cation transfer reactions to the substrate, through the heterolytic Co-C bond dissociation.[13]
Remarkably, an intrinsic difference in the free form of these two cobalamins resides in their
experimentally measured values of Co-C BDE. In AdoCbl, this value corresponds to 31.4 ± 1.5
kcal/mol [14], while for MeCbl it corresponds to 36.3 ± 3.0 kcal/mol. [15] The ca. 5 kcal/mol
BDE difference is one of the observable consequences related to the differential nature of the
Co-C bond between these cofactors.
The balance between the stability of the cofactor and the weak-nature/high-reactivity of the
Co-C bond leads to consider cobalamins as an energy rich agent. It is highly probable that the
enzymatic environment exploits not only the structural but the BDE difference leading to two
completely different dissociation mechanisms. Considering that only the experimental homolytic
dissociation is available for the free form, it can be used as a guide to determine the source of the
nature of bond differences in these cobalamins. Different types of β-axial ligand and their
influence on the geometry and the electronic structure properties of cobalamins have been
thoroughly explored by computational studies. For a revision of latest improvement on the field
of cobalamins computational studies, two recent reviews have been published [16,17].
During the last two decades huge efforts have been devoted on reproducing BDE for these
cofactors using different levels of theory and model sizes. The results pointed out the minor
contributions of the solvent effects and zero point energy corrections to the BDE, together with
the best suited level of theory.[18,19] The relevance of using a complete model to obtain
accurate BDE values has also been discussed. [20-23] Important contributions into this field
have been done by Maseras et. al. [22,24,25] exploring the differences between AdoCbl and
MeCbl that could yield insights into the corresponding bond dissociation mechanisms.
Nevertheless, the answer to the question on how the β-axial ligand leads to different bond
dissociation energies still needs a deeper understanding.
From the structural point of view, the normal and an inverse trans-axial effects between the
Co-C and the Co-Nax bonds (opposite side effects) [26-28] together with the cis steric effect on
the Co-C bond (same side influence) [29] have been observed. Both effects lead to the
conclusion that the behavior of these bonds mainly depends on the balance between the size and
electron donor ability of the β-axial ligand. In addition, the effect that β-axial ligands with
different electron donor abilities have on the electronic structure have been studied by means of
10
Introduction
a complete molecular orbital (MO) picture [27], showing a relationship between the increase of
the electron withdrawal ability and a general orbital energy stabilization.
Cobalamin-dependent Enzymes
There are enzymatic chemical transformations occurring in nature that can be performed in a
laboratory without the aid of enzymatic machinery, but with a considerable effort and at a
reduced rate compared to the catalyzed analogous. However, there are catalytic reactions that are
not chemically plausible without the assistance of an enzyme, because of the increased
complexity involved in the chemical transformation. As the rate of the uncatalyzed reaction is
zero because it is not viable, it can be argued that the rate acceleration is theoretically infinite.
Cobalamin dependent enzymes belong to this fascinating group of enzymes, being able to
catalyze chemically improbable reactions.
AdoCbl is known to be involved in the catalysis of about a dozen enzymatic reactions, such
as substrate isomerization rearrangements. On the other hand, MeCbl acts as an intermediate
methyl donor catalyzing methyl transfer reactions. At this point, our discussion will be focused
on the study of AdoCbl-dependent enzymatic reactions.
These enzymes are classified into three main classes; distinguished by the nature of the
migrating group of the substrate and the nature of the substituent on the carbon atom to which
this group migrates. Firstly, the class I enzymes also known as mutases, includes enzymes such
as methylmalonyl-CoA mutase, glutamate mutase, 2-methyleneglutarate mutase, and
isobutyrylCoA mutase, being all able to catalyze carbon skeleton rearrangements. Secondly, the
class II enzymes or the eliminases, includes the diol dehydratase, glycerol dehydratase, and
ethanolamine ammonia lyase. This class incorporates the AdoCbl-dependent ribonucleotide
reductases
into
the
group,
which
catalyzes
the
reduction
of
ribonucleotides
to
deoxyribonucleotides. Thirdly, the class III enzymes or aminomutases. The two known
aminomutases are the D-lysine-5,6-aminomutase and the D-ornithine mutase, enzymes able to
catalyze amino group migrations. It should be stated that this last group needs piridoxal
phosphate in addition to AdoCbl. All of these enzymes share a common feature on their
enzymatic mechanisms, which is that the first event in their catalytic cycle corresponds to the
homolytic dissociation of the Co-C bond that forms part of the cofactor. As pointed above, the
energy needed to afford the Co-C bond cleavage of the free cofactor is of 31.4 kcal/mol, with an
11
Introduction
associated rate constant of ~ 10-9 s-1 [30]. However, when the cofactor is in complex with the
enzyme, the catalytic rate for the dissociation process is of ~ 2-300 s-1 [31], suggesting that the
enzyme is able to accelerate the Co-C homolytic bond dissociation reaction by a factor of ~1012
[32,33] in order to start the catalytic cycle.
A striking difference between the mutases (class I) and the eliminases (class II) enzymes is
the intolerance of the mutases toward structural modifications of the cofactor, such as in the
adenosyl moiety, the corrin ring and its side chains. The only changes that are allowed for any
activity were on the nucleosidic loop [34]. Modifications of the adenosyl moiety yielded to
completely inactive systems [35], which expose the relevance of this moiety on the mutases
catalytic mechanism. The intolerance to structural modifications should be related to the
formation of very specific and tight intermolecular interactions between the cofactor and the
enzyme. This represents an advantage when studying these systems, because it makes easier to
relate small conformational changes to mechanistic insights so as to define their role on the
process.
Class I enzymes has a vast amount of experimental data available, including crystallographic
structures. This makes them an attractive focus of study through computational models for the
search of a deeper understanding of the cobalamin-dependant enzymes mechanisms.
Class I enzymes are commonly defined as carbon skeleton isomerases and their mechanisms can
be generalized by a 1,2 interchange between a hydrogen atom bonded to a carbon atom and an
alkyl group bonded to a vicinal carbon atom. The reactions catalyzed by class I enzymes are
shown in Figure 3, where is denoted that three of the four carbon skeleton isomerases involve a
migrating group with a sp2-hybridized carbon. Meanwhile, glutamate mutase catalyzes the
migration of a sp3-hybridized glycil moiety.
As stated before, the first catalytic event is the Co-C bond dissociation which gives origin to
two radical species, the adenosyl radical and the metal centered Cbl(II) radical, where the former
subsequently abstracts a hydrogen atom from the substrate generating the substrate-derived
radical. The latter rearranges until the product-related radical is obtained, which retrieves the
hydrogen atom from the adenosyl radical with the formation of the product and the posterior
reconstitution of the Co-C bond that defines the end of the catalytic cycle. This corresponds to
the generalization of the mechanism for the carbon skeleton isomerases and it is displayed on
Figure 4.
12
Introduction
Methylmalonyl-CoA mutase
CH3
CO2
-
CO2-
CoA-S
O
CoA-S
O
Isobutyryl-CoA mutase
CH3
CH3
CoA-S
CH3
O
CoA-S
O
Glutamate mutase
-
H3C
CO2-
O2 C
NH3+
H3C
CO2-
CO2-
O2 C
NH3+
-
Methyleneglutarate mutase
-
O2 C
CO2-
-
O2 C
Figure 3. Reactions catalyzed by class I enzymes.
The migrating groups of the isomerization reactions are highlighted in red (adapted from
Banerjee, Chem Rev 2003 [12]).
The way that nature solves how to accomplish these highly complex chemical
transformations by using free radicals as intermediates species is remarkably. However, it is
even more impressive how the enzymatic environment is able to precisely control the radical
intermediates avoiding unwanted side reactions that might inactivate the enzyme, which is a
question that has fascinated the scientific community to a large extent.
13
Introduction
X
H
X
.
Ado
Co+3
Co+2
.
Ado
+
H
Ado
H
X
X
.
Figure 4. Generalized accepted mechanism for AdoCbl-dependent isomerization reactions.
The red X represents the migrating group, and the blue H represents the hydrogen with which
the carbon skeleton group is interchanged (adapted from Radon, Acc Chem Res 2009 [36])
The rearrangement reaction from substrate-derived to product-related radical follows
sophisticated mechanisms to surmount the energetic barriers involved in these transformations,
and to propagate the reaction into the forward direction of the catalytic cycle. Some of the most
accepted proposed mechanisms according to Radon et. al. [36] are depicted on Figure 5.
These mechanistic pathways require of an unsaturated bond to be plausible, and that is the
main reason why it is expected some resemblance between the mechanisms of methylmalonylCoA mutase, 2-methyleneglutarate mutase, and isobutyrylCoA mutase. Nevertheless, neither of
these proposed mechanisms is completely accepted, and changes can be introduced during the
course of years from future research works. For the particular case of glutamate mutase, the
proposed mechanism involves a particular pathway due to its sp3-hybridized carbon from its
migrating group. This pathway detailed on Figure 6, can be described by the initial cleavage of
the carbon-carbon bond that holds the migrating group to the rest of the substrate structure,
generating two species, a glycyl radical and an acrylate ion [37]. These two fragments recombine
by the attack of the glycyl radical to the adjacent carbon atom to give the rearranged productrelated radical. This is defined as fragmentation-recombination (F/R) mechanism [38,39]. Even
though there is evidence that suggest that it could also be related to 2-methyleneglutarate mutase
enzymatic mechanism, the only model system for which has been proved to be energetically
plausible is for the glutamate mutase reaction.
14
Introduction
X
C
X
CR
C
C
.
.
C
C
X
C
H+
X
C
+
CRH+
HX
C
C
.
.
C
C
X
C
X
A/E
C
C
C
X
C
.
.
C
C
F/R
C
X
.
C
C
X
X
XH+
.
.
.
X
C
C
.
C
C
C
X
C
.
C
C
X
C
C
X
C
Figure 5. Proposed reaction mechanism for the free-radical based 1,2 migrations.
The red X corresponds to the migrating group. CR = Concerted Reaction; CRH+ = Concerted
Reaction assisted by protonation of the substrate; A/E = Addition-Elimination reaction; F/R =
Fragmentation-Recombination reaction.
Among the class I enzymes, glutamate mutase represents an interesting problem system for
the study of AdoCbl-dependent enzymes, due to the availability of the crystal structure of the
AdoCbl-enzyme-substrate complex in several stages of the catalytic cycle, as it will be describe
next. This will allow us to explore thoroughly several mechanistic insights regarding to the
catalytic process of glutamate mutase, with focus in the Co-C bond dissociation mechanism.
15
Introduction
Figure 6. Reaction mechanism for the isomerization of glutamate to methylasparte
catalyzed by glutamate mutase.
Glutamate mutase
Glutamate mutase (GM) catalyzes the reversible interconversion of (S)-glutamate to (2S,3S)3-methylaspartate [40]. Its molecular architecture [41,42] displayed on Figure 7, denotes a stable
heterotetramer constituted by two larger subunits termed ε, connected with each other primarily
by hydrophobic contacts. The remaining two subunits are defined as σ, and they are located at
opposite ends of the ε2 dimer, without direct contact between them. The σ subunits are smaller
than the ε domain, and they are responsible for the AdoCbl binding, which take place inside the
εσ-interfaces. As there are two εσ-interfaces, each enzyme allows for the binding of two
16
Introduction
cofactors simultaneously, staying apart by a distance of 42.5 Å with regards to their cobalt
centers. On the other hand, the ε subunit holds the catalytic site for the substrate binding.
The σ peptide folds as α/β domain with a β sheet consisting of five parallel strands encased
by six α helices. On the other hand, the ε subunit consist of an (α/β)8-barrel (TIM barrel) domain,
where one end of the barrel locates against the upper face of the AdoCbl cofactor when it is in
complex with the enzyme, thus forming the cavity for the substrate corresponding to the
catalytic site.
Figure 7. Molecular Architecture of Glutamate mutase.
The enzyme is bound to two AdoCbl cofactors. Also, the substrate binding site (catalytic site)
and the hydrophobic region that holds for the DMB ligand are denoted on the image.
A fascinating feature of GM is that when the cofactor binds to the σ subunit, the DMB group
intramolecularly coordinated to the metal center is displaced toward a hydrophobic pocket, being
replaced by the imidazole ring of a histidine residue (His16) of the same subunit. This residue
17
Introduction
coordinates with the cobalt through its Nε [43-45]. This new configuration is known as “His-on,
base-off” mode and it is a common feature between the class I and class III enzymes [46,47].
Additionally, the nucleosidic loop is located in a region of the enzyme where it is stabilized by
the interaction with several water molecules. Meanwhile, the class II enzymes bind the cofactor
keeping the DMB ligand coordinated to the cobalt center. For the latter case, it is said that they
bind in a “base-on” conformation.
The histidine involved in the base replacement is part of a consensus sequence (DxHxxG)
present in all the enzymes that binds in the “His-on, base-off” mode. Additionally to the
histidine, this sequence includes an aspartate residue which in glutamate mutase corresponds to
Asp14, and forms a hydrogen bond with the hydrogen from the Nδ of His16 (see Figure 8). The
role of the highly conserved His-Asp pair in class I enzymes has been studied by March and
collaborators [48], using glutamate mutase as a model enzyme. They found that the mutation of
either Asp14 or His16 lead into a 1000-fold decrease in the reaction rate. The mechanistic role of
this pair in the Co-C bond labilization is still unclear and needs to be studied.
Figure 8. Molecular representation of the Histidine16 - Aspartate14 Hydrogen Bond.
In this particular case R = deoxyadenosyl.
18
Introduction
The crystal structure of glutamate mutase in complex with the cofactor and its substrate is
only available with the Co-C bond of AdoCbl in its dissociated form [41]. Interestingly, the
adenosyl moiety displayed two different conformations within the same crystal structure (see
Figure 9). The first conformation was with the Ado-C5’ from the Co-C bond, positioned directly
above the metal center at ~ 3.2 Å with the rybosyl moiety in a C2’-endo conformation. This
seems to be the activated form of the cofactor immediately after the Co-C bond cleavage, which
is defined as conformation-A (confA). The second termed as conformation-B (confB), is
characterized by the rybosyl moiety in a C3’-endo conformation. The C5’ atom is pointing
directly to the catalytic site, at ~ 4.5 Å from the metal center and it is within the van der Waals
distance with regards to the substrate, being properly positioned for hydrogen abstraction. These
two conformational states of the adenosyl group are controlled through the variation of the
torsion angle about the glycosidic bond by ~ 25°, leading into two conformers where the C5’
from each conformation are separated by 1.7 Å of distance, which are thought to be
intermediates of the catalytic reaction.
The stabilization of the ribosyl conformers are assisted by the tight interactions of the
adenosyl group with the enzymatic environment, which are similar between the two conformers.
These interactions comprise of hydrogen bonds between the adenine and the backbone atoms of
Gly68 and Asn123, interactions with the c-acetamide side chain from the corrin ring, and
hydrogen bonds formed by the rybosyl hydroxyl groups with Glu330, Lys326 and a water
molecule (see Figure 10).
Normally, the enzyme mechanism is expected to be defined by the stabilization of the
transition state of the reaction, as stated by Pauling in 1946 [49], leading to an increase of the
reaction rate. Meanwhile, in addition to the transition state stabilization, the catalytic mechanism
of GM involves the control of highly reactive radical intermediates formed in response to these
hard chemical transformations. This statement implies that GM is more devoted to the chemical
control rather than the reaction rate acceleration. This unusual catalytic mechanism is an
example of what is defined as “negative catalysis” firstly proposed by János Rétey in 1990 [50].
In essence, the low barrier for the transition between confA and confB allows to restrain the
trajectory of the radical centered on C5’ atom formed directly after the Co-C bond cleavage,
avoiding undesired reactions [51].
19
Introduction
C2-endo
confA
confB
glm
C5’
C3-endo
C5’
Figure 9. Molecular Representation of the Conformational change from confA to confB.
These two conformations of Ado are included in the GM crystal structure. (Left) On green it is
the conformation adopted after the Co-C bond dissociation. Then, the ribose suffered a
conformational change adopting the conformation displayed on light blue, preparing the Ado•
radical for hydrogen abstraction. (Right) Ribose moiety from C2-endo to C3-endo conformation.
After the cleavage of the Co-C bond and the abstraction of a hydrogen atom from the
substrate, GM catalyzes the carbon skeleton rearrangement of glutamate (glm) to
methylaspartate (masp) by a fragmentation-recombination mechanism. To achieve such complex
reaction mechanism on which a carbon-carbon single bond is first cleavaged and then
recombined in a different fashion, the catalytic site needs to be capable of two tasks. First, it
should be able to induce on the substrate a non-favored extended conformation by strongly
attaching it. Second; the catalytic site has to control the fragmentation stage which by no means
is a trivial task.
20
Introduction
Figure 10. AdoCbl-Glutamate Mutase complex.
The residues of the binding site that interact directly with the cofactor are highlighted in light
green and the substrate (glm) is in dark green.
Proposed mechanisms for the Co-C bond weakening
There are several hypotheses regarding how the enzyme manages to cleave the Co-C bond.
Nevertheless, there are two dominant trends of research. The first involves the idea of the
destabilization of the cofactor after its binding to the enzyme. This trend also contemplates the
possibility of a minor destabilization after the cofactor binds to the enzyme, followed by a larger
destabilization after substrate binding, which should trigger the Co-C bond dissociation. The
second trend considers the stabilization of the radical species formed as product of the bond
21
Introduction
cleavage. Presently, the idea of a mechanism with contributions from both hypotheses it is also
supported.
The first mechanism proposed for the Co-C bond dissociation process on Cobalamins and
one of the most studied corresponds to the mechano-chemical trigger mechanism, which is
related to the first trend. Several theoretical reports have focused their attention into this
mechanism either to confirm or to discard its contribution into the process [52-58]. This
mechanism corresponds to an initial compression of the cobalt axial-nitrogen bond (Co-Nax)
mediated by the enzymatic environment. This contributes to the Co-C bond weakening and to
the posterior cleavage, due to the increase of the electron density on the cobalt induced by the αaxial ligand, decreasing the σ-donation ability of the β-axial ligand into the Co bonding orbitals.
The initial Co-Nax compression should also induce an upward folding of the corrin ring pushing
away the β-axial ligand by a cis steric effect, assisting the Co-C bond dissociation. A schematic
representation of the mechanism is showed on Figure 11. Results obtained by means of
molecular mechanics [59,60] and semiempirical methods [60,61] have revealed that the effect of
the side chains and the nucleotide loop on the general geometry is negligible, arguing against
this mechanism.
Figure 11. Schematic representation of the Mechano-Chemical Trigger mechanism.
The main events associated to this mechanism are numbered according to the order of
occurrence. The first (1) corresponds to the initial contraction of the Co-Nax bond, followed by
the corrin folding (2), which finally pushes the Ado group until the Co-C bond is dissociated.
22
Introduction
After the first reports confirming the finding of the base replacement, it was thought that this
was evidence capable of supporting the mechano-chemical trigger mechanism. The substitution
by a smaller size ligand could afford the needed compression of the Co-Nax bond length that
could not be achieved with the larger size DMB due to its steric repulsion with the corrin
macrocycle. It also allows for the possibility of a compression of the Co-Nax bond driven by the
interactions of the cofactor with the residues of the binding site, only possible with the histidine
as the lower ligand. Eventually, the relevance of the base replacement on catalytic mechanism
and particularly, its role in the mechano-chemical trigger was mostly discarded after a theoretical
study [62] established that the α-axial ligand replacement did not contribute to a great extent to
the weakening of the Co-C bond, even though it should be noted that these studies were done
using reduced models of free cobalamin. However, its complete role remains unknown.
Regarding to the second trend, Brunold and cols. [63,64] have published reports supporting
the idea of the stabilization of the post-homolysis product of the Co-C bond cleavage. These
studies had been mainly focused on glutamate mutase and methylmalonyl-CoA mutase, being
the latter proposed to follow a similar mechanism as glutamate mutase due to their structural
similarities. They present a complete spectroscopic characterization of these systems using
techniques as UV-Vis, circular dichroism (CD) and magnetic circular dichroism (MCD)
determinations of the free cofactor, the free form of the enzyme (Apo-enzyme), the enzyme
bounded with the cofactor (Holo-enzyme), and in complex with the substrate. Their results
revealed meaningless modifications of the spectrum after formation of the holo-enzyme,
revealing no perturbations on the electronic structure of the cofactor by the enzymatic
environment. Meanwhile, after substrate binding, the region of the spectrum that corresponds to
the metal-ligand charge transfer (MLCT) was blue shifted probably due to Co-d atomic orbitals
stabilization, and a unique double peaked feature appeared in the MCD spectrum of glutamate
mutase at 19 500 cm-1. Additionally, a theoretical study of a small cluster model of
methylmalonyl-CoA mutase in complex with AdoCbl revealed an electrostatic stabilization of ~
4 kcal/mol of the adenosyl radical by the enzymatic environment [65] which also supports the
idea of post-homolysis product stabilization.
Finally, Ryde et. al. [66] studied the origin of the catalytic effects on the Co-C bond cleavage
by the quantum mechanics – molecular mechanics (QM/MM) approach obtaining insightful
results. They defined four main components involved in the energetic decrease of the Co-C bond
caused by the enzyme. The first was related to an incomplete Co-C bond dissociation due to the
23
Introduction
cage effect imposed by the enzymatic environment, which allows the formation of stabilizing
interactions between the partially formed adenosyl radical and the Cbl(II) fragment affording for
~ 4.8 kcal/mol of the BDE reduction. Then, a second component was associated to the geometric
distortion of the AdoCbl cofactor in its Co+3 state, mainly focused on the ribosyl moiety strain.
This was considered the dominant effect on the Co-C bond labialization phenomenon,
decreasing its BDE by ~ 14.6 kcal/mol. The third component corresponds to a differential
stabilization carried out by the enzymatic environment of the Cbl(II) fragment formed after the
Co-C bond cleavage, and considers a ~ 10 kcal/mol stabilization. The fourth component was
associated to a slight stabilization of ~ 2.6 kcal/mol of the residues that constitute the binding
site managed by the Cbl(II) fragment.
Another subject that needs clarification corresponds to the real pathway followed along the
catalytic cycle. The definition of the specific stages that constitute the whole process, with
special emphasis on the aspects associated to the transition states and intermediates of the
beginning of the cycle, will lead into the right direction to finally unravel the cobalamins
problem. There is experimental evidence pointing out that the homolytic Co-C bond cleavage is
not the rate-limiting step and that it is kinetically coupled to the hydrogen transfer from the
substrate [67-71]. Even though, at present there is still no consensus with regards to the exact
catalytic reaction pathway, being this subject a problem of great debate. Two major proposals
are the main focus of research. The first suggests a stepwise route where after the Co-C bond
dissociation, an adenosyl radical intermediate is formed, and then it abstracts a hydrogen atom
from the substrate. The second corresponds to a concerted route where the transition state should
couple the Co-C bond dissociation with the formation of the adenosyl-hydrogen bond by
hydrogen abstraction from the substrate.
An important contribution into this field was made by Kozlowski and cols. [72], by using
quantum chemical models of glutamate mutase. Their calculations revealed that the concerted
route involves a transition state ~ 7 kcal/mol lower in energy than in the stepwise route.
Nonetheless, caution should be taken because the models did not consider the enzymatic
environment at any level, and they only used a reduced model of the substrate, which in this
particular case was glutamate. Warshel and cols. [73] obtained similar findings by using
empirical valence bond (EVB) simulations of methylmalonyl-CoA mutase. On the other hand,
Morokuma and cols. [74] have studied by QM/MM hybrid method the mechanism for Co-C
24
Introduction
bond dissociation, arriving to completely different conclusions, where the stepwise mechanism
seemed to be more plausible than the concerted route.
Thesis work
Despite the amount of studies performed in this matter, the necessity to integrate all the
available information to finally understand the cobalamin-dependant enzymatic mechanisms has
become imperative.
The nature of the Co-C bond strength of cobalamins, and how the structural moieties of this
complex system affect the cleavage process were studied by means of computational models. In
this thesis work we present the optimized geometries together with a Co-C BDE study using full
atom models of AdoCbl and MeCbl. These studies were accompanied by the comparative
analysis with their corresponding reduced models, which were rationally designed to quantify
not only the influence of the β-axial ligand, but also the effect that the α-axial ligand, the side
chains and the nucleoside loop have on the Co-C bond strength. The former allowed us to get
new insights regarding to the α–axial base replacement. Additionally, the analysis of the
electronic structure by means of a MO approach is discussed as a new perspective intended to
explain and support the understanding of the experimental differences on their BDEs. This was
partly explored using the hardness (η) principle [75] under the interpretation given by Pearson
[76], using the grounds of the density functional theory framework with the purpose to establish
an explanation about the higher Co-C bond stability of MeCbl compared to AdoCbl.
After establishing the main aspects that dominates the basal strength of the Co-C bond, the
next stage was to study the homolytic Co-C bond dissociation process. This was carried out
through the calculation of the potential energy profile along the reaction coordinate using full
atom models of AdoCbl and MeCbl cofactors, and selected reduced models. These models
allowed us to analyze the main structural changes, together with energy changes and the
electronic structure properties along the dissociation process. These new insights lead us to
specific features of the free cofactor that are affected by the enzymatic environment. We also
discussed the main reasons for the experimental differences on the BDE between AdoCbl and
MeCbl defined by the behavior of the homolytic bond dissociation process. Within the same
scope, the energy contribution due to the corrin ring flexibility on the AdoCbl Co-C bond
25
Introduction
dissociation process was covered in order to shed light on the mechano-chemical trigger
mechanism.
The next section was devoted to pursue the understanding of the catalytic Co-C bond
dissociation process of the cofactor inside the enzyme. Here we discussed the effect of the
neighboring residues on the Co-C dissociation process. Also, we revealed the role of the His16Asp14 pair in the cleavage process. New insights regarding to the reaction pathway connecting
the Co-C bond dissociation and the hydrogen abstraction are presented.
Finally, in the last section we revealed important aspects associated to the reaction
mechanism for the isomerization reaction of glutamate to methylaspartate and the role of the
enzymatic environment on this intriguing mechanism. The goals of the last two sections were
accomplished by means of the cluster approach, using the largest quantum chemical models of
these systems reported at this date
26
Introduction
1.1 HYPOTHESIS
The activation mechanism for the Co-C bond dissociation process involves the perturbation of
the ground state electronic structure of AdoCbl and the stabilization of the products of the Co-C
bond cleavage, both as a consequence of the neighboring enzymatic environment and the
substrate binding. In addition, the main driving forces involved in the propagation of the
catalytic cycle are associated to the stabilization of key stages of its catalytic mechanism.
1.2 MAIN GOAL
To obtain a series of quantum chemical models able to explain the activation mechanism of
AdoCbl Co-C bond dissociation and to define the driving forces involved in the catalytic
mechanism of GM by using computational chemistry methodologies.
1.3 SPECIFIC GOALS
1.3.1
Free Cofactor Study
a. Geometry optimization of AdoCbl and MeCbl cofactors.
b. Design and geometry optimization of a set of reduced model based on AdoCbl and
MeCbl.
c. Analyses of the geometry, bond dissociation energy (BDE) and electronic structure of
AdoCbl, MeCbl and their reduced models.
d. Potential energy surface calculation of the Co-C bond dissociation process of AdoCbl,
MeCbl and some selected reduced models.
e. Analyses of geometry variations, energy profiles, spin densities and charge
redistribution along the dissociation process.
1.3.2
Geometry optimization of the main intermediates of GM catalytic cycle
a. Cluster model of the AdoCbl-GM-glm.
b. Cluster model of the AdoCbl-GM-glm confA.
c. Cluster model of the AdoCbl-GM-glm confB.
27
Introduction
d. Cluster model of the AdoCbl-GM-masp confB.
e. Cluster model of the AdoCbl-GM-masp confA.
1.3.3
Role of the His16-Asp14 pair
a. Potential energy surface calculation of the Co-C bond dissociation process of a reduced
model of AdoCbl-His16 towards confA.
b. Potential energy surface calculation of the Co-C bond dissociation process of a reduced
model of AdoCbl-His16-Asp14 towards confA.
1.3.4
Role of the enzyme’s environment on the Co-C Bond Dissociation Process
a. Potential energy surface calculation of the Co-C bond dissociation process of a reduced
model of AdoCblHis16-GM towards confA.
b. Potential energy surface calculation of the Co-C bond dissociation process of a reduced
model of AdoCbl-His16-Asp14-GM towards confA.
1.3.5
Co-C bond dissociation reaction pathways calculation
a. Potential energy surface calculation of the Co-C bond dissociation process of AdoCblGM-glm complex towards confA.
b. Potential energy surface calculation of the Co-C bond dissociation process of AdoCblGM-glm complex towards confB.
1.3.6
Isomerization Reaction Mechanism: glm to masp
a. Geometry optimization of a cluster model of the substrate binding site in complex with
glm and Ado•.
b. Geometry optimization of a cluster model of the substrate binding site in complex with
glm• and AdoH.
c. Geometry optimization of a cluster model of the substrate binding site in complex with
masp• and AdoH.
d. Geometry optimization of a cluster model of the substrate binding site in complex with
masp and Ado•.
28
Introduction
1.3.7
Fragmentation stage study
a. Potential energy surface along the carbon-carbon bond cleavage on the fragmentation
stage of the catalytic process.
b. Geometry optimization of the intermediate (IF) formed as a product of the fragmentation
process.
c. Geometry optimization of the transition state (TS) of the fragmentation process.
1.3.8
Role of the enzyme’s environment
a. Geometry optimization of the six states above described, namely (glm + Ado•), (glm• +
AdoH), (masp• + AdoH), (masp + Ado•), (IF) and (TS), without the “arginine claw”
(Arg66, Arg100 and Arg149) as part of the model of the binding site.
b. Geometry optimization of the six states above described, namely (glm + Ado•), (glm• +
AdoH), (masp• + AdoH), (masp + Ado•), (IF) and (TS), without the Glu171 as part of
the model of the binding site.
29
Methodology
2 METHODOLOGY
2.1 FREE COFACTOR SYSTEMS
2.1.1 Free Cofactor: Method of Calculation
All calculations were performed using the Turbomole5.9.1 software version [77] under the
density functional theory (DFT) with the Becke [78] and Perdew [79] (BP86) nonhybrid
functional. This level of theory is reliable for geometry and bond dissociation energy (BDE)
calculations according to previous studies, where it was concluded the superior performance and
accuracy of this functional over others.[18,19] In particular, the B3LYP hybrid functional fails
when studying energy differences between qualitatively different electronic configurations.
[80,81] Also, due to the Hartree-Fock exchange energy included in this functional, it favors
open-shell configurations more than BP86, which give rise to artifacts when studying the
catalytic mechanism of cobalamins because of the homolytic nature of the Co-C bond
dissociation. In order to improve the computational efficiency, the resolution of identity
approximation (ri) [82-86] was used in all calculations. Also, the unrestricted formalism was
employed for all the systems here studied.
2.1.2 Free Cofactor: Basis Sets
For cobalt the Stuttgart small-core pseudorelativistic effective core potentials (Stuttgart-ECP)
were used for the 10 inner core electrons.[87] The outer shell electrons were modeled according
to the contraction scheme of the type (8s7p6d2f1g) / [6s5p3d2f1g].[88] Carbon, nitrogen and
oxygen atoms were also treated with the Stuttgart-ECP [89] for the 2 inner core electrons with
exception of phosphorous in which they were used for the 10 inner core electrons. All the outer
shell electrons were modeled by a double-zeta basis set under a 31G scheme, adding a d-type
polarization function (αC=0.600, αN=0.864, αO=1.154, αP=0.340) to each atom. [90] For the
hydrogen atoms a valence-double-zeta basis set was used.
2.1.3 Free Cofactor: Geometry Optimizations
The starting coordinates for the geometry optimization of AdoCbl and MeCbl were obtained
from the most recently reported crystallographic structures of AdoCbl [91] and MeCbl [92].
Then, all the reduced models were derived from these optimized geometries and they are
molecularly constituted according to the structures showed on Figure 12.
30
Methodology
Figure 12. Schematic representation of AdoCbl and MeCbl with their corresponding
reduced models.
31
Methodology
Frequency calculations performed at the same level of theory as geometry optimizations were
carried out for some of the reduced models to verify that the optimized structures corresponds to
stable minima. In all of them a stable minimum was confirmed. The full systems were excluded
from this analysis because of their computational inaccessibility due to their size.
The models of Cbl(II) fragment were obtained directly from their respective parent complete
model by removal of the β-axial ligand. Even for pairs of models where the Cbl(II) fragment
were the same, they were individually optimized.
The use of a solvation model was discarded on this section of the study, due to the instability
observed on some geometric parameters of the optimized structures, which are thought to be a
consequence of the use of ECPs on the whole model.
2.1.4 Folding Angle
In order to measure the distortion from the planarity underwent by the macrocycle induced by
their respective axial R-ligands and the corrin side chains, a previously reported procedure was
implemented [29] quantifying a set of 8 dihedrals angles. Then, their absolutes values were
averaged as a measure of the degree of distortion of the corrin ring.
2.1.5 Co-C Bond Dissociation Energy
To understand the nature of the Co-C bond and to describe how the modifications previously
described affect it, the strength of the Co-C bond was evaluated calculating the BDE of all the
systems here studied. This was computed through the calculation of the energy difference
between the corresponding to the optimized model of the cofactor and the energy of the
optimized fragments according to the following process:
[Co(III)(Cbl)-R] [Co(II)(Cbl)] + R
2.1.6 Chemical Hardness
The chemical hardness [2,75] (η) of the complete set of models was calculated from:
η≅
( IP − EA)
2
(10)
32
Methodology
where IP corresponds to the ionization potential and EA to the electron affinity. According to the
Koopmans theorem, these two quantities can be defined as a function of the frontier orbital
energies as follows:
IP = −ε HOMO
EA = −ε LUMO
(11)
Leading us to:
η≅−
(ε HOMO − ε LUMO )
2
(12)
2.1.7 Free Cofactor: Potential Energy Surface Calculation
A series of relaxed scans were carried out for AdoCbl, MeCbl, AdoCblredIMI and MeCblredIMI
setting up the Co-C bond as the reaction coordinate.
In order to check if the potential energy surface was consistent with a minimum energy
pathway, several scans were performed for each model under study until negligible energetic and
geometric changes, namely corrin distortion angle and Co-Nax bond distance, were obtained.
The Co-C bond distance of the models was systematically increased by 0.05 Å up to ~ 4.26 Å
constraining the Co-C bond length at each point. The starting Co-C bond distance for the Ado
models corresponds to the obtained on the ground state. For the Me systems, the first distance
step of the scan was calculated in order to match in the second point the Co-C bond distance of
the AdoCbl full system ground state. Then, the potential energy profiles, the spin density and the
electronic charge distribution analysis were more easily comparable at each point. Also, to
determine the variations on the electronic charge distribution and spin densities along the
reaction coordinate, the Natural Population Analysis (NPA) [93] was computed as implemented
in Turbomole5.9.1.
Additionally, a scan of the AdoCbl full system was performed with spatial restrain on all the
heavy atoms of the corrin ring. The purpose of this was to study of the energetic contribution due
to the flexibility of the macrocycle in the Co-C bond dissociation process.
33
Methodology
2.2 AdoCbl-GLUTAMATE MUTASE COMPLEX
2.2.1 GM-AdoCbl complex: Model Design
The mechanism of the Co-C bond dissociation process inside the enzyme’s active site was
studied by using a series of quantum chemical models constructed on the basis of the X-ray
structure of AdoCbl in complex with Glutamate mutase. This structure was obtained from the
Protein Data Bank (PDB entry 1I9C) which was described in detail at the introduction section.
The cofactor was modeled by truncation of the side chains that interact with the binding site (b,
d, e, f and g according to Figure 2) to methyl groups which were kept fixed during geometry
optimizations. This approach allowed to include the spatial restrains imposed to the corrin
macrocycle because of its interactions with the binding site, but without fully restricting the
corrin ring flexibility. The rest of the corrin ring substituents were included in the model, namely
the side chains a and c; which interact with the Ado group. In addition, some methyl substituents
of the macrocycle were considered as part of the model depending on their sterical relevance. As
an example, the methyl group pointing to the α-side of the corrin ring was considered important
and consequently included in the model due to its sterical interaction with the α-axial ligand that
is probably affecting its bonding character with the metal center.
Glutamate mutase is intolerant towards structural modifications of the cofactor because it
needs to control its catalytic reaction pathway to manage the unstable radical intermediates that
are part of the glutamate mutase mechanism. This is accomplished by forming very specific
interactions with the cofactor. As a consequence, large global conformational changes should be
avoided by this type of enzymes in order to restrict the spatial reaction coordinate and avoid
unwanted reactions, and only minor conformational changes are probably allowed. Thus, in
order to model the binding site that surrounds the cofactor, the cluster approach was used. This is
a suitable method for studying the glutamate mutase system as it is able to include minor local
conformational changes without the need of including the complete enzyme.
The model was constituted in part by the residues of the binding site that interact with the
Ado group. Among these, there are included the Gly68, Asn123, Lys326 and Glu330 residues,
all from the B chain of the enzyme.
It should be noticed that according to the crystal structure, Gly68 and Asn123 interact with
the -NH2 group of the adenosine moiety through their backbone carbonyl groups. Aiming to
keep the model size as small as possible, these interactions were modeled by reducing the
residues to their C=O groups from the backbone and extending their structures into an
34
Methodology
“aldehyde-amine” intending to preserve part of the dipolar moment generated by the backbone.
The carbon of the C=O group and the nitrogen from the amine were spatially restricted during
geometry optimizations.
Meanwhile, Lys326 and Glu330 were truncated at their γ- and α-carbon, respectively. The αaxial ligand was modeled by the incorporation of His16 accompanied by Asp14 with which it
forms a hydrogen bond. Both residues belongs to the A chain of the enzyme and were truncated
at their α-carbon. Additionally, two water molecules were included to the model to evaluate their
role on the stability of the binding site architecture, because they could work as a structural
bridge between the residues from the first shell of interaction with the cofactor and the outer
residues of the binding site.
The first water molecule interacts with the ammonium group of Lys326 and the second water
forms two hydrogen bonds, one with the carboxylate group of Glu330 and the other with a
hydroxyl group of Ado ligand. The water molecules were kept fixed at their crystallographic
coordinates unless otherwise specified.
To asses the role of the substrate binding on the Co-C bond dissociation process, the
complete substrate molecule was incorporated to the model. Its heavy atoms positions were kept
fixed at their crystal structure coordinates. The resulting model defined as AdoCbl-GM-glm
represents the complex formed between the cofactor, glutamate mutase and the substrate. To
study the influence that the different components of the catalytic site model have on the cofactor,
the AdoCbl-GM-glm model was dissected into four additional systems, leading to a total of five
models.
The first two models were designed to individually assess the role of the Asp14 residue on
the electronic structure of the cofactor, and therefore in the Co-C bond dissociation process.
These models consider the cofactor with His16 as their α-axial ligand, where the only difference
between them relies on the presence and absence of the Asp14 residue, leading to the models
defined as AdoCbl-H16/D14 and AdoCbl-H16. Secondly, two additional models were built by
removing the substrate from AdoCbl-GM-glm. As before, their difference resides in the presence
and absence of the Asp14 residue. These models were defined as AdoCbl-GM-H16/D14 and
AdoCbl-GM-H16, and they represent the state before the substrate binds to the catalytic site.
To avoid artificial movements of the residues belonging to the binding site so as to obtain
geometries consistent with the experimental structure, the truncated atoms on each model were
fixed at their X-ray positions during the geometry optimizations.
35
Methodology
All the hydrogen atoms were added manually and they were freely optimized in all the
models considered in this study.
2.2.2 GM-AdoCbl complex: Method of Calculation
All calculations corresponding to this section were carried out using the nonhybrid DFT
functional BP86 as implemented on the ORCA v2.8.0 software [94]. As denoted above, this
level of theory is adequate to obtain models of acceptable accuracy. The computational
efficiency was increased by the use of the ri- approximation in all calculations and in order to
model the bi-radical nature of the products of the Co-C bond dissociation, an unrestricted
formalism was employed.
The basis set is analogous to the used for the free systems, where the basis set were adapted
to the ORCA format, which actually correspond to the ‘GAMESS’ format in the EMSL library.
Due to the super-molecular nature of the models here studied the need to include dispersion
forces into the calculations become imperative in order to accurately model the intermolecular
interactions between the cofactor and the enzyme. Unfortunately, it is well known that DFT
functionals do not include dispersive effects. Despite the fact that post-Hartree Fock methods
consider the dispersion forces, they are computationally unaffordable because of the model size.
Nowadays, it is available an empirical correction of the dispersion forces for DFT calculations
(DFT-D) [95-97], which has been increasingly used in the field of molecular biophysics and
quantum bioinorganic chemistry [98-100]. Due to the increased accuracy that the DFT-D
approach implies, it was used in the modeling of AdoCbl-GM complexes. This approach
corrects the total energy according to the following equation (adapted from [95]):
EMF − D = EMF + Edisp
(13)
with EMF-D as the corrected energy, EMF as the mean-field energy (i.e., DFT) and Edisp as an
empirical dispersion correction given by:
N at −1 N at
Edisp = − s6 ∑
i =1
C6ij
∑ 6 f dmp ( Rij )
j =i +1 Rij
(14)
36
Methodology
where Nat is the number of atoms in the system, C6ij denotes the dispersion coefficient for the
atom pair ij, s6 is a global scaling factor and Rij is the interatomic distance. Finally, a damping
function fdmp is included into the equation in order to avoid near-singularities for small R, and is
given by:
f dmp ( R ) =
1
1+ e
−α ( R / R0 −1)
(15)
where Ro is the sum of the atomic vdW radii and α is determined by requiring
f damp ( R ) = 0.99 at R = 1.2 Rm .
For the same reason exposed on the methodology for the study of free cofactor models, the
use of a solvation model was also discarded in this section of the study.
2.2.3 GM-AdoCbl complex: Potential Energy Surface Calculation
The Co-C bond dissociation reaction was studied by performing a series of scans along the
Co-C bond using the five models above presented, i.e. AdoCbl-GM-glm, AdoCbl-GM-H16/D14,
AdoCbl-GM-H16, AdoCbl-H16/D14 and AdoCbl-H16.
A similar procedure to the above explained was implemented in this study, where the Co-C bond
distance was systematically increased by 0.05 Å starting from the ground state geometry. The
ending point of the scan was defined by the position of the Ado-C5’ atom on the confA
conformation as found on the crystal structure. As described above, this conformation has been
related to the first stage of the dissociation process, preceding the Ado conformational change
that leads to the hydrogen abstraction from the substrate (confB). It is important to state that the
scan was carried out on the Cartesian space, aiming to restrain the reaction pathway to the
dissociated conformation observed on the crystal structure. Also, to shed light into the
controversy between the concerted and step-wise mechanism for the substrate hydrogen
abstraction, an additional scan was performed using the AdoCbl-GM-glm model, with ending
point at the Ado-C5’ atom coordinates relative to the confB, modeling the potential energy
surface associated to a concerted mechanism.
To determine the variations on the electronic charge distribution and spin densities along the
reaction coordinate, the Natural Population Analysis (NPA) was computed using the stand alone
version of the NBO software [101].
37
Methodology
2.3 ISOMERIZATION REACTION: GLUTAMATE TO METHYLASPARTATE
2.3.1 Enzyme’s Catalytic Site: Model Design
The substrate binding site is constituted by residues complementary to the glm structure,
most of which are charged accordingly with the charged nature of the substrate. The initial
model for the catalytic isomerization reaction considered the substrate and the residues of the
first shell interacting with it. This group of residues is comprised of Arg66, Arg149, Arg100,
Glu171, Tyr177, Tyr181, Phe216 and Thr94 (Figure 13).
Figure 13. Glutamate Mutase-substrate (glm) complex.
The residues belonging to the substrate binding site are highlighted in light green.
The first three arginines constitute the arginine-claw, where Arg66 and Arg149 interact with
the α-carboxylate from the substrate with a Y-shaped conformation, while Arg100 interacts with
the γ-carboxylate of glm. Interacting with the α-ammonium from the substrate, there is the
38
Methodology
carboxylate moiety of Glu171, residue proposed to act as a general base on the positively
charged amine. In addition, the arginine claw together with Glu171 seems to be the responsible
of keeping the substrate in the needed extended conformation Tyr181 forms a hydrogen bond
with the γ-carboxylate and with the α-ammonium from the substrate. Meanwhile, Tyr177
interacts with the α-ammonium of the substrate and also with the carboxylate moiety of Glu171,
probably acting as a structural support. Phe216 interacts with α-ammonium of the substrate by a
cation-π interaction. A water molecule that interacts with the substrate would be relevant for the
stabilization of some intermediate species and was also included in the model. As it spatial
location seems to be determined by Thr94, this residue was also included.
Finally, from the assumption that after Co-C bond dissociation, the corrin macrocycle
remains as a spectator and it is not involved in the subsequent stages associated to the
transformation from substrate to product, we only considered the Ado ligand as part of the model
using the conformation relative to confB available from the X-ray structure. The positions of its
heavy atoms were kept fixed at their crystal structure coordinates.
Four stages were initially modeled. The first corresponds to the state with Ado• radical and
glm before hydrogen abstraction. The second was the system after hydrogen abstraction with
AdoH and glm• radical. The third model was associated to the stage after the
Fragmentation/Recombination process, with the masp• radical as the intermediate and AdoH.
Finally, the fourth model represents the stage after reformation of the Co-C bond, with masp as
the final product and Ado• radical.
To assess the role of the different parts of the catalytic site, a series of models were designed
by removing a residue or a group of residues from the model in order to determine the impact on
the energetic of the energy profile of the reaction mechanism.
2.3.2 Enzyme’s Catalytic Site: Method of Calculation
The quantum chemical calculations related to this section were accomplished using the
hybrid DFT B3LYP functional as implemented in ORCA v2.8.0 program. For this particular
case, this was the functional of choice due to the nature of our system and the lack of a carbonmetal bond on which B3LYP normally fails. The ri- plus chain of spheres [102](RIJCOSX)
approximation was used in order to decrease the computational cost. The dispersive effects were
39
Methodology
taken into account by the empirical dispersion correction by Grimme. Solvation effects were
considered by using the COSMO model [103] with a dielectric constant (ε) set to 4.
2.3.3 Basis Set
The substrate, intermediates, transition state and product were modeled by using a triple zeta
plus double polarization function of the type def2-TZVPP [104]. The same was used for the
ribosyl moiety of the Ado ligand on which the radical state is initially located. For the rest of the
model a double zeta plus polarization functions of the type def2-SVP [105] was used.
2.3.4 Transition State and Intermediate of the Fragmentation process
According to the proposed mechanism showed on Figure 6, the hydrogen abstraction is
followed by a Fragmentation/Recombination stage. Because the geometry of the fragmented
intermediate constituted by the glycyl radical and the acrylate ion is unknown inside the catalytic
site, a scan was performed along the carbon-carbon bond that is broken after the fragmentation
until an intermediate was located. This allows finding an initial guess for the transition state
geometry of the fragmentation stage. Once the intermediate and transition state starting
structures were obtained, they were optimized. For the transition state saddle point optimization,
a hybrid Hessian was calculated in order to save computational time. The intermediate was
optimized by the same protocol used for the rest of the systems.
The gOpenMol [106,107] and VMD [108] software were used for structural and molecular
orbital analysis.
40
Results and Discussion
3 RESULTS AND DISCUSSION
3.1 GROUND STATE MODELS
3.1.1 AdoCbl and MeCbl cofactors: Full Atom Models
In order to validate the obtained theoretical results, the ground states geometries of AdoCbl
and MeCbl were compared with their corresponding crystal structures. The geometry analysis
was carried out considering bond distances Co-C, Co-Nax, Co-Neq together with the corrin ring
distortion. This data is summarized in Table 1.
Table 1. Selected geometric parameters of the Cobalamin Models presented in this study a
Co-Ncb
Corrin distortion
Co-C
Co-Nax
AdoCbl
AdoCblcrystalc
AdoCblwoSC
AdoCblwoloop
AdoCblwoloopIMI
AdoCblred
AdoCblredIMI
MeCbl
MeCblcrystald
MeCblwoloop
MeCblwoloopIMI
MeCblred
MeCblredIMI
2.007
2.031
2.009
2.002
2.003
2.005
2.009
1.968
1.979
1.965
1.966
1.971
1.973
2.242
2.239
2.253
2.265
2.191
2.304
2.208
2.231
2.163
2.273
2.196
2.274
2.185
1.93-1.88
1.91-1.87
1.93-1.88
1.93-1.88
1.93-1.88
1.93-1.87
1.94-188
1.92-1.88
1.92-1.88
1.93-1.88
1.93-1.88
1.93-1.87
1.93-1.88
9.3
9.1
9.7
9.7
9.8
7.8
8.0
9.5
9.1
9.7
9.1
7.6
6.8
a
Co-C, Co-Nax and Co-Nc bond distances are presented in Å and the corrin ring distortion in
deg.
b
Co-Nc corresponds to the bond distances between the equatorial nitrogens and the metal center,
presented as two averaged bond distances according to their equivalence
c
Data from structure reported on ref 91
d
Data from structure reported on ref 92
41
Results and Discussion
The BDE values listed in Table 2 were calculated to assess how the different structural
moieties namely side chains, nucleoside loop and the identity of the β–axial ligand contribute to
the strengths of Co-C and Co-Nax bonds. The AdoCbl Co-C bond distance is 2.007 Å in the
model, 0.024 Å shorter than the corresponding bond distance in the crystal structure. On the
other hand, the obtained Co-Nax bond distance is 2.242 Å, with a negligible difference with
regards to the experimental value (0.003 Å), where differences of the order of 10-3 Å are
considered not significant. When it comes to the MeCbl model, the Co-C bond distance is 1.968
Å, 0.011 Å shorter than the crystal structure, whereas the obtained Co-Nax bond distance is 2.231
Å, 0.068 Å longer than the corresponding distance at the crystal structure. Thus, the calculated
MeCbl Co-C distance is 0.039 Å shorter than the obtained for AdoCbl after optimization. The
geometrical distances for both models are in close agreement to the experimental values.
Table 2. Bond Dissociation Energies (BDE) of the Co-C and Co-Nax axial bondsa
Model
EDE Co-C
BDE Co-Nax
31.4 ± 1.5b
------
AdoCbl
28.2
11.2
AdoCblwoSC
22.7
10.1
AdoCblwoloop
29.6
14.0
AdoCblwoloopIMI
32.2
16.4
AdoCblred
24.4
14.5
AdoCblredIMI
25.8
AdoCblexp
MeCblexp
36.0 ± 3.0
16.2
c
------
MeCbl
31.3
12.4
MeCblAdoCbl
29.0
12.0
MeCblwoloop
31.4
12.3
MeCblwoloopIMI
32.0
13.5
MeCblred
31.1
16.1
MeCblredIMI
31.8
17.3
a
The energy values are presented in kcal/mol
Data from ref 14
c
Data from ref 15
b
42
Results and Discussion
The calculated BDE for AdoCbl Co-C bond was 28.2 kcal/mol, meanwhile for MeCbl is of
31.3 kcal/mol. The energies are ~ 3.2 kcal/mol and ~ 4.7 kcal/mol lower than their corresponding
experimental BDE values, respectively. The calculated BDE for MeCbl is 3.1 kcal/mol higher
than AdoCbl, which compared to the 4.6 kcal/mol experimentally obtained it follows the same
trend. Inside the scope of this study, a BDE difference higher than 1 kcal/mol is considered as a
meaningful difference. These results support the fact that the calculated electronic structure for
both models includes the intrinsic differences between the two active cofactors, making them
appropriate for exploring the nature of the observed differences induced by the change of the βaxial ligand from Ado to the Me group, which leads to a decrease of the Co-C bond distance
with the consequent increase on the BDE.
The α-axial ligand showed a BDE of 11.2 kcal/mol for AdoCbl and of 12.4 kcal/mol for
MeCbl, 1.2 kcal/mol higher than the former, in accordance to the slight decrease in the Co-Nax
bond length described above. In both cases, the Co-Nax BDEs values are approximately one third
of the Co-C BDE. As the bond distances between the cobalt and the equatorial nitrogens did not
vary appreciably with the nature of the β-axial ligand, so they are not further discussed in this
thesis.
The measured distortion of the corrin ring in the ground state for both cofactors is close to the
experimental value as showed in Table 1. Also, when this distortion is compared between the
calculated models of AdoCbl and MeCbl, a similar angle between them is obtained (∆ = 0.2°),
revealing no meaningful steric effects of the Ado group on the corrin ring.
Considering the resemblance of AdoCbl and MeCbl structures, it is possible to argue that the
differences between their theoretical conformations are predominantly dominated by the identity
of the β-axial ligand. In order to test this statement, a model of MeCbl was calculated modifying
the optimized structure of AdoCbl as starting point instead of MeCbl crystal structure. In this
way, the influence due to the interchange of the β-axial ligand from Ado to Me on the rest of the
molecule should be able to provide the needed perturbation to reach the geometry and electronic
structure of the MeCbl model obtained using the MeCbl crystal structure as the starting
coordinates. A BDE of 29.0 kcal/mol was obtained, 1.3 kcal/mol below the value of the
optimized crystal structure, and 0.8 kcal/mol above the corresponding value calculated for
AdoCbl. This result narrows the BDE difference between AdoCbl and MeCbl, revealing that the
crystal structure conformations of the macrocycle and its substituents bear intrinsic information
that contributes in a considerable extent to the observed BDE differences. In addition, this result
43
Results and Discussion
makes clear the dependence that BDE has on the starting geometry of the cofactors thus
remarking the need for high resolution crystal structures. According to the recent analysis, the
Co-C bond strength depends on the β–axial ligand structure as well as on the conformational
changes induced by itself on the rest of the cofactor. Thus, questions like how the Co-C bond
display different chemical behavior toward chemical catalysis lead us to investigate those
relevant conformational differences between AdoCbl and MeCbl that are directly related to the
β-axial ligand. As a starting point, the previously reported intramolecular interactions of the Ado
ligand with the corrin macrocycle and its contributions to the calculated BDE were studied.
3.1.2 Ado-Corrin side chains intramolecular hydrogen bonds
One of the most relevant conformational differences between AdoCbl and MeCbl is the
presence of a set of intramolecular hydrogen bonds on AdoCbl. These are formed between one
of the hydroxyl groups (denoted as OH1 in Figure 2) of the deoxyribose moiety and the carbonyl
group of the acetamide from to the a-side chain with a bond distance of 1.918 Å. In turn, the
amide moiety of the acetamide group forms a hydrogen bond with an oxygen carbonyl of a
propionamide from b-side chain with a bond distance of 1.982 Å, both acetamide groups belong
to the corrin macrocycle. To study these contributions to the Co-C bond strength, a model was
built from the optimized structure of AdoCbl, without consideration of the side chains involved
in the interactions, namely a- and b-side chain. This model defined as AdoCblwoSC, showed a CoC bond distance close to the obtained for AdoCbl (2.009 Å). It seems that the Co-C bond
distance depends essentially on the chemical nature of the β-axial ligand and not in the presence
of these side chains. In contrast, the BDE for AdoCblwoSC was 22.7 kcal/mol, 5.5 kcal/mol lower
than AdoCbl. This energy difference represents the contribution of the intramolecular hydrogen
bond interactions on the Co-C bond strength, being the 22.7 kcal/mol the intrinsic strength of the
AdoCbl Co-C bond. In addition, these results revealed that the calculated BDE for the full model
of AdoCbl does not represent the intrinsic strength of this bond and explains why several
previously reported studies using reduced models (naked corrin) usually underestimated the
BDE. Even though these interactions are not as stable in explicit water as they are in vacuum, it
is not possible to discard that in the measured experimental BDE, these interactions should
weight on the observed energies.
44
Results and Discussion
Regarding to the Co-Nax bond, a distance of 2.253 Å was obtained. This value implies a small
increase of 0.011 Å compared to the full atom model. This increase was accompanied by a slight
decrease of 1.1 kcal/mol on its interaction energy. In general, the corrin geometry was
maintained without major changes but with negligible increase on the planarity distortion of 0.4º.
Finally, with these results at hand, it is possible to establish within the framework of this
research that the total increase on the Co-C bond strength going from AdoCbl to MeCbl was of
8.6 kcal/mol. This implies that the electronic structures associated to the Co-C bond of each
cofactor differs more than it was thought from the BDE difference experimentally determined
due to the contribution of the AdoCbl hydrogen bonds. This value quantifies the pure
contribution of the difference on the chemical nature of the bond formed by methyl and Ado
ligands with the metal center.
3.1.3 Modifications in the Nucleosidic Loop and its effect on the cofactor
In order to assess the contribution of the nucleosidic loop on cobalamin models so as to
establish the existence of differences on its role on both active cofactors, the truncation of this
moiety to a propionamide side chain bonded to the corrin ring was performed. These models
were defined as AdoCblwoloop and MeCblwoloop.
The optimized geometries of both models revealed that the Co-C bond distance underwent
only a slight decrease as a consequence of the removal of the nucleosidic loop. Even though the
two cofactors showed an increase on the corrin ring distortion angle, it was not a meaningful
variation. Interestingly, while MeCblwoloop Co-C BDE was unaffected, AdoCbl Co-C BDE
increased from 28.2 to 29.6 kcal/mol, despite of the small changes observed on its geometry.
This indicates that MeCbl Co-C bond is rather insensitive to modifications on the nuclesidic
loop, meanwhile on AdoCbl the presence of the loop destabilizes the bond by 1.4 kcal/mol. On
the other hand, the Co-Nax bond was elongated by 0.023 and 0.042 Å on AdoCblwoloop and
MeCblwoloop, respectively. These results reveal that the nucleosidic loop forces the α-axial ligand
to locate itself at a closer distance of the corrin ring than the energetically preferred in absence of
this moiety. This fact is supported by the increase on the AdoCbl Co-Nax BDE after the removal
of the loop from 11.2 to 14.0 kcal/mol, even considering that this stronger bond has a longer
bond distance. A probable explanation of this observed bond elongation is due to the steric
effects provided by the size of the DMB group. In the AdoCbl optimized model, the closest atom
45
Results and Discussion
of DMB to the macrocycle corresponds to a benzyl hydrogen, at a distance of 2.731 Å from the
nearest carbon atom of the corrin ring. This distance is shorter than the sum of their van der
Waals radius (RH=1.20 Å; RC=1.70 Å; RH+RC=2.90 Å) inducing repulsion between the π cloud
of the corrin ring and the dimethylbenzyl moiety. The role of the nucleosidic loop seems to be
related to the anchoring of DMB to the metal center because of the low BDE of the Co-Nax bond.
The absence of the nucleosidic loop may result in a displacement of DMB by another ligand
(e.g. water) without the need of special conditions.
3.1.4 Effects of α-Axial Base Interchange
The low BDE on the Co-Nax enables the α-axial ligand to dissociate from the Co center which
due to the nucleosidic loop can be achieved only upon proper conditions. These conditions are
fulfilled inside the binding site of some enzymes, where DMB is replaced by an imidazole ring
of a histidine residue. Actually, the α–axial ligand substitution can occur in AdoCbl and MeCbl
cofactors. However, the role of this replacement on the differential mechanisms observed for the
Co-C bond dissociation of the two cofactors is far for being completely clarified. Thus, to get a
better understanding of this phenomenon in the ground state, several models were designed.
Aiming to model the base replacement on more realistic models and to study its effect on the
Co-C bond strength, two models were optimized. These systems were derived from AdoCblwoloop
and MeCblwoloop, replacing the DMB group by an imidazole ring and were defined as
AdoCblwoloopIMI and MeCblwoloopIMI. The models represent the cofactor bound to the enzyme and
were analyzed through direct comparison with their respective full atom models which represent
the free cofactor. In order to compare our models with the commonly used reduced models
known as naked corrin models, four additional systems were also obtained. The first two,
defined as AdoCblred and MeCblred, were built by complete removal of the corrin side chains and
the nucleoside loop. The remaining two, were the models needed to assess the effect of the base
replacement within the scope of the naked corrin models, and were designed from the latter two
models by replacing the DMB group with an imidazole ring, and defined as AdoCblredIMI and
MeCblredIMI.
The Co-C bond distance of AdoCblwoloopIMI displayed a negligible length decrease regards to
AdoCbl, whereas its Co-Nax bond length was reduced by 0.051 Å. Despite of the lack of
influence of the base replacement on AdoCbl Co-C bond length, its BDE was increased by 4.0
kcal·mol-1, attaining a final BDE of 32.2 kcal/mol. This energy increase is mainly attributed to
46
Results and Discussion
the shortening of the Co-Nax bond length, resulting on a Co-Nax BDE increase of 5.2 kcal/mol,
and to a decrease of the steric strain as a consequence of the removal of the nucleosidic loop.
Despite similar trends on the geometric variations were observed between MeCblwoloopIMI and
MeCbl, namely the Co-C remained unchanged and the Co-Nax bond shortening, it did not
displayed a meaningful increase on its Co-C bond strength.
The Co-C bond distances of the naked corrin models were also barely affected by the base
replacement, with increments on the Co-C bond strength of 1.4 and 0.7 kcal/mol for Ado and
methyl related models, respectively. These results denote that AdoCblredIMI presents a lower
increase on its Co-C BDE than the larger models, and that MeCblredIMI shows a slight increase on
its Co-C BDE, whereas the larger models did not increased its Co-C bond strength after α-axial
ligand substitution. Thus, the naked corrin models are not completely adequate to represent the
energetic variations produced by the base replacement. Regarding to the Co-Nax bond lengths,
AdoCblredIMI and MeCblredIMI displayed a decrease of 0.096 and 0.089 Å, respectively. These
bond contractions were higher than the observed on the larger models. Nevertheless, the
calculated Co-Nax BDE increase of AdoCblredIMI was modest compared to the obtained for the
larger model. This is explained by the fact that the comparative study of AdoCbl and
AdoCblwoloopIMI altogether considers the stabilizing effect due to the removal of the nucleosidic
loop as discussed above, which not only affects the Co-C BDE but also the Co-Nax bond
strength. Meanwhile, AdoCblred do not have the nucleosidic loop in its structure, being unable of
exhibit the complete stabilizing effect after the base replacement. In addition, the smaller
imidazole ring probably forms stabilizing interactions with the side chains of the α-side of the
corrin ring that were prevented in the case of DMB due to its bigger size. Therefore, the shorter
Co-Nax bond distance displayed by AdoCblwoloopIMI compared to AdoCblredIMI may indicate a
stronger influence of the α-axial ligand on the Co-C bond strength explaining the larger increase
observed on the former. Finally, this reveals that larger Co-Nax bond contractions will lead to
higher Co-C bond strengths.
Remarkably, the MeCbl derived models did not show meaningful variations neither in the
Co-C nor the Co-Nax bond strength in spite of the bond length decrease observed on the latter.
This is most likely related to the stronger Co-C bond, which does not present major BDE
variations after base replacement, independently of the set of studied models. This also exposes a
minor influence on the Co-C bond strength due to the corrin ring substituents. A plausible
explanation for this is the lack of strong intramolecular interactions between the methyl ligand
47
Results and Discussion
and the corrin ring. Meanwhile on AdoCbl there is a balance between the steric repulsion of the
β-side side chains with the Ado group, and the intramolecular hydrogen bonds, the methyl group
only has the repulsion contribution, but it should not be high due to its smaller size.
With regards to the possible role of Co-Nax bond length decrease after the change of the αaxial base, it can be attributed to the adoption of a more favored conformation for the charge
transfer expected to occur in the Co-C bond dissociation process. From an energetic point of
view it can be remarked that the Co-Nax bond length decrease is always accompanied by a CoNax BDE increase reinforcing the idea that this is related to avoid the bielectronic reduction of
the metal center which is favored on a square planar configuration which lacks of an α-axial
ligand.
The Co-C BDE increase obtained on the AdoCbl related models would be related to a safety
mechanism in the case of a mechanical elongation of the Co-C bond after binding to the enzyme,
in order to avoid the Co-C bond dissociation before substrate binding. The substrate binding may
release this safety mechanism allowing the Co-C bond dissociation and the beginning of the
catalytic cycle. The absence of this behavior on MeCbl related models might be associated to the
impossibility of the methyl ligand of forming interactions with the binding site of the
corresponding enzymes, making difficult a mechanical elongation and therefore is unnecessary a
safety mechanism like the suggested for AdoCbl. Additionally, one of the catalytic mechanisms
proposed for the MeCbl dependant enzymes involves a SN2 reaction mechanism which is
unlikely to involve a mechanical elongation of the Co-C bond supporting this proposal.
From Table 2 it can be noticed that for the larger models, the distortion angles of the corrin
varied between 9.1° and 9.8°, displaying no major differences between the models with the Ado
and Methyl moieties. This small variation reveals an independent behavior of the macrocycle
regarding to the identity of the β-axial ligand. For all the naked corrin models, the distortion
angles of the corrin varied between 7.2 – 7.7°. The same independent behavior was observed
among these models. In addition, it is apparent that the set of side chains together with the
nucleosidic loop are responsible for a larger distortion angle. In general, upon base replacement,
the models derived from AdoCbl showed a slight increment on the distortion angle, whereas the
opposite was observed for the MeCbl related models. Nevertheless, the magnitudes of the
variations are not larger enough to get a clear picture of this behavior.
48
Results and Discussion
3.1.5 Sterical Strain between Ado and the Corrin Ring
Although the modification or removal of the structural moieties of cobalamin structure seems
to affect the Co-C bond strength in a larger extent on AdoCbl than MeCbl models, the former
was never close to the values obtained for the methyl related systems, no matter how much the
Ado systems were modified. Also, it was noticeable that in spite of all the structural
modifications made it to AdoCbl, the Co-C bond distance was almost unchanged among its
reduced models. The same was observed among the MeCbl derived models. The direct
correlation between the Co-C bond distances and their corresponding BDE, allows concluding in
a first approach that the main difference between the AdoCbl and MeCbl Co-C BDE are related
to the direct interaction between the Co and C atoms. This points out that the Co-C bond strength
differences are mostly due to the hypothetical substitution of one of the hydrogen atoms of the
methyl β-axial ligand by a deoxyadenosyl derivative, which affects the C reactivity. One
problem with this proposal is the possibility of a steric contribution to the Co-C bond distance
and consequently the BDE. This could be caused by the corrin ring pushing up the Ado ligand
due to its bulkier size, and thus lengthening the Co-C bond distance and weakening the Co-C
bond. In order to discard this steric effect and continue with our proposal, two reduced models
were built and optimized. The first was constituted by a cobalt center with four amines as the
equatorial ligands, and an imidazol ring with Ado group as the axial ligands. The second was the
similar but with a methyl group instead of Ado. The results showed Co-C bond distances of
1.978 and 2.007 Å for the methyl and the Ado model, respectively, very close to the
corresponding on the full atom models. This reveals no steric effect between the corrin ring and
the Ado group, and that the longer Co-C bond distance on AdoCbl models is completely related
to the intrinsic features of the interactions between Co and C centers.
3.1.6 Structural Fragments Contributions to the Co-C bond Strength on AdoCbl
At this point, we have described the effects that the structural moieties of cobalamins have on
the Co-C BDE. We are in position to define the energetic contributions to AdoCblred BDE due to
the stepwise addition of the structural components of cobalamins. According to the results
describe vide supra, the Co-C BDE for AdoCblred was of 24.4 kcal/mol, 1.7 kcal/mol stronger
than AdoCblwoSC. This small difference corresponds to the stabilizing effect due to the removal
of the nucleosidic loop above described (~ 1.4 kcal/mol) and probably due to the lost of a
destabilizing steric effect between the corrin side chains and the β-axial ligand, which account
49
Results and Discussion
for the remaining 0.3 kcal/mol. It should be consider that the magnitude of this value is in the
error range of the methodology used. With regards to AdoCblwoloop, AdoCblred was 5.2 kcal/mol
lower than it. The only difference between these two models is the absence of all side chains of
the corrin with regards to the former. This analysis allows explaining the energy difference by
the absence of the stabilizing contributions of the intramolecular hydrogen bonds (5.5 kcal/mol)
and the destabilizing steric effect of the side chains quantified as 0.3 kcal/mol, which is the
difference of 5.2 kcal/mol obtained when the two models are compared directly.
The difference obtained between the AdoCbl and AdoCblred BDE is 3.8 kcal/mol favoring
AdoCbl, value that comes from the decomposition of the contributions between the stabilizing
effect of the intramolecular interactions (5.5 kcal/mol) and the destabilizing effects accounted by
the nucleosidic loop and the steric effect of the side chains (1.4 kcal/mol + 0.3 kcal/mol) which
results in the 3.8 kcal/mol of difference between these two models.
Bellow, on Figure 14 there is a schematic representation of the optimized geometries and the
energy contributions of the structural modifications carried out on AdoCbl and MeCbl cofactors.
50
Results and Discussion
Figure 14. Schematic representation of the optimized geometries of AdoCbl, MeCbl and
their reduced models.
Arrows represent how the systems were compared, and the numbers correspond to the change on
the bonding dissociation energy (BDE) values between the models. A positive value indicates an
increase on the BDE in the direction of the arrow, a negative value specify a decrease.
51
Results and Discussion
3.1.7 Electronic Structure and Co-C bond Strength Relationship
The electronic structure analysis of these systems is often difficult and elusive due to the
complexity of the molecular orbital (MO) composition of these cofactors. A reasonable strategy
is to choose from the whole set of MOs those that are able to describe at least on a qualitative
basis the reactivity of the systems.
On a first approach it seemed logical to study the hardness (η) of the systems using the
extension based on the frontier molecular orbital (FMO) energy gap. Despite it is not intended to
study the reactivity of AdoCbl and MeCbl toward another molecule, this parameter would allow
us to discuss the differences on the Co-C bond strength through the concept of susceptibility of
the electron density toward its reorganization, which is related to the absolute chemical hardness.
For purposes of the forthcoming analysis, the η will be defined as ηHOMO-LUMO.
The results listed on Table 3 denote that the ηHOMO-LUMO values obtained for AdoCbl and
MeCbl derived models were very similar when compared pairs of analogous models, being the
MeCbl models slightly higher in magnitude than their respective AdoCbl analogous in several
cases. The large ηHOMO-LUMO values variations observed between the full atom models and their
respective reduced models indicate the influence of the structural moieties on this chemical
parameter, being unable to describe the insensitivity of the MeCbl Co-C bond strength toward
structure modifications, and the important but modest variations obtained among the AdoCbl
models. In addition, AdoCblwoloop and AdoCblwolopIMI models presented comparable ηHOMO-LUMO
values with their respective equivalent model derived from MeCbl. This would be interpreted as
systems with comparable resistance toward its electron density reorganization, contrary to what
is expected from the experimental and theoretical results, at least regarding to the region
between Co and C atoms.
The failure of ηHOMO-LUMO on the discrimination between AdoCbl and MeCbl bond strenghts
comes from the fact that this is only able to point out the global susceptibility of the system
toward its density deformation, and not necessarily represent the direct resistance of the electron
density located between the two atoms of our interest. When large molecular systems are
studied, the reactivity is not necessarily dominated by the frontier orbitals. [109] In this
particular case as depicted on Figure 15 for the full atom models, these MOs do not contain the
Co(dz2) atomic orbital with the C(sp3) and N(sp2) hybrid orbital components expected to be
directly involved in the dissociation process. While the LUMO has M-L π*, L-L π and π*
52
Results and Discussion
character in all the models here studied, due to the π orbital interaction located on the corrin ring
which helps to stabilize it; the HOMO varied depending on the presence of the nucleoside loop.
Table 3. The Energetic associated to the Co-C bond and its Electronic Structure
ηBOND-ANTIBc
∆Eσd
∆Eσ*e
ηHOMO-LUMOb
Model
AdoCbl
0.34
2.14
1.73
2.56
AdoCblwoSC
0.34
2.15
1.66
2.64
MeCbl
0.44
2.40
1.93
2.87
AdoCblwoloop
0.97
2.10
1.66
2.64
MeCblwoloop
0.97
2.39
1.88
2.90
AdoCblwoloopIMI
0.95
2.12
1.60
2.64
MeCblwoloopIMI
0.96
2.42
1.64
3.19
AdoCblred
0.91
2.15
1.43
2.87
MeCblred
1.02
2.39
1.74
3.04
AdoCblredIMI
0.90
2.14
1.38
2.90
MeCblredIMI
1.03
2.40
1.75
3.06
a
The energy values are presented in eV
The chemical hardness calculated from the frontier molecular orbital energies
c
The Co-C bond resistance calculated from the orbital energies of the Co-C bonding, Co-Nax
antibonding MO, and its corresponding antibonding MO
d
Stabilization energy of the Co-C bonding, Co-Nax antibonding MO
e
Destabilization energy of the C-Co-Nax antibonding MO
b
When the nucleoside loop was included as part of the model, the HOMO was dominated by a
combination of p atomic orbitals belonging to the four oxygen atoms of the PO4- group. This MO
presents a completely antibonding character mostly defined as a π* interaction, being the most
unstable occupied MO. As a consequence, the Co-C bonding MOs are displaced to lower energy
levels. After the nucleosidic loop removal, the Co-C bonding orbitals are located closer in
energy to the HOMO. However, the closest orbital to the HOMO was not the dominant Co-C
bonding MO due to its large delocalization on the different nuclear centers of the molecule.
53
Results and Discussion
Figure 15. Representation of the frontier molecular orbitals belonging to the full atom
models of AdoCbl and MeCbl.
The HOMO is highly located on the PO4 group, whereas the LUMO is located on the metal
center and the corrin ring.
From the distribution of Co-C MOs, the most important regarding the dissociation process is
the MO with Co-C bonding and Co-Nax antibonding character, depicted on Figure 16, and it will
be the focus of our study. This is higher in energy than the C-Co-Nax bonding MO, and its
relevance comes from the fact that the MO analysis of all the Cbl(II) models revealed that the
SOMO where the unpaired electron is located after the Co-C bond dissociation is derived from
this MO. This means that the main energy changes associated to the Co-C bond dissociation
process, are correlated with the increase in energy of this MO until it is located as the HOMO on
the Cbl(II) models without nucleoside loop, or as the HOMO-1 on models with this moiety.
54
Results and Discussion
Figure 16. Molecular orbital (MO) and Co-C bond strength relationship diagram.
∆ε depicts the energy difference between the MOs related to the Co-C bond strength. The half of
its value was interpreted as the resistance of the Co-C bond toward its electron density
deformation and defined as ηbond-anti, showing a larger value for MeCbl than AdoCbl.
A common feature of cobalamins is the Co-Nax bond length shortening after Co-C bond
breaking, observed theoretically and experimentally from crystal structures. If stated that the CoNax bond length variations after Co-C bond cleavage are mostly defined by the two MOs which
relates the Co-C with the Co-Nax bond. Then, the orbital part of the Co-Nax interaction will be
defined by the energy balance between the stabilizing C-Co-Nax bonding MO which is the one of
lower energy, and the destabilizing MO that has Co-C bonding and Co-Nax antibonding
character. After Co-C bond dissociation, the destabilizing component of the Co-Nax is raised to
the highest occupied MO energy in most cases. Thus, the stabilizing component becomes
dominant and leads to a shorter Co-Nax bond distance.
After defining the MO able to describe the behavior of AdoCbl and MeCbl, its antibonding
counterpart was identified. Then, the stabilization and destabilization energies (∆Eσ and ∆Eσ*,
respectively) of these were obtained as a first approach for a measure capable of distinguish the
differences between AdoCbl and MeCbl Co-C BDE. The ∆Eσ was defined as the energy
difference between the energy of the bonding MO and the energy of the SOMO from their
55
Results and Discussion
corresponding Cbl(II) counterpart, while the ∆Eσ* was calculated using the energy of the
antibonding MO. The results listed on Table 3 exposes that the ∆Eσ of the MeCbl models were
lower when each model was compared with its respective AdoCbl system. This denotes that
MeCbl models allow for a better accommodation of the electron pair that occupies this MO,
probably decreasing their electronic repulsion. From a MO picture, this is appreciated through
the analysis of the shape of the Co-C bonding and Co-Nax antibonding MO of AdoCbl, which
presents a destabilizing orbital interaction between two sets of contributions to this MO. The
first set corresponds to the σ-bonding contribution of the Co-C together with the corresponding
to the two C1-H bonds, and the second is formed between the σ-bonding contribution of C2-C3
and C2-H (according to the numeration depicted on Figure 2). These two groups interact with
each other by orbital phases with opposite signs, which might explain the lower stabilization of
the Co-C bond, which consequently leads to a longer Co-C bond.
Regarding to the ∆Eσ* energies, the MeCbl related models displayed higher values than most
of their AdoCbl model counterpart. These is explained by the shorter Co-C bond distance of
MeCbl models which leads into a higher destabilizing effect than the obtained for the AdoCbl
models caused by the interaction between the Co and C atomic orbitals with opposite atomic
orbital phases.
The problem arises when we tried to extend this analysis to explain the constantly higher
MeCbl BDE with regards to AdoCbl when external contributions to the Co-C bond strength are
discarded. The ∆Eσ values of any of the AdoCbl models should be lower than each of the values
obtained for the MeCbl models, but the calculated ∆Eσ for AdoCbl and AdoCblwoloop, which were
1.73 and 1.66 eV, respectively; are both higher than the 1.64 eV obtained for MeCblwoloopIMI.
Despite this, the latter model has a higher BDE than AdoCbl and AdoCblwoloop and the ∆Eσ value
can not describe the differences on the Co-C bond strengths by itself. Even though ∆Eσ* was
generally higher for the MeCbl models, this value alone is unable to explain the BDE
differences.
From the interpretation of hardness as the half of the energy gap between FMOs, and its
relationship with the resistance of the molecular system to reorganize its electron density, it
seemed interesting as a second approach, to focus on the molecular orbitals directly involved on
the reactivity toward the Co-C homolytic bond dissociation, namely the bonding and
antibonding MOs above defined. Thus, their energy difference will define the energetic
resistance of the Co-C bond toward its cleavage. In order to associate this idea with the chemical
56
Results and Discussion
hardness, we associated the half of their energy difference to the resistance toward the
reorganization of the overlapping density needed for the bond breaking, and it was defined as
ηBOND-ANTIB just for discussion purposes. The results showed a clear trend, where all the MeCbl
models were about 0.24 to 0.30 eV higher in magnitude than the AdoCbl models, which
corresponds to a range of 5.5 to 6.9 kcal/mol. Interestingly, this range of energy accounts for
great part of the 8.6 kcal/mol of BDE difference obtained between AdoCbl and MeCbl. The
integration of these results with the explanation associated to the different behavior observed for
the ∆Eσ and ∆Eσ* values on AdoCbl and MeCbl, shed light into the origin of the differences
observed between these two catalytically active cofactors.
Finally, the ηBOND-ANTIB exposes the nature of the intrinsic difference between AdoCbl and
MeCbl Co-C bond, revealing that systems with methyl as β-axial ligand have a bonding density
harder to reorganize, which make its Co-C bond stronger toward its dissociation. As both
cofactors share the same Cbl(II), the differences are allocated on the β-axial ligand.
57
Results and Discussion
3.2 CO-C BOND DISSOCIATION PROCESS
The discussion presented so far has been focused on the ground state properties of AdoCbl
and MeCbl models. The aim of this section is to shed light on the main differences between the
electronic and structural properties of each cofactor arising along the homolytic dissociation
process using these models. Additionally, to gain a deepest understanding into the role of the
base replacement on the dissociation process, the AdoCblredIMI and MeCblredIMI models were
included.
3.2.1
Potential Energy Surface and Spin Density Analysis
The potential energy surfaces (PES) for the Co-C bond dissociation of the four models are
presented on Figure 17. All of them showed a smooth behavior along the whole process. The
PES displayed on Figure 17-A exposes that the Co-C bond dissociation process associated to
MeCbl is more demanding from an energetic point of view than the obtained for AdoCbl. The
energy differences between the PESs of these cofactors were evident from the beginning of the
scan and it increased at longer Co-C bond distances. After 3.21 Å of Co-C bond distance, both
PESs reached a plateau, keeping the energy difference almost invariable during the rest of the
profile, with an averaged energy difference of ca. 7.6 kcal·mol-1. This value corresponds to the
total additional energy needed by MeCbl to achieve the Co-C cleavage with regards to AdoCbl.
The study of the variables that contributes into this difference will aid to reveal the basis of the
higher energy requirements of MeCbl Co-C bond dissociation process.
The PES of the Co-C bond dissociation process can be divided into two main regions of
analysis. The first region corresponds to the energy pathway associated to the energy needed to
carry out the necessary conformational changes to approach to the transition state geometry. The
second region is dominated by the energy changes resulting from the electron density
reorganization that will lead to the Co-C bond dissociation.
The homolytic Co-C bond dissociation mechanism involves the Co reduction by an inner
sphere electron transfer process mediated by the β–axial ligand. Accordingly, the first region of
the PES corresponds to the Co-C bond elongation accompanied by the necessary conformational
changes that allow the beginning of the charge transfer process. Thus, the limit between the first
58
Results and Discussion
and the second regions is defined by the appearance of spin density on the atoms that form part
of the bond. It can be noticed from Figure 18 that the Co and C spin densities appear when the
bond is elongated to ~ 2.46 Å on both cofactors. At this point of the scan, the Co atom displayed
spin densities values of 0.07 and 0.20 e on AdoCbl and MeCbl, respectively.
35
35
AdoCbl
MeCbl
30
Energia [kcal mol ]
25
-1
-1
Energia [kcal mol ]
25
20
15
10
5
20
15
10
5
(A)
0
2,0
2,5
3,0
3,5
4,0
4,5
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond distance [A]
35
AdoCblredIMI
MeCblredIMI
30
(B)
0
Co-C bond distance [A]
35
MeCbl
MeCblredIMI
30
25
25
-1
Energia [kcal mol ]
-1
Energia [kcal mol ]
AdoCbl
AdoCblredIMI
30
20
15
10
5
20
15
10
5
(C)
0
2,0
2,5
3,0
3,5
Co-C bond distance [A]
4,0
4,5
(D)
0
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond distance [A]
Figure 17. Comparison of the potential energy surfaces for the Co-C bond dissociation
process of the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
The diminished rate for the spin density generation in AdoCbl when is compared to MeCbl,
can be interpreted as an intrinsic resistance of the Ado group to transfer large amounts of charge
at the beginning of the bond dissociation process. This resistance leads to the fact that the energy
needed to elongate the Co-C bond to ~ 2.46 Å on AdoCbl is 9.9 kcal/mol, which is 3.5 kcal/mol
59
Results and Discussion
lower than for MeCbl. This reveals that a slower charge transfer process on cobalamins
stabilizes the system through a more controlled charge transfer process, as observed for AdoCbl.
After a Co-C bond length of 2.71 Å, the Co atom of both cofactors attained similar spin densities
with negligible differences along the rest of the reaction coordinate. This behavior exposes that
starting from this bond length, Cbl(II) does not play a major role on the larger destabilization
observed on the MeCbl energy profile when compared to AdoCbl. The energy needed to reach
the 2.71 Å Co-C bond distance is 18.2 and 23.2 kcal·mol-1 for AdoCbl and MeCbl, respectively.
This implies an increase of the difference between the stability of these cofactors from 3.5 to 5.0
kcal/mol.
1,6
1,6
Co
C
Co
C
1,2
0,8
0,4
0,0
-0,4
-0,8
0,4
0,0
-0,4
-0,8
-1,2
-1,2
(A)
-1,6
2,2
2,6
2,8
3,0
2,2
Co-C bond distance [A]
0,8
2,4
0,8
Spin Density [e]
0,0
-0,4
-0,8
2,8
3,0
2,8
3,0
Co AdoCbl
C AdoCbl
Co AdoCblredIMI
C AdoCblredIMI
1,2
0,4
2,6
Co-C bond distance [A]
1,6
Co MeCbl
C MeCbl
Co MeCblredIMI
C MeCblredIMI
1,2
(B)
-1,6
2,4
1,6
Spin Density [e]
Co AdoCblredIMI
C AdoCblredIMI
Co MeCblredIMI
C MeCblredIMI
1,2
Spin Density [e]
Spin Density [e]
0,8
AdoCbl
AdoCbl
MeCbl
MeCbl
0,4
0,0
-0,4
-0,8
-1,2
-1,2
(C)
-1,6
(D)
-1,6
2,2
2,4
2,6
Co-C bond distance [A]
2,8
3,0
2,2
2,4
2,6
Co-C bond distance [A]
Figure 18. Comparison of the spin density profiles along the Co-C dissociation process of
the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
60
Results and Discussion
Regarding to the charge transfer process, it apparently ends at a Co-C bond distance of 3.41
Å. From this, it can be stated that the core of the reaction occurs between the 2.46 and 3.41 Å of
Co-C bond distance, where most of the charge transfer takes place. Considering that Cbl(II) is
the electron acceptor and it is the same on both cofactors, as it should be their ability to receive
charge, the charge transfer process is mainly controlled by the electron donor ability of the βaxial ligand toward the metal center. Meanwhile, the C atom of MeCbl reaches higher values of
spin density than AdoCbl. This difference seems to be related to the lower capacity of spin
polarization of the methyl radical leading to the formation of a more unstable radical than Ado•.
At Co-C bond distances longer than 3.41 Å, only minor energy increases are observed. This
energy changes are related to the formation and stabilization of the new radical species product
of the reaction. This process involves slight electronic structural and geometric changes such as
the transition from a tetrahedral sp3 C centered radical to a trigonal planar sp2 centered radical.
This region determines the end of the energy increasing which defines the final barrier high.
When the Co-C bond distance is close to ~ 3.01 Å, the spin density on the Co atom is 0.80 e,
~ 89% of the total spin density to be transferred to it from the carbon, which in all models was
close to 0.90 e, value obtained at the end of the scan . On the one hand, the amount of charge
already transferred at 3.01 Å indicates that the Co-C bond is close to be cleaved. After this Co-C
bond length, the energy and the geometry of the dissociated complexes do not change in a
noticeable extent. On the other hand, the spin density value obtained at the end of the scan is the
same obtained for the isolated Cbl(II) model, suggesting that at the end of the scan the Co-C
bond is completely dissociated. At the same point where the charge transfer process ends
completely indicated by the maximum values of the spin densities, the PES reaches a plateau
too.
Close to the final stage of the bond breaking process, AdoCbl underwent a slight decrease on
the energy barrier caused by the stabilization of the supramolecular complex because of the
hydrogen bonds between Ado and the corrin side chains. The energy at the end of the scan was
22.6 kcal/mol. Remarkably, the energy barrier associated to AdoCbl is very close to the BDE
calculated for AdoCblwoSC (22.7 kcal/mol), and not to the one obtained for the full atom model.
This supports the suggestion that the BDE calculated for the full atom model includes the
contribution of the intramolecular hydrogen bonds. This observation encourages us to test a new
hypothesis concerning the protocol for the AdoCbl Co-C BDE calculation. The main idea was to
consider that the conformations of the products of the bond dissociation are influenced by the
61
Results and Discussion
presence of the opposite fragment due to the intramolecular hydrogen bonds. This would affect
the geometry and energy of its counterpart. Additionally, this phenomenon could be part of the
events that occurs in the experimental determination of the BDE. Hence, to explore this
possibility, the BDE was recalculated using the energy of Ado and Cbl(II) at the adopted
geometries at a Co-C bond distance of 4.26 Å. The recalculated BDE values for AdoCbl was
31.2 kcal/mol, 0.2 kcal/mol lower than and in good agreement with the experimental; supporting
the hypothesis proposed. Even though the previous proposal seems to be reasonable and
reformulates the AdoCbl Co-C BDE calculation procedure, the latter result needs to be
considered with caution because it did not include the solvation and zero point energy
corrections. Nonetheless, these interactions seem to be related to the stability of the Co-C bond
of the cofactor when it is not bounded to the enzyme. When the cofactor is bonded to the
enzyme (e.g. glutamate mutase), these intramolecular interactions are completely dissociated due
to the fact that the ribosyl moiety and the corrin ring side chains interact with specific residues of
the enzyme and not between them. This would weaken the Co-C bond through the elimination of
this external component leading to a BDE close to the obtained for AdoCblwoSC.
To sum up, the final energy needed to achieve the Co-C bond cleavage corresponded to 22.6
kcal/mol for AdoCbl and 30.1 kcal/mol for MeCbl, with a maximum energy difference of 7.5
kcal/mol.
The comparison between the PESs of AdoCbl and AdoCblredIMI depicted in Figure 17-B,
showed that only AdoCbl presents the decrease at the final stage of the dissociation process. This
is because AdoCblredIMI does not have corrin side chains and it can not form the intramolecular
hydrogen bonds. Additionally, this supports the fact that the energy decrease observed on
AdoCbl is actually caused by these interactions. The energy difference between these two
models started to appear at a Co-C bond distance of 2.96 Å. From this point, a small but
increasing difference appeared through the rest of the process with a final difference of 2.3
kcal/mol. This value accounts for the total stabilization due to the intramolecular interactions
when the bond is completely cleaved.
With regards to the rest of the PESs, both models showed similar energy values. The spin
density profile of AdoCblred showed that it started to appear at a Co-C bond length of 2.41 Å,
distance slightly shorter than the observed for AdoCbl. At the beginning of the ET process, it
was also appreciable a larger increase of the spin density on AdoCblred than the observed on the
full atom model. After a Co-C bond length of 2.81 Å, both models equals their spin density
62
Results and Discussion
magnitudes. As discussed above for AdoCbl and MeCbl, this difference in the spin density
behavior involves an energy increase at the end of the PES for this type of processes. This is also
observed when it is compare the BDE of AdoCblwoSC, model that represents the intrinsic strength
of the Co-C bond, and AdoCblredIMI. The latter has a BDE of 25.8 kcal/mol, which is 3.1
kcal/mol higher than the obtained for AdoCblwoSC.
MeCbl and MeCblredIMI showed very similar PESs with only sligth differences along the
process. The comparison of their spin densities depicted on Figure18-C, expose that the spin
density appears earlier and in a larger extent on MeCblredIMI, similar to what was described above
for the models with Ado as the β-axial ligand. The Co-C bond distance where it appears on
MeCblredIMI is at 2.31 Å, 0.15 Å shorter than the observed for MeCbl.
AdoCblredIMI and MeCblredIMI showed an associated Co-C cleavage energy barrier of 24.9 and
31.3 kcal/mol, respectively. These energies correlated well to the corresponding BDE calculated
for those models. The energetic differences displayed by these models have a behavior similar to
the observed between AdoCbl with MeCbl. In both reduced models, spin densities on Co and C
atom appear at shorter Co-C distance regarding to the full atom models.
The results reveal that the nature of the methyl group is neither able to transfer charge
gradually nor the ability to lower the energy needed to achieve the charge transfer.
At this point, the base replacement does not show any significant modification on the
dissociation process that could lead us into its role.
3.2.2 Geometric Distortion
The geometric parameters which exhibited the most meaningful variations on its behavior
along the dissociation process were the corrin ring distortion angle and the Co-Nax bond length.
The latter is depicted on Figure 19. All the studied models showed an increment in the corrin
distortion angle during the dissociation process, with an attained value at the longest Co-C bond
distance similar to the obtained for its isolated Cbl(II) subunit. This indicates that the distortion
angle at 4.26 Å of Co-C bond length is close to the obtained upon complete bond dissociation; in
turn no further corrin distortion is expected at longer Co-C bond distances. Along with this, the
maximum corrin distortion points out that the Cbl(II) fragment is favored by a more folded
conformation of the macrocycle. To estimate the degree of distortion of the coordination
geometry around the Co center, we superimposed the four Neqs of AdoCbl ground state with the
63
Results and Discussion
Neqs of the AdoCbl structure with a Co-C bond length of 4.26 Å. The results exposed a
displacement of 0.18 Å of the Co center toward downwards, out of the Neqs plane, resulting in a
distorted square pyramidal coordination geometry.
2,26
2,26
Co-Nax AdoCbl
Co-Nax MeCbl
2,22
2,20
2,18
2,16
2,14
2,12
2,20
2,18
2,16
(B)
2,12
2,5
3,0
3,5
4,0
4,5
2,0
Co-C bond distance [A]
2,26
2,5
3,0
3,5
Co-Nax bond distance [A]
2,20
2,18
2,16
2,14
4,5
Co-Nax MeCbl
Co-Nax MeCblredIMI
2,24
2,22
4,0
Co-C bond distance [A]
2,26
Co-Nax AdoCblredIMI
Co-Nax MeCblredIMI
2,24
Co-Nax bond distance [A]
2,22
2,14
(A)
2,0
2,12
Co-Nax AdoCbl
Co-Nax AdoCblredIMI
2,24
Co-Nax bond distance [A]
Co-Nax bond distance [A]
2,24
2,22
2,20
2,18
2,16
2,14
(C)
2,0
2,12
2,5
3,0
3,5
Co-C bond distance [A]
4,0
4,5
(D)
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond distance [A]
Figure 19. Comparison of the Co-Nax bond length variations along the Co-C bond
dissociation process of the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
The unpaired electron located on the Co center after the Co-C bond cleavage is not
symmetrically distributed in the Co-dz2 atomic orbital because of its delocalization on the α-side.
The spin density analysis showed that it is also localized on the sp2 atomic orbital of the nitrogen
atom of the imidazole ring (See Figure 20). This may cause a destabilization of the system by the
electronic repulsion between the spin density localized on the lower side and the corrin π-cloud
coming from the Neqs. This induces an upward folding with the subsequent distortion of the
coordination geometry. According to these results, we suggest that the flexibility of the corrin
64
Results and Discussion
ring allows a conformational change able of minimize the electron repulsion, stabilizing the
system towards the electron reduction. In addition, our results indicate that pushing the β-axial
ligand upwards, as previously proposed with the mechanochemical trigger mechanism, it is not
viable because it would need an even larger folding of the corrin ring.
Figure 20. AdoCbl and MeCbl Spin Density distribution.
Both cofactor models have a Co-C bond length of 4.26 Å, being this bond is completely
dissociated. The spin density is mainly located on the C, Co and Nax.
To evaluate the energetic contribution of the corrin folding on the Co-C bond dissociation
process, a PES along the Co-C bond was calculated, where the position of the heavy atoms of
the corrin ring were kept fixed at the ground state coordinates. The results depicted on Figure 21,
showed an increase of 5.4 kcal/mol on the energy barrier for the dissociation process, when
compared with the PES obtained for AdoCbl with a flexible corrin ring.
The distortion angle variations for all the models were similar but not identical. However, we
believe that the corrin ring flexibility contributes on the Co-C bond dissociation process in a
comparable extent on all the studied systems. If the enzyme would be able to distort the corrin
ring in a similar fashion as the observed on the free form upon Co-C bond cleavage, it should be
65
Results and Discussion
able to facilitate the charge transfer at the ground state avoiding the electronic repulsion arising
from the incoming electron.
30
Energy [kcal/mol]
25
20
15
10
5
AdoCbl Free Corrin
AdoCbl Fixed Corrin
0
2,0
2,5
3,0
3,5
4,0
4,5
Co-C Bond Distance [A]
Figure 21. Energetic contribution of the corrin ring flexibility to the Co-C bond
dissociation process.
Here, the potential energy surfaces of a model of AdoCbl with the corrin ring spatially restricted
was compared with the completely unrestricted model.
The behavior of the Co-Nax bond distance along the dissociation process on all the models
started by an initial stage dominated by the Co-Nax bond contraction until a minimum Co-Nax
bond distance is reached. The point of the scan where the Co-Nax bond minimum is located
corresponds to the Co-C bond length in which the charge transfer process starts. Then, the CoNax bond is elongated until a maximum length is reached at a Co-C bond distance close to
3.01 Å on all models. This maximum coincides with the region where 89% of the total spin
density was transferred to the Co center indicating that the Co-C is almost completely cleaved.
Finally, the maximum is followed by a slight decrease on the Co-Nax bond length, which is
66
Results and Discussion
probably related to a relaxation process, particularly on the reduced models. The average
distance of this final stage is shorter than the initial Co-Nax bond length. This is noticed on all the
studied models.
The Co-Nax bond contraction leads to the conformation needed to prepare the system to
receive the electronic charge from the β-axial ligand to the metal center. The Co-Nax bond length
of AdoCbl and MeCbl is shortened by 0.09 and 0.07 Å, respectively; and the subsequent
elongation increase its length by 0.09 Å on AdoCbl and 0.05 Å on MeCbl. The final distances
obtained for Co-Nax were close to their corresponding isolated Cbl(II) fragments, but they were
not identical. The reason for this may rely on the dependence on the initial geometries and
consequently of the designed model, as established on the first part of this report.
The reduced models showed a Co-Nax bond contraction of 0.06 for AdoCblredIMI and 0.04 Å
for MeCblredIMI, lower than the obtained for the full models. As stated above, after the maximum
is reached, a slight decrease of the Co-Nax bond length is observed on the reduced models at the
final stage of the Co-C dissociation process. This is provided by the smaller size of the α-axial
ligand with regards to the full atom models. Both reduced models showed almost the same CoNax bond at the end of the studied reaction coordinate, with a bond length close to 2.18 Å.
The effect of the β-axial ligand on the reduced models Co-Nax bond length is visualized in the
region between the ground state geometry and the corresponding with a Co-C bond distance of
3.01 Å. MeCblredIMI presents a higher propensity to Co-Nax bond contraction at earlier stages of
the Co-C bond elongation, leading to shorter Co-Nax bond distances until the 3.01 Å Co-C bond
length is reached. This shorter Co-Nax bond distance observed at earlier stages of Co-C bond
elongation on MeCblredIMI compared to AdoCblredIMI, correlates with the earlier beginning of the
charge transfer process observed on it This implied an energetically more demanding process
compared to it counterpart with the Ado group, in agreement with our previous findings.
3.2.3 Electronic Charge Rearrangement due to the Co-C bond Dissociation
The Co-C bond dissociation process involves the reduction of the metal center through an
electron transfer process, which was mainly studied by the spin density analysis. The addition of
electron density on the Co also implies the redistribution of the electronic density on the cofactor
as part of the mechanism for the Co-C bond dissociation. In order to obtain an accurate picture
of the main processes associated to this charge rearrangement, an analysis of the variations on
the partial charges along the reaction coordinate is presented. This analysis is focused on the
67
Results and Discussion
specific sections of the cofactor that are directly involved on the events associated to the
dissociation process. These are the cobalt center, the carbon from the β-axial ligand bonded to
the Co and the Neqs.
The full atom models showed very similar variations of their total partial charges after the
Co-C bond cleavage. Meanwhile, minor differences were observed when compared the full atom
models with their respective reduced models. Initially, in both cases the reduced model
presented a lesser positive and negative charge on Co and C, respectively; than their
corresponding full atom models. However, after a Co-C bond distance of 3.01 Å their partial
charges converged to similar values. This indicates that beyond this Co-C bond length the αaxial ligand has no influence, which is also related to the fact that at this stage the Co-C bond is
almost completely cleaved. The influence of the base replacement on the dissociation process
seems to be related to slightly decreasing the magnitude of the electron density rearrangement
needed for the Co-C cleavage.
The total variations on the partial charges on the four models were defined by a decrease of ~
0.25e on C accompanied by a charge decrease on the Co of ~ 0.16e. Finally, an increase of ~
0.23e was observed on the Neqs. These values were slightly lower for the reduced models. As
expected, the decrease of the charge on the C atom indicates that it is transferring charge to the
metal center during the dissociation process. However, the Co atom also loses charge, opposite
to what was expected (see Figure 22). After the analysis of the natural electronic configuration,
the apparent lost of charge on Co is caused by the decrease of charge donated by the Neqs to the
Co-dx2-y2 atomic orbital. This is accompanied by the increase of charge on the Neqs-px and Neqs-py
atomic orbitals. This reveals a gradual diminution of the charge donation towards the metal
center and probably of the strength of these Co-Neqs bonds; although no length variations were
observed. This facilitates the process associated to the upward folding of the corrin ring after the
reduction of the Co. In addition, it is observed that the Co-dxz and Co-dyz atomic orbitals donate
charge to the Neqs-pz atomic orbitals by π-retrodonation. This probably aids to diminish the
electronic repulsion between the electron densities from Co-dxz and Co-dyz with the incoming
electron density to the Co-dz2 atomic orbital. As the charge transferred to the Neqs by the Co
center is larger than the received by it from the β-axial ligand, the total charge located on Co
diminishes after the Co-C bond dissociation.
68
Results and Discussion
0,96
0,96
Co AdoCbl
Co MeCbl
0,92
0,88
NPA charge [e]
0,88
NPA charge [e]
Co AdoCbl
Co AdoCblredIMI
0,92
0,84
0,80
0,76
0,84
0,80
0,76
(B)
(A)
0,72
0,72
2,0
2,5
3,5
4,0
4,5
2,0
3,0
3,5
4,0
4,5
0,96
Co AdoCblredIMI
Me AdoCblredIMI
0,92
2,5
Co-C bond Distance [A]
Co-C bond Distance [A]
0,96
Co MeCbl
Co MeCblredIMI
0,92
0,88
0,88
NPA charge [e]
NPA charge [e]
3,0
0,84
0,80
0,76
0,84
0,80
0,76
(D)
(C)
0,72
0,72
2,0
2,5
3,0
3,5
Co-C bond Distance [A]
4,0
4,5
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond Distance [A]
Figure 22. Electronic Charge Redistribution on Co atom along the Co-C dissociation
process of the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
The variation on the Co charge started with a slow and near to linear decrease of it on the
region associated to Co-C bond elongation with no electron transfer from C. At a Co-C bond
length of 2.41 Å a breakpoint is observed, after which a fast decrease on the Co charge occurred
because of the beginning of the electron transfer. This behavior continues until a plateau is
reached after 3.01 Å of Co-C bond distance. At this point most of the electron from C was
already transferred as pointed out before. The partial charge variations on the C atom followed
exactly the same behavior described for the Co atom along the Co-C dissociation reaction (see
Figure 23).
Despite the fact that the variations of the partial charges between models were similar, there
is an interesting feature of methyl systems when compared to Ado models. MeCbl and
MeCblredIMI showed more negative charges on the C atom than their analogous Ado models
69
Results and Discussion
during the whole process. Meanwhile, the magnitude of the Co partial charge is similar between
Ado and methyl related models, indicating that the charge on Co is independent of the β-axial
ligand identity. Then, the difference on the C charge is due to the nature of the β-ligand and it
could be related to the inability of the methyl molecule to distribute its negative charge on more
centers as the Ado ligand does. More importantly, the larger difference between the Co and C
partial charges of MeCbl than AdoCbl, results in a stronger electrostatic contribution to the Co-C
bond strength.
-0,2
-0,2
C AdoCbl
C MeCbl
-0,3
NPA charge [e]
-0,4
NPA charge [e]
C AdoCbl
C AdoCblredIMI
-0,3
-0,5
-0,6
-0,7
-0,4
-0,5
-0,6
-0,7
(A)
(B)
-0,8
-0,8
2,0
2,5
3,0
3,5
4,0
4,5
2,0
2,5
3,5
4,0
4,5
Co-C bond Distance [A]
Co-C bond Distance [A]
-0,2
-0,2
C AdoCblredIMI
C MeCblredIMI
-0,3
C MeCbl
C MeCblredIMI
-0,3
-0,4
NPA charge [e]
NPA charge [e]
3,0
-0,5
-0,6
-0,7
-0,4
-0,5
-0,6
-0,7
(C)
(D)
-0,8
-0,8
2,0
2,5
3,0
3,5
Co-C bond Distance [A]
4,0
4,5
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond Distance [A]
Figure 23. Electronic Charge Redistribution on C atom along the Co-C dissociation
process of the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
The comparison between the Neqs partial charges of AdoCbl and MeCbl cofactors (Figure 24)
displayed identical profiles both in magnitude and behavior, meaning that they do not
discriminate between β-axial ligands. The same was observed between the reduced models. The
70
Results and Discussion
differences arise when the full atom models were compared with their reduced models. The full
atom models present more positive charges along the complete reaction coordinate, although
both follow the same trend. A probable explanation is related to the larger planarity of the corrin
ring present on the reduced models, which may allow a larger π-retrodonation on its ground
state. According to this, the base replacement affects the Neqs partial charges on the ground state,
but it does not affect the amount of charge that is received from the metal center during the
dissociation process.
Regards to the Neqs partial charge behavior along the dissociation reaction, it follows the same
variations as described for Co and C, but in the opposite direction, which in this case represents
a process of charge increase.
-1,80
-1,80
Neqs AdoCbl
Neqs MeCbl
-1,85
-1,90
NPA charge [e]
-1,90
NPA charge [e]
Neqs AdoCbl
Neqs AdoCblredIMI
-1,85
-1,95
-2,00
-2,05
-2,10
-1,95
-2,00
-2,05
-2,10
(A)
(B)
-2,15
-2,15
2,0
2,5
3,0
3,5
4,0
4,5
2,0
3,0
3,5
4,0
4,5
-1,80
Neqs AdoCblredIMI
Neqs MeCblredIMI
-1,90
2,5
Co-C bond Distance [A]
Co-C bond Distance [A]
-1,85
Neqs MeCbl
Neqs MeCblredIMI
-1,85
NPA charge [e]
NPA charge [e]
-1,90
-1,95
-2,00
-2,05
-2,10
-1,95
-2,00
-2,05
-2,10
(C)
(D)
-2,15
-2,15
2,0
2,5
3,0
3,5
Co-C bond Distance [A]
4,0
4,5
2,0
2,5
3,0
3,5
4,0
4,5
Co-C bond Distance [A]
Figure 24. Electronic Charge Redistribution on Neqs atom along the Co-C dissociation
process of the free models.
(A) AdoCbl v/s MeCbl (B) AdoCbl v/s AdoCblredIMI (C) AdoCblredIMI v/s MeCblredIMI (D) MeCbl
v/s MeCblredIMI
71
Results and Discussion
3.3 INSIGHTS INTO THE GM CATALYTIC CYCLE
To explore the driving forces involved in the catalytic cycle of GM and how the different
intermediates are related to them, most of the stages of the catalytic cycle were modelled (see
Figure 25). The results are depicted on the energy diagram from Figure 26.
The results showed that the binding of the substrate to the complex AdoCbl-GM is
energetically favored by 17.1 kcal/mol, process represented in the energy diagram by the
transition from A to B. The subsequent stage triggered by the substrate binding is the Co-C bond
breaking process represented by the transition from B to C, which displayed an energy barrier of
18.5 kcal/mol. The conformation adopted in this stage corresponds to the confA. The barrier
height is related to the instability of the Ado radical formed after the Co-C bond cleavage.
Figure 25. Schematic representation of the steps of the GM-catalytic cycle modelled in this
study.
72
Results and Discussion
Figure 26. Energy Diagram of the steps of the GM-catalytic cycle modelled in this study.
A-I represents the steps described in Figure 24.
The next stage corresponds to the ribose conformational change from confA to confB (C to
D), where the carbon radical goes from above the metal center to a position on which it points
toward the reactive hydrogen of glm. This conformational change involves an energy decrease of
11.2 kcal/mol.
If we consider the paradigm between the step-wise and the concerted mechanisms, the latter
should involve a pathway on which the Co-C bond dissociation process is coupled to the
hydrogen abstraction from the substrate, where the confB is the reference state for this pathway.
The energy barrier between the ground state and the confB is 7.3 kcal/mol (B to D), close to
the ~ 8.0 kcal mol-1 proposed as the apparent BDE of the Co-C bond after the formation of the
AdoCbl-GM/glm complex. This decrease on the energy barrier supports the idea of a concerted
mechanism.
73
Results and Discussion
After the conformational change of the ribose, the hydrogen abstraction (D to E) takes place
with a large energy decrease of 16.4 kcal/mol. This energy is related to the difference between
the stabilities of the Ado• radical and the more stable glm• radical. The formation of glm• is the
main driving force within the catalytic cycle, and represents a critical step in which the catalytic
cycle is propagated towards product formation.
The transformation from the substrate-derived to the product-related radical (E to F) shows
an energy increase of ~ 8.1 kcal/mol. The subsequent hydrogen abstraction from the AdoH
ligand by the masp• radical (F to G) is favored by 3.8 kcal/mol. This indicates that the masp•
radical is more unstable than the Ado radical, difference that promotes the hydrogen abstraction
from AdoH in order to form the final product. This result expose the complexity of the F/R
mechanism, responsible of forming an unstable specie in order to promote the catalytic cycle
towards the product formation, providing the necessary driving forces.
To finish the catalytic cycle, the ribose needs to undergo the conformational change from
confB to confA (G to H). This stage resulted to be an endergonic pathway, at least in presence of
the product, with an energy difference of 12.6 kcal/mol.
An alternative pathway could be going directly from stage G to H (Figure 26), which implies
a drop of 6.5 kcal/mol in the energy difference. Another possibility to be tested is that the
pathway from confA to confB and the subsequent Co-C bond formation is favored only in
absence of masp, which should leave the binding site after hydrogen abstraction from AdoH.
74
Results and Discussion
3.4 CATALYTIC CO-C BOND DISSOCIATION PROCESS
3.4.1 Role of the Aspartate-14 Residue on the Dissociation Process
The role of the base replacement on the catalytic Co-C bond dissociation reaction is still
unclear. Despite the fact that previous theoretical studies discarded its role on the process
because no decrease on Co-C bond strength was observed after replacing DMB by an imidazole
ring, a 1000-fold decrease rate of the reaction rate has been experimentally observed after
mutation of this residue. Also, it should be considered that the previous theoretical studies were
performed using small models and without considering the enzymatic environment as part of the
quantum systems.
The main difference between DMB and His16 as α-axial ligands corresponds to the
interaction of the latter with an aspartate residue through a hydrogen bond, giving rise to the
His16-Asp14 pair. This interaction somehow modifies the electron donor and acceptor ability of
the His16 as a ligand with regards to DMB, modifications that certainly could provide the
necessary properties that lead into different Co-C bond dissociation mechanisms.
His-Asp pair has been found to be a critical component of several enzymatic reactions. At
present, the role that these pairs play in catalysis is best understood in enzymes such as serine
and trypsin-like proteases, where structural and biochemical NMR studies have revealed
important pKa values and hydrogen bonding patterns within the catalytic pocket. However, the
role of the His-Asp pair in metal assisted catalysis is less clear [110].
Herein, we studied the role of the His16-Asp14 pair through the comparative study of
AdoCbl-H16/D14 and AdoCbl-H16 models displayed on Figure 27, to shed light into the base
replacement problem and to provide new insights regarding to the mechanism by which the HisAsp pair contributes to metal assisted enzyme catalysis. It should be noticed that this models
only includes the His16 and Asp14 (only AdoCbl-H16/D14) from the enzyme, and they do not
include the rest of the residues of the catalytic site.
The obtained PESs and spin density profiles are displayed on Figure 28. The calculated
energy barriers for the dissociation process were of ~ 20.0 and ~ 13.5 kcal/mol for AdoCbl-H16
and AdoCbl-H16/D14, respectively. These results exposed that the inclusion of the Asp14 residue
as part of the model induces a decrease of 6.5 kcal/mol of the energy barrier, revealing that the
His16-Asp14 pair is far from innocent during the Co-C bond dissociation process.
75
Results and Discussion
Figure 27. Optimized geometries of the AdoCbl-H16/D14 and AdoCbl-H16/D14 models after
the Co-C bond dissociation process.
22
16
AdoCbl-H / D
16
AdoCbl-H
20
16
14
18
0,6
0,4
14
Spin Dens [e]
Energy [kcal/mol]
16
14
Co AdoCbl-H / D
16
14
C AdoCbl-H / D
16
Co AdoCbl-H
16
C AdoCbl-H
0,8
12
10
8
6
4
(A)
0,2
0,0
-0,2
-0,4
(B)
-0,6
2
-0,8
0
-1,0
-2
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 28. AdoCbl-H16/D14 versus AdoCbl-H16. A) Potential energy surfaces of the Co-C
bond dissociation process B) Spin density profiles along the Co-C dissociation
76
Results and Discussion
The energy difference between the PES of both models appeared from the beginning of the
elongation process, stage that was less energetically demanding for AdoCbl-H16/D14. The energy
difference was increasing along the subsequent stages of the bond cleavage, denoting that Asp14
affects not only the elongation process, but also the electron transfer occurring from the C to the
Co center.
To establish the relationship between the energetic changes and the electron transfer
processes, we compared the energy profiles with the spin densities centered on the Co and C
atoms where the unpaired electrons are mainly located after the Co-C bond cleavage process.
According to the spin density profiles, AdoCbl-H16 started the electron transfer at a shorter Co-C
bond distance than AdoCbl-H16/D14. As stated before on the study of the free cofactors, the
electron transfer process is energetically favored when it occurs at longer Co-C bond distances,
explaining the lower energies of AdoCbl-H16/D14 on the region where the electron transfer takes
place. At the end of the scan, the spin density centered on the carbon (C5’) of both models
reached the same magnitude. This is the expected result because in both cases the same Ado•
radical is generated. Regarding to the Co center, it was observed a larger delocalization of the
unpaired electron on the model with Asp14 exposed by its lower spin density values along the
scan. This increase of the delocalization could be related to the mechanism responsible of
delaying the electron transfer and decreasing the energy barrier of the Co-C bond cleavage
process of AdoCbl-H16/D14.
Despite the fact that the role of the His16-Asp14 seems to be focused on the electron transfer
region of the PES, the energy difference between the PESs of these models increased at the final
stages of the process. This is caused by the stabilization of the Cbl(II), where the His16-Asp14
pair also plays an important role.
The most noticeable geometric variation observed along the Co-C bond dissociation was the
Co-Nax bond length (Figure 29). From the ground state geometry of AdoCbl-H16/D14 model it is
possible to observe that Asp14 acts as an acceptor of the His16 δ-proton. This partial proton
transfer induces a decrease on the Co-Nax bond distance from 2.133 to 2.096 Å. Meanwhile, the
resulting Co-C bond length was of 2.030 Å and 2.006 Å for AdoCbl-H16/D14 and AdoCbl-H16,
respectively. Remarkably, the model without the Asp14 residue showed the same Co-C bond
length obtained for the free models of the AdoCbl. This supports the idea that the histidine
residue by itself does not modify significantly the properties of the Co-C bond. The model with
77
Results and Discussion
the His16-Asp14 exposed a slight increase of the Co-C bond distance, which confirms that these
residues together are able to affect the Co-C bond.
The difference on the Co-Nax bond distance between the two models persisted almost
constantly along the dissociation process, where the AdoCbl-H16/D14 always showed shorter CoNax bond lengths due to the Asp14 residue. The first stage associated to the Co-C bond
elongation, was defined by a sustained decreased of the Co-Nax bond lengths on both models
until a minimum was reached. The minimum coincides with the Co-C bond distance on which
the charge transfer starts, as described for the free cofactor. Then, the Co-Nax bond distance
started to increase, ending with a slightly higher bond distance than the observed on the ground
state. The Co-Nax bond elongation is induced by the beginning of the electron transfer which
increases the electronic charge of the Co-dz2 and consequently returns part of the charge donated
by Nax back to it.
2,15
16
14
AdoCbl-H / D
16
AdoCbl-H
2,14
Co-Nax Bond Distance [A]
2,13
2,12
2,11
2,10
2,09
2,08
2,07
2,06
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 29. AdoCbl-H16/D14 versus AdoCbl-H16.Comparison of the Co-Nax bond length
variations along the Co-C bond dissociation process.
78
Results and Discussion
The analysis of the partial charges of the models at their ground state exposed that the
His16-imidazole atoms of the model with Asp14 suffered an increase of 0.05e on the Nax and of
0.24e on their total charge. This supports the formation of a strong hydrogen bond with partial
deprotonation of His16 carried out by Asp14 that increases the negative charge of the former.
Additionally, we evaluated the variation on the amount of charge donated by His16 to the metal
center by calculating the natural electron configuration of His16 and the His16-Asp14 pair as
isolated fragments. The results showed that the His16-Asp14 pair donates two times more charge
to the metal center than the His16 alone. The charge is donated through the Nax-pz atomic orbital
to the Co-dz2. It should be noticed that the Co retrodonates charge through the Co-dyz to the Naxpy atomic orbital. The increase of the charge donated by the α-axial ligand triggered a series of
changes on the rest of the electronic structure of the cofactor. The C5’ atom from the Ado group
increased its negative charge by 0.04e, while the total negative charge of the Neqs was decreased
by 0.06e. Also, the Co center suffered a slight increase of its electronic charge (less positive). To
sum up, the strong hydrogen bond between the His16 and the Asp14 increase the negative
charge of His16-imidazole ring together with the electrostatic attraction between the Co and the
Nax, thus shortening the Co-Nax bond distance. The higher proximity between these two atoms
increases the overlap between the Nax-pz and the Co-dz2 atomic orbitals promoting the charge
transfer toward the metal center. As a consequence, the Co accepts a lower amount of charge
from the Ado-C5’ atom to balance the occupancy of the Co-dz2. Additionally, the shortening of
the Co-Nax bond rises in energy the C-Co-Nax bonding-antibonding MO described in the first
section of this thesis, decreasing the destabilizing component of the Co-Nax bond which also
decreases the bonding orbital component of the Co-C bond.
The initial Co-C bond elongation stage was less energy demanding for AdoCbl-H16/D14 as the
result of the decrease of the amount of charge donated by C to the Co on the ground state, which
decreased the orbital nature of the Co-C interaction, also related with what was explained above.
This allowed for the elongation of the Co-C bond at a lower energy cost than the observed for
the model without the Asp14.
The electronic retrodonation from Co to Nax leads to a loss of charge outside the z axis where
the Co-dz2 and Nax-pz are located. This promotes the increase of charge donation from the Neqs to
the Co-dx2-y2 atomic orbital to avoid a highly positive charge on the Co region. The increased
overlapping between the Co-dz2 and Nax-pz denoted by the shorter Co-Nax bond distance of
AdoCbl-H16/D14, promotes the delocalization of the unpaired electron in the Nax-pz atomic orbital
79
Results and Discussion
during the electron transfer process. This aids to decrease the spin density on the Co center
stabilizing the system and decreasing the energy of the process.
0,90
16
-0,53
14
Co AdoCbl-H / D
16
Co AdoCbl-H
0,88
-0,54
0,86
-0,55
NPA charge [e]
NPA charge [e]
0,84
-0,56
-0,57
-0,58
-0,59
0,82
0,80
0,78
0,76
-0,60
16
(A)
14
Nax AdoCbl-H / D
-0,61
Nax AdoCbl-H
2,0
2,2
2,4
(B)
0,74
16
0,72
2,6
2,8
3,0
3,2
2,0
2,2
Co-C Bond Distance [A]
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
-0,30
16
14
C AdoCbl-H / D
16
C AdoCbl-H
-0,35
16
14
Neqs AdoCbl-H / D
16
Neqs AdoCbl-H
-1,80
-1,85
NPA charge [e]
NPA charge [e]
-0,40
-0,45
-0,50
-0,55
(C)
-0,60
-0,65
-1,90
-1,95
-2,00
(D)
-2,05
-2,10
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 30. AdoCbl-H16/D14 versus AdoCbl-H16. Comparison of the electronic charge
redistribution along the Co-C dissociation process (A) Nax (B) Co (C) C (D) Neqs
It is noticed from Figure 30 that the Asp14 from AdoCbl-H16/D14 prevents the charge
reorganization of the C atom together with the electron transfer (Figure 28) until a longer Co-C
bond distance than the observed on AdoCbl-H16 is reached. This again is caused by the larger
amount of electronic charge donated by Nax to the metal center on the AdoCbl-H16/D14 model,
which prevents the C to transfer charge to the Co at shorter Co-C bond distances. After
AdoCbl-H16/D14 reaches a Co-C bond distance of 2.53 Å, the charge on C begins to decrease,
80
Results and Discussion
accompanied by an increase of the electronic charge on the Neqs. The electronic charge of the
Neqs plays an important role in the dissociation process, helping to maintain a charge balance on
the metal center. Meanwhile, the AdoCbl-H16 model showed slightly more positive charges on
Co than its counterpart, but both models ended with similar partial charge values. This points out
that the effect of the His16-Asp14 pair on the Co center is located on the first stages of the
dissociation process. With regards to the Neqs partial charges, both models presented exactly the
same behavior along the Co-C cleavage, with the only difference that the model without Asp14
showed more negative charges, meaning that it donated less charge to the Co center. The C atom
seems to reorganize its electronic charge from the beginning of the process, also showing less
negative charges. This is because the His16 does not saturate the Co-dz2, allowing the Co to
reorganize some charge from the Co-C bond region. Finally, the Nax partial charges were less
negative as expected.
3.4.2 Role of the Enzymatic Environment on the Co-C Bond Dissociation Process
To shed light into the influence of the enzymatic machinery on the catalytic Co-C bond
dissociation process, we performed a comparative study using the AdoCbl-GM-H16/D14 and
AdoCbl-His16/Asp14 models depicted on Figure 31. These models differ on the fact that
AdoCbl-GM-H16/D14 includes a set of residues as part of the model, which represents the
neighboring residues of the binding site.
The resulting PESs are depicted on Figure 32. Both models started the dissociation process
following similar energy pathways, indicating that the first stage of the elongation process is the
same for the two systems. This was expected because both models included the Asp14 residue.
After a Co-C bond length of 2.30 Å the PESs followed separated energy pathways. The energy
difference between them increased only slightly along the dissociation process, with an average
value near to 1.5 kcal/mol until a Co-C bond distance of 2.75 Å was reached. After this,
AdoCbl-GM-H16/D14 showed a noticeable decrease of the energy barrier with regards to the
model without the enzyme as part of the model. The maximum energy difference between the
two models at the end of the scan was ~ 3.0 kcal/mol, and the energy barrier calculated for
AdoCbl-GM-H16/D14 was of 10.5 kcal/mol.
81
Results and Discussion
Figure 31. Optimized geometries of the AdoCbl-H16/D14 and AdoCbl-GM-H16/D14 models
after the Co-C bond dissociation process.
16
14
AdoCbl-GM-H / D
16
14
AdoCbl-H / D
14
16
0,6
12
0,4
10
0,2
Spin Dens [e]
Energy [kcal/mol]
14
Co AdoCbl-GM-H / D
16
14
C AdoCbl-GM-H / D
16
14
Co AdoCbl-H / D
16
14
C AdoCbl-H / D
0,8
8
6
4
(A)
2
0,0
-0,2
-0,4
(B)
-0,6
-0,8
0
-1,0
-2
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 32. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14. A) Potential energy surfaces of the
Co-C bond dissociation process. B) Spin density profiles along the Co-C dissociation
process
82
Results and Discussion
The interactions between the Ado group and the Lys326-Glu330 pair induce the stretching of
the Co-C bond to ~ 2.075 Å of bond length, because the Lys326-Glu330 pair tends to stabilize
the conformation associated to the dissociated form of the cofactor rather than the ground state
of the cofactor. As a result, these interactions facilitate the final stage of the Co-C bond
elongation process before the charge transfer begins. Regarding to the energy decrease observed
at the end of the scan, it reveals that the main role of the enzymatic environment is related to the
stabilization of the Ado• radical formed as a product of the Co-C bond dissociation process.
According to the spin density profile, the electron transfer process started at a Co-C bond
distance close to 2.51 Å on the two models, as depicted on Figure 32. The main difference was
that AdoCbl-GM-H16/D14 transferred a slightly lower amount of charge than AdoCbl-H16/D14 at
the beginning of the electron transfer process. Even thought, the energy difference at this stage is
mainly due to the previous energy decrease at the elongation process before the charge transfer.
After a Co-C bond length of 2.75 Å the spin densities on both models have similar values,
contrary to the results obtained for Co on the study of AdoCbl-H16/D14 versus AdoCbl-H16. This
indicates that the main effect of the enzymatic environment is mostly related to the stabilization
of the resulting products of the process, and not to the transfer process. The delocalization of the
unpaired electron received by the Co was similar on both systems because the two models
included the Asp14 residue.
The model including the enzymatic environment has a shorter Co-Nax bond length at the
ground state. The stretching of the Co-C bond mediated by the enzymatic environment leads to
the contraction of the Co-Nax bond length, keeping shorter Co-Nax bond distances than
AdoCbl-H16/D14 until the charge transfer process begins (Figure 33). The Co-Nax bond
contraction is the result of the mechanism to facilitate the Co-C bond elongation carried out by
the residues of the catalytic site; namely Gly68, Asn123, Lys326, Glu330. This mechanism leads
to the energy decrease observed on the region of the PES associated to the elongation process
that precedes the electron transfer. Despite the contraction process was larger for the
AdoCbl-GM-H16/D14 model, after the elongation of the Co-Nax bond both systems reached very
similar Co-Nax bond lengths at the end of the scan. The reason for this is that after the Co-C bond
is cleavage, the residues of the catalytic site lost their connection with the α-axial ligand, which
is the same on both models.
83
Results and Discussion
16
2,11
Co-Nax Bond Distance [A]
14
AdoCbl-GM-H / D
16
14
AdoCbl-H / D
2,12
2,10
2,09
2,08
2,07
2,06
2,05
2,04
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 33. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14.Comparison of the Co-Nax bond
length variations along the Co-C bond dissociation process
The charge redistribution analysis (Figure 34) showed that on the AdoCbl-GM-H16/D14 model
the charge on the C atom barely changed until the electron transfer process started. It is possible
to infer that the enzyme manages to avoid affecting the charge on C as a mechanism for
decreasing the energy demand when the elongation and the posterior electron transfer take place.
Then, the C displayed a large decrease on its charge which is related to the electron donation to
the metal center. It was accompanied by the decrease of charge on Co, which showed a similar
behavior for both models, even though the AdoCbl-GM-H16/D14 model displayed more positive
partial charges.
The total charge on the Neqs started with the same magnitude in both models. During the first
stages of the dissociation, the two models showed a similar increase of their negative charge.
After the region where the electron transfer starts, the total partial charge on the Neqs from
AdoCbl-GM-H16/D14 showed values more negative than the obtained for AdoCbl-H16/D14. This
84
Results and Discussion
is related to the behavior observed for C, where the larger increase of negative charge on the Neqs
is the result of compensating the large amount of charge donated by the C to the Co in a shorter
Co-C bond length range.
-0,55
0,88
-0,56
0,86
14
0,84
-0,58
-0,59
(A)
NPA charge [e]
-0,57
NPA charge [e]
16
Co AdoCbl-GM-H / D
16
14
Co AdoCbl-H / D
0,82
0,80
(B)
0,78
-0,60
16
Nax AdoCbl-GM-H / D
16
-0,61
14
0,76
14
Nax AdoCbl-H / D
2,0
2,2
2,4
0,74
2,6
2,8
3,0
2,0
3,2
2,2
16
2,8
3,0
3,2
-1,80
-0,40
-1,85
-0,45
-0,50
-0,55
(C)
NPA charge [e]
NPA charge [e]
2,6
14
C AdoCbl-GM-H / D
16
14
C AdoCbl-H / D
-0,35
2,4
Co-C Bond Distance [A]
Co-C Bond Distance [A]
-1,90
-1,95
(D)
-2,00
16
-0,60
Neqs AdoCbl-GM-His / Asp
-2,05
16
Neqs AdoCbl-His / Asp
-0,65
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
14
14
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 34. AdoCbl-GM-H16/D14 versus AdoCbl-H16/D14. Comparison of the electronic
charge redistribution along the Co-C dissociation process (A) Nax (B) Co (C) C (D) Neqs
In the ground state, the partial charge of Nax was less negative on the AdoCbl-GM-H16/D14
model. This was due to the shorter Co-Nax bond distance of this model that allowed for a larger
charge transfer from the α-axial ligand to the metal center. During the Co-C bond dissociation,
the negative charge of Nax decreased until the electron transfer started, after which it began to
increase. Both models converge to very similar charge values, again supporting the idea that
85
Results and Discussion
after complete cleavage, the enzyme mainly affects the Ado• radical and not to the rest of the
cofactor.
3.4.3 Understanding the Role of the Enzymatic Environment
Our findings strongly suggest that the role of the enzymatic environment corresponds to the
stabilization of the product of the Co-C bond dissociation, most specifically the Ado• radical.
Meanwhile, the role of the His16-Asp14 pair is related to the decrease of the energy demand for
the electron transfer process. However, it was not completely evident from the study of
AdoCbl-H16-D14 versus AdoCbl-His16. In order to verify the role of the His16-Asp14 pair, we
performed the comparative study of AdoCbl-GM-H16/D14 and AdoCbl-GM-H16 models
(Figure 35), aiming to isolate the effect of the Asp14 on the electron transfer region of the PES
by including the stabilization of the Ado• radical in both models.
Figure 35. Optimized geometries of the AdoCbl-GM-H16 and AdoCbl-GM-H16/D14 models
after the Co-C bond dissociation process
The PESs and the spin density profiles are showed on Figure 36. The PESs analysis exposed
that the role of the Asp14 is actually highly located at the region associated to the electron
transfer. The energy stabilization accomplished by the inclusion of the His16-Asp14 as part of
86
Results and Discussion
the model can be rationalized by the analysis of the spin density profile. The difference between
the electron transfer processes of the two models is remarkable, revealing that the model without
the Asp14 started to transfer the electron charge at shorter Co-C bond distances, where the total
process is noticeable more challenging than the observed for AdoCbl-GM-H16/D14. Also, the
delocalization on the α-axial ligand is evident from the lower spin density observed on the Co
atom of the model with the His16-Asp14 pair.
14
16
1,0
14
AdoCbl-GM-H / D
16
AdoCbl-GM-H
12
16
0,4
8
0,2
Spin Dens [e]
Energy [kcal/mol]
0,6
10
6
4
(A)
14
Co AdoCbl-GM-H / D
16
14
C AdoCbl-GM-H / D
16
Co AdoCbl-GM-H
16
C AdoCbl-GM-H
0,8
0,0
-0,2
-0,4
(B)
-0,6
2
-0,8
0
-1,0
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Disntace [A]
Figure 36. AdoCbl-GM-H16/D14 versus AdoCbl-GM-H16 A) Potential energy surfaces of the
Co-C bond dissociation process B) Spin density profiles along the Co-C dissociation
process.
The final stage of the PES presented a lower energy difference than the obtained among the
other models, which is not related to the Ado• radical stabilization because that effect is included
in both PESs. This energy difference is associated to the stabilization of the Cbl(II) after the CoC bond cleavage.
Finally, the different roles of the catalytic site are easily visualized after the analysis carried
out at this point by the comparison of the PES of AdoCbl-H16/D14 and AdoCbl-GM-H16. As
showed on Figure 37, the PES of the AdoCbl-H16/D14 model is lower in energy at the region
where the electron transfer occurs, and AdoCbl-GM-H16 exposes a larger stabilization at the
final stage of the scan, which is related to the stabilization of the Ado• radical. The spin density
87
Results and Discussion
profile follows the expected behavior with a higher delocalization of the Co spin density on the
model with the Asp14 residue.
16
16
12
0,6
10
0,4
8
6
4
(A)
2
14
Co AdoCbl-H / D
16
14
C AdoCbl-H / D
16
Co AdoCbl-GM-H
16
C AdoCbl-GM-H
0,8
Spin Dens [e]
Energy [kcal/mol]
1,0
14
AdoCbl-H / D
16
AdoCbl-GM-H
14
0,2
0,0
-0,2
-0,4
(B)
-0,6
-0,8
0
-1,0
-2
2,0
2,2
2,4
2,6
2,8
Co-C Bond Distance [A]
3,0
3,2
2,0
2,2
2,4
2,6
2,8
3,0
3,2
Co-C Bond Distance [A]
Figure 37. AdoCbl-H16/D14 versus AdoCbl-GM-H16 A) Potential energy surfaces of the CoC bond dissociation. B) Spin density profiles along the Co-C dissociation process.
3.4.4 The Hydrogen Abstraction Pathway Paradigm
The pathway connecting the Co-C bond dissociation with the hydrogen abstraction from the
substrate has been unclear for several years. It has been proposed that the exact pathway may
contribute into the decrease of the energy barrier for the Co-C bond dissociation reaction.
Herein, we calculated the PES for two possible reaction pathways. The first was the step-wise
process, which involves an initial stage for the Co-C bond dissociation, until the conformation
defined as confA is reached. The next stage involves the ribose conformational change towards
confB, preparing the system for the hydrogen abstraction reaction. The second pathway
corresponds to the search of a concerted mechanism. In this case we performed the scan using a
reaction coordinate that goes directly from the ground state to the confB. The models used are
depicted on Figure 38.
88
Results and Discussion
Figure 38. Molecular Representation of the Step-Wise and the Concerted reaction
pathways
The resulting PESs displayed on Figure 39, expose that the two mechanism followed similar
energy pathways during the elongation process. However, the differences between these
mechanisms started to appear at short Co-C bond distances. The energy barrier for the Co-C
bond dissociation process was lower for the concerted mechanism when compared to the
step-wise mechanism, which is very energy demanding. After a Co-C bond length of 3.50 Å, the
bond is completely cleavage and both pathways reach similar energy values. However, the
concerted pathway seems to lead to lower energy values preparing the system for the hydrogen
abstraction.
In spite of the fact that the concerted pathway seems to be plausible as the mechanism for the
Co-C bond dissociation for the GM catalytic reaction, this represent the preliminary results of a
study in progress.
89
Results and Discussion
Step-Wise
Concerted
18
16
Energy [kcal/mol]
14
12
10
8
6
4
2
0
-2
2,0
2,5
3,0
3,5
4,0
Co-C Pathway Distance [A]
Figure 39. Step-Wise versus Concerted reaction pathway. Comparison of the potential
energy surfaces of the Co-C bond dissociation along the respective reaction pathways.
90
Results and Discussion
3.5 ISOMERIZATION REACTION: GLM TO MASP
3.5.1 The carbon Skeleton Rearrangement
In this section we aim to explore the role of the enzymatic environment on the energetic of
the chemical transformation of glm into masp. The only transition state here studied is the
corresponding to the fragmentation stage. This is because it is considered the most energy
demanding due to the nature of the process
The transition states for the hydrogen abstractions and the recombination stage were not
modeled because they are expected to be very low energy barrier reactions as they involve
radical attacks, and they should not represent an energetic challenge within the cycle.
The different states studied in this section are depicted on Figure 40, where the studied
transition state is associated to the process between the C and D stages.
Figure 40. Schematic representation of the steps associated to the isomerization reaction of
glutamate to methylaspartate
91
Results and Discussion
Consistent with our previous results, the hydrogen abstraction by the Ado• radical from glm
resulted in an exergonic reaction, involving a large stabilization of the system with an energy
decrease of 13.6 kcal/mol. The interactions between the substrate and the binding site did not
change much with regards to the description given at the methodology section. As expected, the
carbon where the unpaired electron is located changed from sp3 (tetrahedral) to sp2 hybridization
(planar). The specific conformation adopted by glm• is similar to the conformation of the
subsequent transition state of the fragmentation process, as denoted on Figure 41. This indicates
that part of the mechanism for the isomerization reaction involves restricting the glm•
intermediate to a conformation by its interactions with the binding site. Thus, the energy
difference between these two states is decreased.
Figure 41. Optimized geometries of the full atom model at the different steps associated to
the isomerization reaction catalyzed by glutamate mutase.
For clarity, the residues are not displayed. The distances are given in (Å).
The model of glm• with the binding site was used as the starting point for the scan along the
carbon-carbon bond expected to be broken after the substrate-derived radical is formed. The
purpose of this scan was to search the geometries for the transition state and the intermediate of
92
Results and Discussion
the fragmentation process. The obtained potential energy surface showed a smooth behavior
along the process and it is presented on Figure 42.
The transition state and the intermediate were successfully located. The transition state is
closely related to the product of the fragmentation process, namely the glycyl radical and the
acrylate ion.
In order to accurately analyze the energy differences between the states here studied, we
proceeded with the geometry optimization of these two systems without restraints.
Figure 42. Potential energy surface of the substrate carbon-carbon bond dissociated during
the fragmentation stage of the isomerization reaction
93
Results and Discussion
As depicted on the diagram from Figure 43, the final energy barrier calculated for the
fragmentation process was of 32.9 kcal/mol, followed by a decrease of 1.8 kcal/mol associated
to the formation of the fragmentation intermediate. The energy difference between the glm•
intermediate and the fragmentation intermediate is 31.1 kcal/mol. It seems to be a real challenge
for the enzymatic machinery to overwhelm the energy barrier of this stage of the process.
The recombination stage that follows the fragmentation process involves a considerable
stabilization of the system, with an energy decrease of 18.2 kcal/mol, and the energy barrier of
this process it is expected to be very close to that energy value. The difference between the
stabilities of glm• and masp• was 12.8 kcal/mol, value calculated from their total energy
variation in favor of glm•. These results exposed the same behavior observed for the AdoCblGM complex.
40
Full Model
35
30
25
-1.8
Energy [kcal/mol]
20
C
15
D
10
-18.2
5
32.9
3.0
0
-5
A
F
E
-13.6
-10
-15
B
-20
-25
glm
glm Rad
TS
I
masp Rad
masp
Figure 43. Calculated energy diagram of the mechanism for the isomerization reaction of
glutamate to methylaspartate.
A-F represents the steps described in Figure 39.
94
Results and Discussion
The final step corresponds to the hydrogen abstraction from AdoH carried out by the masp•
radical. The energy for this step was slightly endergonic by 3.0 kcal/mol. This value is opposed
to the results obtained for the models of the AdoCbl-GM complex, on which a -3.8 kcal/mol
energy decrease was obtained for the same step. Considering that the AdoCbl-GM did not
include the substrate binding site as the models of this section, it is the binding site the
responsible of the destabilization of the masp ligand. More important, this slight destabilization
of the product could be related to the mechanism to release the product from the binding site.
3.5.2 Role of the enzymatic environment on the Isomerization Reaction
Due to the complexity of the isomerization reaction catalyzed by GM, it is impossible to
perform it without the right enzymatic machinery, as stated before. Each part of the catalytic site
contributes in a specific way to accomplish this complex chemical transformation. Thus,
establishing the importance that each residue separately has on the process would lead us to
learn how nature has managed to surmount this difficult chemical reaction.
In this section we will focus on two particular effects that could be important for the
enzymatic process. The first is the influence that the Glu171 has on the stabilization of the
different intermediates of the catalyzed reaction. The second is to determine the function that the
arg claw (Arg66, Arg100, Arg149) has on the catalytic cycle.
a) Role of Glutamate-171
The diagram of Figure 44 shows the comparison between the energy profile for the
isomerization reaction of the full atom model and a system without the Glu171 in its structure.
The first step associated to the formation of the glm• intermediate is defined by a lower
stabilization of the total system. This is caused by a decrease of the structural organization of the
binding site. The fragmentation stage is higher in energy for this particular case, because of the
absence of the hydrogen bond between the ammonium from the substrate and the carboxylate
group of Glu171. The function of the hydrogen bond is to augment the donor ability of the amine
from the substrate, helping to stabilize the glycyl radical formed as a product of the
fragmentation. This supports the idea of Glu171 as a general base able to promote the
isomerization reaction. The subsequent steps denote the relevance of Glu171 on the stabilization
of the rest of the intermediates of the reaction.
95
Results and Discussion
40
Full Model
Model wo Glu171
30
Energy [kcal/mol]
20
C
D
10
0
F
A
E
-10
B
-20
glm
glm Rad
TS
I
masp Rad
masp
Figure 44. Role of the Glu171 residue on the energetic of the GM catalytic mechanism.
Comparison between the calculated energy diagrams of the mechanism for the isomerization
reaction of the full atom model and a model without the Glu171 residue. A-F represents the steps
described in Figure 39.
b) Role of the Arginine Claw
The arg claw is a group of arginines that seems to work as the main structural support of the
binding site. However, the exact contribution of these residues into the catalytic mechanism is
still unclear. The diagram of Figure 45 aims to compare the energy profiles between the full
atom model and a system without these set of residues.
The results revealed an interesting behavior along the process. The system without the
arg claw showed a higher stabilization of the glm• intermediate after the hydrogen abstraction.
The reason for this is because the binding site of the model without the arg claw allowed a larger
conformational freedom to glm•. Our results suggest that the binding site, and particularly the
arg claw, restricts the conformation of glm to a higher energy conformation to approach the
glm• intermediate with the transition state.
96
Results and Discussion
40
Full Model
Model wo ARGs
30
Energy [kcal/mol]
20
10
C
D
0
A
E
F
-10
-20
B
glm
glm Rad
TS
I
masp Rad
masp
Figure 45. Role of the Arg claw on the energetic of the GM catalytic mechanism.
Comparison between the calculated energy diagrams of the mechanism for the isomerization
reaction of the full atom model and a model without the Arg claw. A-F represents the steps
described in Figure 39.
The next two stages of the catalytic cycle also denote a decrease of the energy with regards to
the analogous stages for the full atom model. The apparent stabilization of the transition state
and intermediate are caused again by the conformational freedom. However, in this particular
case it leaded to conformations of lower energy which are not suitable for the recombination
stage. Our findings reveal that the arg claw is essential in the control of the conformations of the
transition state and intermediates, restricting the cycle through the unique reaction pathway that
allows for the F/R process.
It seems that the formation of the masp• needs the support of the arg claw for its stabilization.
Finally, the product formation is disfavored in absence of these residues, which indicates that
there are other residues in charge of the release of masp from GM.
97
Results and Discussion
c) Chemical Role of the Binding Site
As explained before, the reaction catalyzed by GM is impossible to perform it without the
proper enzymatic machinery. This is partially because of the great structural requirements
needed to propagate the reaction. However, there is also a chemical role of the environment. To
estimate its role on the energy along the different stages of the cycle, we extracted the
geometries of the substrate and its derivatives, from the full atom model. Then, the hydrogen
atoms were optimized, keeping fixed the rest of the structure. The resulting energy profile is
depicted on Figure 46.
40
Full Model
Free Model
30
Energy [kcal/mol]
20
C
10
D
0
A
E
-10
F
B
-20
glm
glm Rad
TS
I
masp Rad
masp
Figure 46. Role of the binding site on the energetic of the GM catalytic mechanism.
Comparison between the calculated energy diagrams of the mechanism for the isomerization
reaction of the full atom model and a model without the aminoacidic residues from the catalytic
site. A-F represents the steps described in Figure 39.
98
Results and Discussion
The decrease on the stabilization of the glm• is mainly caused by the lack of counter ions to
stabilize the system. This exposes that in the model without Glu171, part of the initial effect is
also caused by the reason here stated. The lower stabilization of the transition state is probably
associated to the absence of Glu171 residue to promote the fragmentation process. Then, the
glycyl radical and the acrylate ion seem to be slightly more stable without the explicit binding
site. Finally, the larger stabilization of the masp• and the product exposes that the enzyme
releases the product by a combined chemical mechanism and structural incompatibility
mechanism with the binding site.
99
Conclusions
4 CONCLUSIONS
In the first section of this thesis, the relevance of the use of full atom models was evaluated by
defining the role that the different structural moieties of cobalamins have on the energetic of the
Co-C bond. Our findings revealed the higher susceptibility of AdoCbl toward its structural
modifications which are almost certainly controlled by the enzymatic environment. On the other
hand, the remarkable insensitivity of MeCbl to modifications on its structure, and the direct
reliance of its Co-C BDE on the interactions between the Co and C centers, supports the idea of
a mechanism where a Co-C bond dissociation is not directly involved as the first step of its
catalytic cycle, but probably is part of a concerted mechanism. The dependence on the accuracy
of the initial structure for the study of these largely complex systems was clearly established.
Also we denoted that the commonly larger Co-Nax bond distances obtained on studies using
naked corrin models are entirely due to the absence of the nucleosidic loop on their structures.
The quantification of the contribution on AdoCbl Co-C bond strength of the intramolecular
hydrogen bonds, formed between the ribosyl moiety of the Ado group and the corrin side chains,
lead us to state that the intrinsic BDE difference between the two cofactors should be larger than
the experimentally determined. A role for the base replacement on AdoCbl dependant enzymes
is suggested. After the possibility of a mechanical elongation of the Co-C bond caused by the
enzyme-AdoCbl complex, and in order to avoid the Co-C bond dissociation in absence of
substrate, the substitution of the α-axial ligand by a smaller group should lead to the shortening
of Co-Nax bond length inducing a slight increase on the Co-C bond strength. We believe that this
would work as a safety mechanism that should be disabled after substrate binding. After defining
all the external contributions to the Co-C BDE, the intrinsic nature of the difference observed
between AdoCbl and MeCbl Co-C bond strengths was studied by assuming that the hypothetical
substitution of a hydrogen atom of the methyl group by the deoxyadenosyl moiety is the
responsible of inducing the destabilization of the Co-C bond, leading into a lower BDE on
AdoCbl. The origin of the Co-C bond destabilization was established by the definition of the
most important components of cobalamins electronic structure, which allowed us to interpret the
BDE differences in terms of AdoCbl and MeCbl differential reactivity toward the Co-C bond
dissociation reaction. The energy gap between the Co-C bonding, Co-Nax antibonding MO and
the C-Co-Nax antibonding MO was larger for MeCbl when compared to the obtained for
100
Conclusions
AdoCbl, which correlates well with the higher Co-C BDE obtained for MeCbl. In addition, the
half of the value for the energy gap was related to the resistance to the electronic density
reorganization on the region comprehended between the Co and the C atoms. Finally, the
destabilization of AdoCbl Co-C bonding, Co-Nax antibonding MO was related to its shape,
which on AdoCbl presents an additional destabilizing interaction between orbital phases of
opposite signs missing on MeCbl.
In the second section of this thesis, the potential energy surfaces along the Co-C bond for
AdoCbl and MeCbl were obtained. The results allowed us to evaluate the Co-C bond
dissociation process of the free forms of these cofactors. The calculated energy barrier of
AdoCbl was in agreement with the values obtained for the intrinsic strength of the Co-C bond of
this cofactor, supporting our suggestion about a lower Co-C BDE of AdoCbl than the
experimentally obtained. We found that the intramolecular hydrogen bonds formed between the
Ado ligand and the corrin side chains contribute to the BDE experimentally determined. In
addition, we reproduced the experimental value of AdoCbl BDE by considering that the
fragments of the Co-C bond cleavage were not completely dissociated from each other, affecting
their energy and leading to the apparently higher Co-C BDE. We exposed that the Co-C bond of
MeCbl has a larger electrostatic contribution than the observed on AdoCbl, as the result of the
larger negative charge located at the C of the methyl ligand. This is because of the lower ability
of methyl group to polarize and reorganize its negative charge when compared to Ado, resulting
in a higher Co-C BDE for MeCbl. The electron transfer process was similar on the systems
studied in this section, but some key differences appeared to be essential on the process. First, a
controlled electron transfer with lower amounts of charge per Co-C bond distance resulted on the
decrease of the energy necessary for the process. Second, the electron transfer is less energy
demanding when it occurs at longer Co-C bond distances. It is expected that after an optimum
Co-C bond distance, this necessarily applies. Here we also revealed the role of the corrin ring
flexibility on the Co-C bond dissociation process, where its function is related to the decrease of
the energy associated to the charge reorganization during the cleavage reaction. The exact
mechanism involved the initial electron transfer from C to Co that increases the occupation of
the Co-dz2 atomic orbital and the Nax-pz atomic orbital. As expected, the spin density distribution
was not symmetrically distributed with regards to the corrin plane, leading to the electronic
repulsion between the spin density located on the lower side of the corrin ring and the π-
101
Conclusions
electrons of the Neqs of the same macrocycle. This induces the decrease of charge donated by the
Neqs to the Co center with the purpose to facilitate the distortion of the corrin ring towards an
upward folding, thus decreasing the electronic repulsion. The study of the base replacement by
the comparison of models with DMB and imidazole as the α-axial ligad are not able to reveal the
role of this particular modification of the cofactor after it binds to the enzyme. This point out that
the histidine residue alone is not the responsible of the changes on the catalytic rate of the GM
reaction experimentally observed.
In the third section of this thesis we defined the role of the enzymatic environment on the
catalytic Co-C bond dissociation process. First, we inferred the functioning of the His16-Asp14
pair, which is a conserved motif present in several enzymes, and whose role on enzymatic
reactions assisted by metals is unclear. We found that the hydrogen bond between His16 and
Asp14 induce the partial deprotonation of the His16, increasing the electronic charge of the
His16-imidazole ring. This leads to the increase of the electrostatic contribution to the Co-Nax
bond visualized by the consequent decrease of its bond length. The Co-Nax bond contraction
involves the increase of the amount of charge donated by His16 to the Co-dz2 atomic orbital,
causing a decrease of the amount of charge donated by the C of the β-axial ligand to the same
atomic orbital when compared to the system without the Asp14 residue. This prevents the C
from transferring additional charge to the Co at short Co-C bond distances which is energetically
more demanding than at longer bond distances. The consequences of these modifications on the
electronic structure of AdoCbl on the different stages of the Co-C bond dissociation were
several. Starting with the Co-C bond elongation process, the decrease of the orbital character of
this bond diminishes the energy needed to elongate it, stage that precedes the electron transfer
process. Once the electron transfer starts, the unpaired electron received by the Co during this
process is delocalized between the Co-dz2 and the Nax-pz atomic orbitals. Interestingly, the
Asp14 increased the magnitude of the delocalization on the Nax-pz atomic orbital, decreasing the
Co spin density and lowering the energy associated to the electron transfer process. Also, as
denoted above the His16-Asp14 manages to control the Co-C bond distance at which the C is
allowed to start the electron charge process. Finally, at the end of the Co-C bond dissociation
process, Cbl(II) is stabilized by the His16-Asp14 pair because of the larger delocalization of the
electronic charge. After we clarified the role of the His16-Asp14 pair, we determined the role of
the catalytic site, more specifically the residues that interact with the Ado group. The
102
Conclusions
interactions between the Ado group and the catalytic site induce the stretching of the Co-C bond
by the mechanical elongation carried out by these residues. This is because the configuration of
the binding site is distributed in such manner that it stabilizes the conformation related to the
dissociated form of the cofactor rather than the ground state of the cofactor. These interactions
lower the energy of the Co-C bond elongation process, but only in the region near to the charge
transfer. The Co-C bond stretching is connected with the Co-Nax bond length decrease, which is
even shorter than the obtained only with the His16-Asp14 pair, facilitating in return the Co-C
bond elongation. Along the whole Co-C bond dissociation process, the enzymatic machinery
leads the Ado moiety towards the final dissociated conformation promoted by the stabilization of
the Ado• radical formed as the product of the Co-C bond cleavage. This stabilization effect is
one of the driving forces of the process rather than promoting the electron transfer process.
Meanwhile, the latter process is mainly driven by the His16-Asp14 pair. All of these effects
contribute together on the Co-C bond weakening. The reaction pathway connecting the Co-C
bond dissociation and the hydrogen abstraction from the substrate has been a long standing
question regarding the complete family of AdoCbl-dependant enzymes. Two general reaction
pathways have been proposed. The first associated to a concerted pathway and the second to a
step-wise pathway. Here we presented new insights about this intriguing problem, exposing that
the concerted seems to be more plausible than the step-wise reaction pathway from an energetic
point of view. Nevertheless, we considered that this complex problem needs a deeper
investigation before stating the final reaction pathway due to the many variables involved in the
process.
The fourth section of this thesis presented a complete study of the isomerization reaction
mechanism and revealed the role of the catalytic site in this mechanism. The Co-C bond
dissociation generates the Ado• radical, which in spite of being stabilized by the enzymatic
environment, it is still unstable and therefore very reactive. The enzyme manages this specie by
the hydrogen abstraction from the substrate leading to the formation of the highly stable glm•
radical. This stage is one of the main driving forces that aid to propagate the cycle to the forward
direction. The next stages are controlled by the binding site through the restriction of the
conformation of the substrate, transition states and intermediates. The fragmentation stage is
favoured by the adoption of a high energy conformation of the substrate and the glm• radical
intermediate that precedes the fragmentation. This also allows the generation of the
103
Conclusions
fragmentation intermediates in the conformation needed to promote the subsequent
recombination stage. The binding site performs various tasks simultaneously in order to
accomplish this complex reaction. As part of the catalytic machinery, Glu171 act as structural
support for the several species formed along the catalytic cycle. It also stabilizes the transition
state and intermediates of the reaction by the stabilization of the glycyl radical, thus promoting
its formation. Meanwhile, the Arg claw as part of the binding site is in charge of the
conformational restrictions. It is essential in the control of the conformations of the transition
state and intermediates, restricting the cycle through the unique reaction pathway that allows for
the F/R process. Regarding to the release of the product, it seems to be mediated by the
destabilization of it by a decrease of the complementarities with the binding site and by chemical
incompatibilities.
Finally, we provided new insights about the catalytic Co-C bond dissociation process and
revealed new features that aid to explain the main driving forces that dominate the catalytic
cycle of GM.
Below, a schematic representation of the main conclusions obtained in this thesis is presented.
104
Conclusions
105
References
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