Genetically Modified Myoglobin As A Mimic For Heme Enzymes

GENETICALLY MODIFIED MYOGLOBIN AS A MIMIC FOR HEME ENZYMES
by
SUBHASH CHAND
Presented to the Faculty of the Graduate School of
The University of Texas at Arlington in Partial Fulfillment
of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
THE UNIVERSITY OF TEXAS AT ARLINGTON
May 2013
Copyright © by Subhash Chand 2013
All Rights Reserved
ACKNOWLEDGEMENTS
I would like to take this opportunity to express my deepest gratitude to my advisor, Dr.
Roshan Perera, for his excellent guidance, support, understanding, patience, inspiration and
constant encouragement which have been instrumental in providing my graduate research
every success. I would also like to thank the members of my advisory committee, Dr.
Subhrangsu S. Mandal and Dr. Jongyun Heo, for their many invaluable opinions towards my
research. I would like to express my deep gratitude to Dr. Krishnan Rajeshwar, and Dr. Daniel
W. Armstrong for being extremely generous with their precious advice and collaborative
research and guidance that kept my research focused. In addition, I would like to thank Dr. Brad
Pierce and Dr. Kayunta Johnson-Winters for allowing me to use the various facilities for my
research and assisting me to learn some very significant techniques.
I am very much indebted to Dr. Brian Edwards, Dr. Norma Tacconi and Dr. Muhammed
Yousufuddin, for sharing their copious knowledge and helping me with many of the
instrumentation parts used in my research. I would like to express my utmost thankfulness to
the Department of Chemistry and Biochemistry and the Graduate school of The University of
Texas at Arlington for supporting my graduate studies here.
It was a pleasure to share my doctoral studies with lab-mates like Dr. Sriparna Ray, Dr.
Sridev Mohapatra, Leticia Loredo, Yanbo Zhang, Sriyani Liyanage, Nam Tran, Brian Stamos,
Tuan Phan, Chris Wilson, Jimmy Saing and Buuh Yousuf. I owe my sincere appreciation to Dr.
Sriparna Ray for her continuous efforts in checking thesis, which required many hours.
Last but not least, I would like to thank my family, my father, Mr. Keshaw Prasad Yadav,
my mother, Mrs. Urmila Devi, my brothers Shrikant Yadav, Sandeep Yadav, Shashikant Yadav
and Manish Yadav, my cousins Mr. Ram Chander Yadav and Sunil Yadav and my friends of
UTA and beyond - Prasad, Piyush, Bibhu, Chetan, Swanand, Megha, Prashant, Aakash, Amit,
iii
Soumyava, Saket, Kirti, Karthik, Irene, Valay, Vaishali, Ajoy, Mrunmayi, Prudhvi and Ajinkya for
their support during my stay in UTA as well as in my research.
I would like to make a special mention of my uncle, Mr. Harendra Kumar Yadav who
has showered me with unconditional love and endless support necessary to succeed in life.
April 16, 2013
iv
ABSTRACT
GENETICALLY MODIFIED MYOGLOBIN AS A MIMIC FOR HEME ENZYMES
Subhash Chand, PhD
The University of Texas at Arlington, 2013
Supervising Professor: Roshan Perera
This study addresses the mechanistic relationships between formation of reactive
oxygen species (ROS) and their catalytic oxidation functions in oxygenation and peroxygenation
reactions in heme enzymes. Even though both cytochrome P450s (CYP P450s) and
peroxidases have different catalytic activities, the involvement of common ROS (Compound 0
and Compound I) have been proposed. Therefore, to understand the generation and activation
of peroxide to form ROS, genetically-engineered myoglobin (Mb) mutants were created by
incorporating redox-sensitive 3-amino-L-tyrosine (NH2Tyr) or L-3, 4-dihydroxyphenylalanine
(DOPA) into its active site. Distal His 64 replaced with redox amino acids mutant Mb showed
excellent turnover rates for thioanisole and benzaldehyde oxidation, compared to the wild-type
protein. A 9-fold and 81-fold increase in activity, respectively, was observed in the presence of
hydrogen peroxide (H2O2). The presence of a redox unnatural amino acid in the active site
enhances the rate of compound I (Cpd I) formation and stabilizes it to form one extra H-bond as
compared to the wild type (WT) Mb. This increased oxidation activity in mutants offer insights
into the role of the distal active site residues which are involved in acid-base catalysis and distal
charge relay “pull” effect in peroxide activation and formation of ROS in heme proteins.
v
Furthermore, cyclic voltammetry (CV) and atomic force microscopy (AFM) were used to
investigate the importance of active site orientation of an immobilized protein for direct electron transfer
(DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on
the modified gold electrode surface in a random multilayered fashion, the Ser 3 replaced with NH2Tyr in
Mb mutant, was immobilized via a Diels-Alder reaction specific to the NH2Tyr residue to form a
homogeneous monolayer. Electrochemical calculations for the number of surface exposed redox-sensitive
molecules on the electrode surface (Γ) and heterogeneous rate constant for DET were 1.29 × 10 –10 mol
cm–2; 2.3 sec–1 for the WT Mb and 1.54 × 10–10 mol cm–2; 1.3 sec–1 for the S3NH2Tyr Mb mutant,
respectively. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover
frequencies (TOF) around 1.9 × 103 sec–1 (with 87% conversion) for the WT Mb, and 1.5 × 10 3 sec–1 for
the mutant Ser 3-amino-L-tyrosine (S3NH2Tyr) Mb (with 81% conversion). These results indicate that
site-directed single monolayer immobilization affords almost the same number of surface exposed Mb
active sites as the random multilayer immobilization strategy, though the latter contains a greater number
of protein molecules on the electrode surface. The microarray concept development provides novelty to
study protein-protein interactions, drug discovery, and biomedical and proteomic research.
vi
TABLE OF CONTENTS
ACKNOWLEDGEMENTS.............................................................................................................iii
ABSTRACT...................................................................................................................................v
LIST OF ILLUSTRATIONS..........................................................................................................xii
LIST OF FIGURES......................................................................................................................xii
LIST OF TABLES........................................................................................................................xv
LIST OF SCHEMES....................................................................................................................xvi
PREFACE..................................................................................................................................xvii
Chapter
Page
1. INTRODUCTION……………………………………………………………………………1
1.1 Heme proteins……………………………………………………………………1
1.1.1 Myoglobin ……………………………………………………………. 5
1.2 Incorporation of unnatural (noncanonical) amino acids into proteins……… 7
1.3 Direct electron transfer and bioelectrocatalysis……………..…………….... 10
1.4 Dissertation overview ............................................................................…..12
2. MECHANISTIC INSIGHTS AND IMPROVED OXIDATION ACTIVITY USING
GENETICALLY-ENGINEERED MYOGLOBIN........................................................... 14
2.1 Introduction..................................................................................................14
2.2 Experimental section...................................................................................17
2.2.1 Chemicals....................................................................................17
2.2.2 Preparation of WT Mb and H64NH2Tyr Mb mutant
construct...…………………………………………………………...17
2.2.3 WT Mb and H64NH2Tyr Mb protein purification………….………18
2.2.4 WT Mb and H64NH2Tyr Mb protein analysis.....………….………18
2.2.5 Spectroscopy………………………………………………..……….20
iii
2.2.6 Preparation of oxyferrous complexes……………………………..20
2.2.7 Effect of H2O2.....................………………………………………...21
2.2.8 Catalytic Sulfoxidation of Thioanisole and Oxidation
of Benzaldehyde………………………………………………………21
2.2.9 Electrochemical Instrumentation…………………………………...21
2.3 Results and Discussion…………………………………………………………25
2.3.1 Genetic design rationale and characterization…………………...25
2.3.2 Catalytic activity……………………………………………………...28
2.3.3 ABTS Peroxidase assay…………………………………………….29
2.3.4 Electrochemical characterization…………………………………..30
2.3.5 Ligand binding study of WT and H64NH2Tyr Mb mutant…….….33
2.3.6 Mechanistic insights………………………………….......…………36
2.4 Conclusions…………………………………………………………………...…37
3. INTRODUCTION OF PEROXIDASE ACTIVITY IN MYOGLOBIN
BY INCORPORATING AN UNNATURAL AMINO ACID AT THE DISTAL
HISTIDINE POSITION..............................................................................................38
3.1 Introduction……………….………………………………………………….…38
3.2 Experimental procedure..............................................................................40
3.2.1 Chemicals………………………………………..............................40
3.2.2 Preparation of WT Mb and H64DOPA Mb mutant
constructs.......................................................................................40
3.2.3 WT Mb and H64DOPA Mb protein purification and analysis ......40
3.2.4 Spectroscopy...…………………………………………..................42
3.2.5 Effect of H2O2..............................................................................42
3.2.6 Preparation of oxyferrous complex..............................................42
3.2.7 Preparation of CO, NO, cyanide, and azide adduct
samples..........................................................................................42
iv
3.2.8 Catalytic sulfoxidation of thioanisole and oxidation
of benzaldehyde.........................................................................43
3.2.9 Electrochemical instrumentation.................................................43
3.3 Results and discussion................................................................................43
3.3.1 UV-visible spectroscopic characterization of WT and
H64DOPA mutant…......................................................................43
3.3.2 Deoxyferrous and oxyferrous species of WT Mb and
H64DOPA Mb...............................................................................46
3.3.3 CO, NO, cyanide, and azide adducts of WT and
H64DOPA Mb..............................................................................47
3.3.4 Redox potentials of WT and mutant Mb......................................49
3.3.5 Thioanisole sulfoxidation and benzaldehyde oxidation.............51
3.4 Conclusion...................................................................................................55
4. FAVOURABLE ACTIVE SITE ORIENTATION IN MYOGLOBIN
FOR DIRECT ELECTRON TRANSFER, ELECTROCATALYSIS, AND
MICROARRAY INVESTIGATION..............................................................................56
4.1 Introduction…………...................................................................................56
4.2 Experimental Section……...........................................................................60
4.2.1 Chemicals...................................................................................60
4.2.2 Preparation of WT Mb and S3NH2Tyr mutant Mb construct......60
4.2.3 WT Mb and S3NH2Tyr Mb protein purification............................60
4.2.4 UV-vis spectroscopy...................................................................63
4.2.5 AFM measurements…………………………...............................63
4.2.6 Ligands adduct formation...............................................….........64
4.2.7 Preparation of microarray............................................................64
4.2.8 Electrochemical instrumentation and procedures………...........65
4.2.9 Electrode modification procédures…………………………… .…65
4.2.9.1 Au/L-Cys/WT Mb………………………………………..65
4.2.9.2 Au/L-Cys/TEGDA/ S3NH2Tyr Mb…….……….............66
v
4.2.9.3 Electrocatalysis.........................................................66
4.3. Results and discussion……………………………………………….....…..66
4.3.1 Monolayer vs. multilayer covalent immobilization of
Mb on Au………………………………………………………….......66
4.3.2 Electrochemical characterization...............................................73
4.3.3 Catalytic studies.........................................................................82
4.3.4 Microarray studies......................................................................83
4.3.4.1 Preparation of functional protein microarrays..........................83
4.3.5 Ligand binding studies................................................................88
4.4. Conclusion……………………………………………………………………..88
5. ELECTRONIC NATURE INVESTIGATION OF HEME CENTER BY
MODIFYING PROXIMAL AND DISTAL ACTIVE SITE ENVIRONMENT OF
SPERM WHALE MYOGLOBIN WITH ELECTRON RICH RESIDUES.......................90
5.1 Introduction..................................................................................................90
5.2 Experimental Section..................................................................................92
5.2.1 Chemicals...................................................................................92
5.2.2 Preparation of WT, H64pNO2Phe and H93pNO2Phe
Mb mutant constructs..................................................................92
5.2.3 WT, H64pNO2Phe and H93pNO2Phe Mb purification and
analysis..........................................................................................93
5.2.4 Electronic absorption spectroscopy............................................94
5.2.5 Preparation of deoxyferrous Mb..................................................96
5.2.6 Preparation of oxyferrous complex..............................................96
5.2.7 CO and NO complex preparation................................................96
5.2.8 Azide and cyanide complex preparation.....................................96
5.3 Results and discussion...............................................................................96
vi
5.3.1 Ferrous-CO, ferrous-NO and ferrous O2 complexes of WT
and H64pNO2Phe and H93pNO2Phe mutants of myoglobin......98
5.3.2 Ferric-cyanide and ferric-azide complexes of WT
and H64pNO2Phe and H93pNO2Phe mutants of Mb...................105
5.4 Conclusion.................................................................................................110
REFERENCES..........................................................................................................................111
BIOGRAPHICAL INFORMATION.............................................................................................128
vii
LIST OF ILLUSTRATIONS
Figures
Page
1.1 Structures of heme a and heme b........................................................................................2
1.2 Structures of naturally occurring iron porphyrines…............................................................3
1.3 Overall structure of sperm whale Mb.......…………………..........................................….....6
1.4 A general method for genetically encoding unnatural amino acids in live cells…...............8
1.5 The list of unnatural amino acids that have been introduced to the genetic
codes of E. coli, yeast and mammalian cells.......................................................................9
1.6 Schematic representations of bioelectrocatalysis involving direct electron
transfer in an enzyme........…..............................................................................……........12
2.1 Mechanistic pathway for generation of the highly reactive Cpd I from
the ferric resting form in heme-proteins……………………………………………………....15
2.2 Schematic representation of the oxyferrous complex of WT Mb..………………………....17
2.3 Coomassie-stained SDS-PAGE analysis of expression of WT Mb and
H64NH2Tyr Mb.............................................…………………………………….………..….19
2.4 The mass spectrum (MALDI-TOF) of the H64NH2Tyr Mb protein…………………...…20
2.5 GC analysis of oxidation products of thioanisole and benzaldehyde ………………..…....24
2.6 Electronic absorption spectra of the H64NH2Tyr Mb..........................................................27
2.7 Effect of H2O2 on WT ans mutant Mb………………………………………………………….28
2.8 Deoxyferrous and oxyferrous spectra of WT and mutant Mb……………………………....28
2.9 Differential pulse voltammetry (DPV) of WT and mutant Mb in presence and
absence of H2O2.….............................................................................................................32
2.10 Characterization of WT and H64NH2 Mb ligand complexes….........................................35
2.11 Mechanism of oxidation of 3-amino-L-tyrosine which releases 2-electrons
and two protons to produce quinone product in the presence of an oxidant……….…….37
3.1 Schematic representation of the formation of Cpd I and Cpd II
viii
in Mb and HRP..…............................................................................................................39
3.2 Analysis of WT and H64DOPA mutant Mb by SDS-PAGE and MALDI-TOF.........……...41
3.3 High-valent heme complexes of WT and H64DOPA Mb...................................................44
3.4 Stability of WT and H64DOPA Mb in presence of H2O2...........................................…………….....45
3.5 Deoxyferrous and oxyferrous spectra of WT and H64DOPA mutant Mb………….….......46
3.6 Study of WT and H64DOPA Mb ligand complexes....…………………………….……........50
3.7 Reduction potential of WT and H64DOPA Mb in presence and absence of
H2O2 at pH 7.0 and at pH 10.0................................................................................….........50
3.8 Gas-chromatography data and MS data of thioanisol and benzaldehyde
oxidation with WT and H64DOPA........................................................................................52
3.9 Proposed acid-base catalytic mechanism for Cpd I formation
in H64DOPA mutant Mb in presence of H2O2 ......................................................................55
4.1 Graphical representation of WT and S3NH 2Tyr mutant Mb attachment on modified
gold electrodes.....................................................................................................................59
4.2 Coomassie-stained SDS-PAGE analysis of S3NH2Tyr Mb and WT Mb..............................61
4.3 Electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS)
analysis of the incorporation of S3NH2Tyr into S3(TAG) Mb …………………..……...........62
4.4 Tapping mode AFM topographic images for chemically modified Au surface
of immobilized S3NH2Tyr Mb mutant and with WT Mb.........................................................72
4.5 CV response of L-Cys-modified, WT Mb and S3NH2Tyr Mb mutant immobilized
gold electrode in nitrogen-purged 100 mM phosphate buffer…...………………….…........73
4.6 CV and peak current response of WT and mutant Mb.....................................….................74
4.7 Plot of peak potentials vs pH for WT Mb and S3NH2Tyr Mb................................................78
4.8 Comparision of immobilized Mb with hemin on modified gold electrode..............................81
4.9 A microarray of five proteins on a single slide displays........................................................85
4.10 Spectroscopic characterization of high spin, low spin and ligand complexes
of WT and mutant Mb..........................................................………....................….............87
5.1 Showing structure of heme b and heme c............................................................................91
5.2 Schematic representations of WT and mutants Mb.............................................................92
5.3 Coomassie-stained SDS-PAGE analysis of expression of WT Mb, H64pNO2Phe
and H93pNO2Phe mutants Mb..........................................................................................94
ix
5.4 Characterization of ferric WT and mutants Mb.................................................................97
5.5 Deoxyferrous electronic absorption spectra of WT, H64pNO2Phe and
H93pNO2Phe mutant Mb..................................................................................................98
5.6 The heme carbonyl complex showing dπ–pπ* backbonding…........................................99
-
5.7 Showing presence of electron rich functional group (NO 2 ) at distal position…………….99
5.8 Comparative studies of carbonyl complexes of WT, H64pNO2Phe,
and H93pNO2Phe ……………………………………......………………………………..…102
5.9 Comparative studies of NO complexes of WT, H64pNO2Phe,
And H93pNO2Phe............................................................................................................108
5.10 Characterization of oxyferrous complexes of WT, H64pNO 2Phe,
and H93pNO2Phe Mb....................................................................................................104
5.11 Proposed structure of Fe (II) O2, Fe (II) CO and Fe (II) NO..........................................105
5.12 Comparative studies of cyanide complexes of WT, H64pNO 2Phe,
and H93pNO2Phe Mb....................................................................................................107
5.13 Characterization of azide oxyferrous complexes of WT, H64pNO2Phe,
and H93pNO2Phe Mb....................................................................................................108
5.14 Two canonical forms of azide bound to heme iron of WT Mb and
H64pNO2Phe Mb mutant.............................................................................................109
5.15 Two canonical forms of cyanide bound to heme iron of WT Mb and
H64pNO2Phe Mb mutant ............................................................................................109
x
.
LIST OF TABLES
Table
Page
1.1 Biological functions of heme proteins……………………………………………………............4
2.1 Rate of sulfoxidation of thioanisole and benzaldehyde oxidation by WT Mb
and mutant H64NH2Tyr Mb...………………………………………………………..……………29
2.2 ABTS oxidation reaction assays of Mb and its mutant in the presence of
5 mM H2O2. Kinetic values are based on the average of at least 2 determinations……..…...30
2.3 Reduction potentials were measured at pH 7.0 and 10.0 in the absence and
presence of 5 mM H2O2………………………………………………………………….….……33
3.1 Reduction potential of WT Mb, H64DOPA Mb mutant and DOPA only at pH 7.0
and pH 10.0 in presence and absence of H2O2 ……………...........…………………..………49
3.2 Rate of sulfoxidation of thioanisole and benzaldehyde oxidation by WT Mb
and H64DOPA mutant Mb..………....……..…….………………………………………….....…..53
3.3 ABTS oxidation reaction assays of Mb and its H64 DOPA mutant Mb in the
presence of 5 mM H2O2.........................................................................................................54
4.1 Comparison of heterogeneous electron transfer rates of myoglobin on gold electrodes…..77
4.2 Comparison of direct electrochemistry of Mb on various electrode systems………………..79
4.3 Electrocatalytic conversion of thioanisole to its oxidized form and turnover frequency
(TOF) using bare, multilayered WT Mb-immobilized and monolayered S3NH2Tyr Mb……..82
5.1 The electronic absorption spectral features of the oxy, carbonyl, NO, cyanide and
azide complexes with H64pNO2Phe Mb, H93pNO2Phe Mb mutants and WT Mb………….100
xi
LIST OF SCHEMES
Scheme
Page
4.1 Schematic representation of Au electrode modification for monolayered
immobilization of S3NH2Tyr Mb mutant on the Au surface; see text for details………...…..68
4.2 Schematic representation of multilayered WT Mb immobilization on the modified
gold electrode surface using EDC catalyzed bioconjugation reaction...................................70
xii
PREFACE
This study “Genetically modified myoglobin as a mimic for heme enzymes” is submitted by
Subhash Chand to The University of Texas at Arlington for the degree of Doctor of Philosophy
(Ph.D.), and has the following kind contributions. Chapter 4 was published as “S. Ray, S.
Chand, Y. Zhang, S. Nussbaum, R. Krishnan, R. Perera, Immobilization of Myoglobin on the
Gold Electrode to Promote Direct Electron Transfer, Electrochimica. Acta. 2013, 99, 85-93”. In
this study Dr. Ray has carried out electrochemistry studies. Figures (4.1, 4.5, 4.6, 4.7, and 4.8)
and schemes (4.1 and 4.2) were kindly contributed by her. The microarrays studies in chapter 4
have been publisted as “B. Stamos, L. Loredo, S. Chand, T. Phan, Y. Zhang, S. Mohapatra, R.
Perera, Biosynthetic Approach for Functional Protein Microarrays, Anal. Biochem. 2012, 424,
114-23”.
xiii
CHAPTER 1
INTRODUCTION
1.1 Heme proteins
Heme proteins are the big family of proteins and are widely distributed in nature. Some
of these proteins act as oxygen storage (e.g., myoglobin) and some as an oxygen carrier (e.g.,
hemoglobin). While other heme proteins play important roles either in mediating electron
transfers (e.g., Cyt b5, Cyt c), or in catalysis (e.g., cytochrome P450, peroxidase, and catalase)
(1, 2). Along with heme, many enzymes contain other cofactors such as molybdopterin, copper,
flavin, and iron-sulfur cluster (1, 2). In these proteins (e.g., flavocytochrome b2, flavocytochrome
c3,
p-cresolmethyl
hydroxylase,
sulfite
oxidase,
fructose
dehydrogenase,
alcohol
dehydrogenase, and cytochrome c oxidase), charge transfer play a major functional role in
heme. Iron porphyrin prosthetic groups are the most common among all heme proteins (1-3).
Different varieties of porphyrins such as heme a, heme b, heme c, heme d, heme d1, heme
P460, heme o, siroheme, and chloroheme are present in heme proteins (Figures 1.1 and 1.2,
and Table 1.1). These porphyrins have a common basic skeleton, but due to substitution at
various positions, they differ in their structural details. In heme proteins, nitrogen from the
porphyrin ring occupy four coordination sites out of six of the heme iron, leaving only two
positions available for further ligand interactions which strongly influence the reactivity and
redox potential of the heme protein (1-3).
In many heme and globin enzymes, penta-coordinated heme iron is found (1, 3). In
globins, the proximal histidine (His) residue act as fifth ligand while axial heme position remains
open for O2 coordination, while the sixth coordination position is used for substrate binding in
some heme enzymes (1, 3). The redox potential of the heme center depends on the variety of
proximal ligands while distal ligands are responsible for the catalytic reaction in general (2, 3).
1
The spin state of the heme center is governed by the nature of axial ligands (1, 2). If a
water molecule is present as the sixth ligand, the center is in a high spin state, while presence
–
–
of carbon monoxide (CO), nitric oxide (NO), O2, azide (N3 ), and cyanide (CN ) as the sixth
ligand makes it in a low spin state (2, 3).
Heme type b is the simplest form of all known hemes and contains methyl group at 1, 3,
5, and 8 positions. At the same time 6 and 7 positions have two propionate groups and also 2
and 4 positions have vinyl groups (1). Energetically favored orientation of heme is the coplanar
structure, where vinyl group and the aromatic heme plane have the capability to interact with the
amino acids scaffold in the proteins (1, 2).
Heme a
Heme b
Figure1.1 Structures of heme a and heme b.
2
Heme c
Heme d
Heme d1
Heme o
Figure1.2 Structures of naturally occurring iron porphyrines.
3
Table 1.1 Listing biological functions, type, axial ligation and oxidation state of heme proteins (2).
Protein function
Type of heme
Axial ligation
Cytochromes c
Electron transfer
c
His
Mb
O2 storage
b
Hemoglobins
O2 transport
b
Peroxidases
A-H2 + H2O2 → A + 2H2O
b
His
Fe(III) (S = 5/2)
Catalases
H2O2 + H2O2 → O2 + 2H2O
b
Tyr
Fe(III) (S = 5/2)
P450 proteins
RH +2e + 2H + O2 → ROH + H2O
b
Cys
Fe(III) (S = 5/2)
Cytochrome bo quinol
O2 + 4 Fe(II)cyt c + 4e + 4H → 4
oxidases
Fe(III)cyt c + 2H2O
o
His
Fe(III) (S = 5/2)
P 460
His
4
Protein family
Hydroxylamine
oxidoreductase
–
+
–
–
Nitric reductases (cyt d1)
Sulfate reductase
–
–
+
–
–
4H + O2 + 4e → 2H2O
+
–
–
Fe(III) (S =1/2)
Deoxy Fe (II) (S = 2)
Oxy Fe(II) (S = 0)
Fe(III) (S = 5/2)
Or (S = 3/2)
–
2H + NO2 + e → NO + 2H2O
+
Fe(II) (S = 0)
+
H2O + NH2OH → NO2 + 4e + 5H
+
His
Oxidation/spin state
+
8H + NO2 + 6e → NH4 + 2H2O
His
d1
Siroheme
Fe(III) (S = 1/2)
His+ Tyr
Cys
Fe(II) (S =1 or 2)
1.1.1 Myoglobin
Myoglobin (Mb) is a relatively small heme protein (17 kDa) that is less affected by
environmental conditions when compared to most of the other heme proteins. Mb is a singlechain globular protein of 153 amino acids, as found in the sperm whale, and serves as oxygen
storage (4, 5). It consists of eight separate right-handed α helices in its apoprotein (Figure 1.3).
Oxygen can bind at the distal site of proteins, directly to the iron atom of the heme. The axial
His (His 93) links protein to the heme center through a bond between the iron atom and the His
(2, 4). As shown in figure 1.2, a wide variety of amino acid residues are present near the active
site of Mb. These residues play a critical role in determining the electronic nature of the heme
center and catalytic nature of the heme proteins (4-6). Studies shows that in Mb, the positions
such as 64, 68, and 93 are the most crucial residues (4). Due to its ability to prepare mixed
ligand complexes with different valences (ferric, ferrous, and ferryl states), Mb is often used as a
model to mimic other heme proteins such as cytochromes, peroxidases, catalases,
chloroperoxidases, and many more (5-10).
The role of the proximal ligand in controlling the reactivity and stabilizing the heme has
been a matter of interest for many years. In Mb, the role of proximal and distal His has been
assessed by replacing it with different residues (11-15) .
Mb is an oxygen storage heme protein; however, in presence of H2O2, it catalyzes a
variety of oxidative reactions such as sulfoxidation and epoxidation. Since Mb does not form a
stable Mb Cpd I in the presence of H2O2, the rate of epoxidation and sulfoxidation are
significantly slow in Mb as compared to those of peroxidases (16-19). Watanabe and coworkers
have created mutations at 64, 43, and 29 positions of Mb, replacing respective amino acids with
Asp, His, and Trp, as well as with Leu and His in double mutants. They have been able to
introduce catalase monooxygenase and peroxidase activity in Mb by generating Cpd I in
different Mb mutants (16-27).
5
A
Heme center
His 64
His 93
B
Leu 29
Val 68
His 64
Ile 107
Heme
Phe 43
His 93
Leu 89
His 97
Ser 92
Figure 1.3 Overall structure of sperm whale Mb. (A) WT Mb showing heme center and His at 64
and 93 positions and (B) schematic representation of active site of WT Mb.
6
Dawson and coworkers have created an H93G Mb cavity mutant and explored it as an
excellent structural model for His-ligated and other heme containing proteins (15, 28-32). They
have successfully compared and used Mb mutants as a model for the study of cytochrome f,
nitrite reductases, peroxidases, cytochrome P450, and many other heme proteins (15, 29, 3343). They have also extensively studied the ligand binding properties of heme proteins (6, 35,
43).
1.2 Incorporation of unnatural (noncanonical) amino acids into proteins
There are two strategies for the incorporation of noncanonical amino acids or unnatural
amino acids (UAA) into proteins: (1) site-specific and (2) residue-specific (44, 45). The sitespecific approach involves replacement of a single amino acid residue (Figure 1.4) while the
residue-specific method allows replacement of all or a fraction of one of the natural amino acids
by UAA (44-52). In the site-specific method, to deliver the analog in response to a stop or fourbases codon, a heterologous tRNA/aminoacyl-tRNA synthetase pair is used (53). Amber codon
suppression in mammalian cells has been used to incorporate unnatural amino acids by using
the E. coli tRNA/aminoacyl-tRNA synthetase pair; none of the suppressor tRNA was charged by
any of the mammalian aminoacyl-tRNA synthetases (54). For the site-specific incorporation of
UAA, amber codon suppression is the most frequently used method (Figure 1.3) and to date
more than 50 UAA have been introduced in recombinant proteins using these methods (Figure
1.4) (44, 52). For the incorporation of structurally, chemically and spectroscopically important
amino acids, Schultz and coworkers have produced orthogonal suppressor tRNA/aminoacyltRNA synthetase pairs and used it to incorporate many UAA in recombinant proteins (55-60). By
using both four base codon and nonsense codon suppression, Dougherty and coworkers have
successfully incorporated two UAA into an ion channel protein (61-63). Four bases codons,
commonly known as frameshift suppression, have also been used for the site-specific insertion
of UAA into fluorophore proteins and in many more recombinant proteins (50, 63-65).
7
Figure 1.4 A general method for genetically encoded unnatural amino acids in live cells.
8
9
Figure 1.5 The list of unnatural amino acids that have been introduced to the genetic codes of E. coli, yeast, and mammalian cells.
Furthermore, for the site-specific incorporation of UAA, analogous five bases codon and
reassignment of sense codons can also be used, but fidelity of the method is much lower than
that of anti-sense or frameshift suppression methods (66-68). It is possible in the genetic code
that one amino acid can be coded by more than one codon, as there are 61 codons for 20
natural amino acids; this makes the genetic code highly degenerate. In E.coli, many amino
acids are coded by more than one codon. Phe is coded by UUU and UUC. Only one tRNA has
been assigned for both codons. The tRNA uses base pairing and wobble interaction to decode
UUC and UUU, respectively. tRNA synthetases from different organisms have been used for
the reassignment of UUU into host E.coli. The discriminating capability of synthetases between
unnatural and natural amino acids plays a crucial role in the incorporation of UAA and yield of
recombinant proteins.
Similar to post-translational modifications, chemical methods for protein modifications
are equally important and they have been used to enhance the performance of therapeutic
proteins and labeling with fluorescent dyes (69, 70). Highly reactive Cys and Lys residues have
been most commonly used in the process of protein modifications (70).
1.3 Direct electron transfer and bioelectrocatalysis
Electrochemical methods are very useful in determining the redox properties of proteins
and kinetics of electron transfer (71). Electrochemistry can be used as a powerful tool in the
biocatalytic study of a protein and in biosensors and biofuel cell production (72). In general, to
facilitate the electron communication in between electrode and redox-sensitive protein, small
soluble mediators were used (72, 73). Due to the possibility of their unspecific side reaction,
these mediators lead to erroneous results; to overcome this problem, direct electron transfer
(DET) methods were developed. For the electron communication, the DET methods do not
require a mediator between the enzymes and the electrodes. These methods at times have
their own limitations such as denaturation of protein upon adsorption on electrodes, and
10
unspecific immobilization, which can be solved by using different varieties of linkers and
insulators.
The acceleration of the electrochemical reactions by a biological enzyme comes under
the realm of bioelectrocatalysis, and electrons can be transferred between an enzyme active
site and the electrode without any mediator. Electrodes for fuel cells and for the biosensors are
the two most common applications of bioelectrocatalysis. It was essential to achieve DET in
redox enzymes on solid electrode for its further development. In addition, to achieve the most
effective electron transfer, the importance of protein orientation on the solid electrode surface
has been investigated (71-73).
In principles, bioelectrocatalysis involves acceleration of the electrochemical reactions
by biological catalysis (72). Figure 1.6 indicates the involvement of the enzymes in direct
bioelectrocatalysis. The communication of electrons with the electrode was provided by enzyme
catalyzing redox reactions. Oxidation and reduction reactions are the two halves of redox
reactions and one of these half reactions can be substituted in bioelectrocatalysis (72).
Heme is among those prosthetic groups which can communicate directly with the
electrode. Heme can be used both in formation of the enzyme active center as well as for the
communication with the redox mediator, thereby leading to communication with the electrode.
This makes heme one of the most important constituents of direct bioelectrocatalysis systems
and hence heme proteins are frequently used in bioelectrocatalysis (74, 75).
11
Figure 1.6 Schematic representations of bioelectrocatalysis involving direct electron transfer in
an enzyme. One of the two coupled half-reactions is substituted by the electrochemical reaction.
1.4 Dissertation overview
Creation of genetically engineered Mb mutants by incorporating a non-cannonical
redox-active NH2Tyr into its active site for the mechanistic insight and oxidative activity has
been extensively explored in chapter 2. This study proposed the importance of the distal active
site residues in acid-base catalysis and formation of reactive oxygen species (ROS) in heme
proteins.
In chapter 3, I reported the incorporation of another unnatural amino acid DOPA to a
specific position in the active site of Mb. The mutant Mb protein could carry out the oxidation of
thioanisole and benzaldehyde and support the idea that the distal His is important for the
formation of Cpd I to exhibit peroxygenase activity in Mb.
In chapter 4, I reported monolayer and multilayer immobilization of Mb using two unique
immobilization techniques, onto a chemically-modified gold electrode surface. Further, using
12
DET method bioelectrocatalysis of thioanisole oxidation was carried out successfully with both
the protein immobilized electrodes. The results described in chapter 4 suggest the importance
of proper orientation of recombinant heme proteins containing UAA in bioelectrocatalysis and
effective electron transfer as comparison to the random multilayered WT Mb case. I have also
analyzed the protein-protein interactions of five different proteins which have been covalently
attached onto a solid support. Functional protein microarray concept development was
confirmed by catalytic activity assay using spectroscopic characterization, which will be of huge
importance in the field of drug discovery, and biomedical and proteomic research.
In chapter 5, I have summarized the importance and significance of electron rich
residues on the electronic nature of heme center. I have observed that the axial His attached to
the heme center plays a more crucial role in dictating the electron cloud near the heme center
and hence in formation of reactive species. When axial heme residue was replaced by electron
rich residue like pNO2Phe, it contributed to higher energy and red shift of the spectra due to the
higher electron density near the heme center. This study also explored the oxidation state of the
metal center, the nature of the ligand, the type of residues near the heme center that play an
important role in determining of backbonding, the direction of charge transfer, and the possibility
of hydrogen bonding. I also underlined that the size of UAA residue plays a crucial role in the
stability of ligand adducts.
Overall in this study I have used Mb as a model for the heme protein to study their
monooxygenase and peroxidase activity. No prior studies were reported using mutant protein
that utilizes its UAA acid residue to immobilize site specifically onto the electrode surface. I also
explored the DET and bioelctrocatalysis and importance of the proper orientation of protein
using Mb mutants as a model for heme proteins. This study also offers a similar orientation
concept while dealing with the protein microarray problems. The Mb mutants have also been
used to explore the role and effect of different varieties of residues near the heme center and
their effect in ligand binding spectra.
13
CHAPTER 2
MECHANISTIC INSIGHTS AND IMPROVED OXIDATION ACTIVITY USING GENETICALLY
ENGINEERED MYOGLOBIN
2.1 Introduction
Peroxidase enzymes couple substrate oxidation to H 2O2 reduction (as generically
represented by Reaction 1). They are widely distributed throughout the plant and animal
kingdoms and are frequently isolated from bacteria, mold, and microorganisms (76-78). Their
main biological function is to act as antioxidants by protecting cells, tissues, and organs against
the oxidation of a variety of organic and inorganic compounds by H2O2, organic hydroperoxides,
peracids, or inorganic oxysalts, such as periodate or chlorate (79-85).
AH2 + H2O2  A + 2H2O
(1)
Most heme-containing peroxidases (e.g., horseradish peroxidase, HRP) have a
+
proximal His and a polar distal pocket, with a non-ligated His as an H acceptor/donor and a
cationic arginine to stabilize developing negative charges during O-O bond cleavage.
Peroxidases transform the oxidizing power of H2O2 into high-valent, protein-bound oxidants. The
general peroxidase pathway (Figure 2.1) involves addition of H2O2 or other oxygen atom donors
IV
([O]) to the ferric resting state (1) to form Cpd I (5), a ferryl (Fe ) radical species where the πcation radical is usually centered on the porphyrin (86). Cpd I typically oxidizes substrates by
one electron to yield an organic radical and Cpd II (6), a ferryl heme complex (79, 87). Cpd II
then oxidizes another substrate molecule and returns to the ferric state (1). A ferryl state-like
peroxidase, Cpd I, is thought to be the active species in the catalytic cycle (88, 89). Involvement
of a hydroperoxo-ferric intermediate (Cpd 0) has also been proposed (6, 82). Alternatively,
addition of H2O2 to ferric-heme protein can generate Cpd 0 (4) via a peroxide “shunt” pathway.
14
Heme-containing peroxidases such as HRP form Cpd I from Cpd 0 using distal catalytic
arginine amino acids to “pull” the O-O bond apart heterolytically.
Figure 2.1 Mechanistic pathway for generation of the highly reactive Cpd I (5) from the ferric
resting form (1) in heme-proteins.
Lacking this distal machinery, WT Mb undergoes nearly equal amounts of homo- and
heterolytic cleavage upon H2O2 addition. Cpd II, the stable product of homolytic cleavage of the
O-O bond of (6), is the prime product of Mb but has no catalytic activity. Previous research
shows that site-directed mutagenesis is a versatile technique to change the functional
properties of Mb. Thus the distal His (H64) has been replaced by amino acids such as Ala, Ser,
Leu and Asp, and the corresponding effect on the activity of Mb has been probed (90). Recent
15
reports evaluated the His93Gly (H93G) “cavity” mutant of Mb as a versatile scaffold for
modeling heme states (29, 35, 36). The difference in accessibility of the two sides of the heme
have frequently made it possible to prepare mixed ligand adducts in ferrous, ferric, as well as
ferryl, oxidation states with this mutant. In addition, the protection provided to the heme by the
protein environment allows for the preparation of stable oxyferrous and oxo-iron (IV) complexes
at near-ambient temperatures with sperm whale Mb. Similar studies have been carried out to
obtain spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome
P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at –55 °C in presence of 70/30 (v/v)
glycerol/buffer cryosolvent (91).
Taking the above facts into account, I can effectively model the heme protein active site
using a natural probe. Here in this study, Mb mimic of peroxidase enzymes using unnatural
amino acids was used to characterize the active species in the catalytic cycle, investigate the
catalysis mechanism, and to model the heme protein active sites (44, 92). To this end, Mb, an
oxygen storage and carrier protein, was converted into a catalytically active peroxygenase using
a genetically incorporated redox-active NH2Tyr into its active site (Figure 2.2) (44, 93-95). The
mutant H64NH2Tyr Mb was then employed to investigate the structure-function relationship in
heme proteins (based on acid-base catalysis and distal charge relay effect).
A Mb mutant in which His 64 was replaced with NH2Tyr (H64NH2Tyr) showed high
turnover rates for thioanisole and benzaldehyde oxidation (9-fold and 81-fold), when compared
to WT, in the presence of H2O2. Our results indicate a possible situation where acid-base
catalysis of NH2Tyr coupled with distal “pull” effect via the hydrogen bonding network, play an
important mechanistic role to mimic peroxidase. Furthermore, peroxygenase activity of this
mutant exhibited remarkable stability against heme bleaching with the generation of active
catalytic species.
16
Figure 2.2 Schematic representation of the oxyferrous complex of Mb. (A) active site of WT Mb.
(B) active site of H64NH2Tyr, (PDB number 1MBO).
2.2 Experimental procedure
2.2.1 Chemicals
The gases (O2 and N2) were purchased from Air Liquide USA while NH2Tyr, sodium
dithionite
(Na2S2O4),
thioanisole,
benzaldehyde,
30%
H2O2,
and
2,2’-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich USA, and
used without any further purification. All chemicals were at least analytical grade.
2.2.2 Preparation of WT Mb and H64NH2Tyr Mb mutant constructs
The WT Mb expression constructs, H64 (TAG) Mb pBAD expression vector was
prepared by Dr. Roshan Perera and aminoacyl tRNA synthetase (aatRNA S) plasmids were
gifts from Dr. Peter Schultz (The Scripps Research Institute, La Jolla, CA USA). The H64 (TAG)
Mb expression pBAD (Ampicillin) vector and tRNA synthetase plasmid (Tetracycline) were cotransformed in DH10B E. coli bacteria cells. The double antibiotic resistant (ampicillin and
tetracycline) colonies were picked, grown, and the cell stocks were stored at –80 °C prior to
protein expression and purification.
17
2.2.3 WT Mb and H64NH2Tyr Mb protein purification
The H64NH2Tyr Mb was expressed in E. coli grown in glycerol minimal media (GMML)
containing 25% Luria Broth (LB) media and suitable antibiotics (ampicillin and tetracycline).
Both the WT Mb and mutated (H64NH2Tyr) Mb proteins were induced by adding 0.02%
arabinose to bacterial cultures when optical density at 600 nm (O.D600) was 0.5. Cells were
harvested by centrifugation at 13000 g and lysed in lysis buffer (Tris HCl buffer at pH 8.0) by
addition of RNase, Dnase, and lysozyme to the cell contents, followed by three freeze (liquid
N2) thaw (37 °C) cycles and 10 rounds of 40 sec sonications on ice. The crude lysate was
mixed thoroughly with Ni-NTA resin and the protein-bound nickel-nitrilotriacetic acid (Ni-NTA)
was collected in a column. Purified proteins were eluted from the column using an elute buffer
(300 mM NaCl, 250 mM imidazole, 50 mM phosphate buffer, pH 7.0). The purified WT Mb and
mutant proteins were stored at –80 °C until further use.
2.2.4 WT Mb and H64NH2Tyr Mb protein analysis
The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
were shown in figure 2.3. The mass of mutant H64NH2Tyr Mb protein was analyzed by matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) and was observed to be 18397
Da, (Figure 2.4) which matched with the theoretically calculated mass.
18
Figure 2.3 Coomassie-stained SDS-PAGE analysis of expression of WT Mb and H64NH2Tyr
Mb. (A) Lane 1, Molecular weight standards as indicated in kiloDalton (kDa); Lane 2, expressed
WT Mb; Lane 3, expressed H64NH2Tyr Mb, (B) Lane 1, Molecular weight standards as
indicated in kDa; Lane 2, expressed H64NH2Tyr mutant Mb in the absence (–) of 1 mM NH2Tyr;
Lane 3, expressed H64NH2Tyr Mb in the presence (+) of NH2Tyr. No Mb protein was found by
SDS−PAGE analysis in the absence of NH2Tyr.
19
Figure 2.4 The mass spectrum (MALDI-TOF) of the H64NH2Tyr Mb protein. A mass of 18397
Da matches the calculated mass for the Mb mutant. No WT Mb protein (predicted mass, 18356
Da) was observed.
2.2.5 Spectroscopy
UV-visible electronic absorption spectra of WT Mb and mutant Mb proteins were
acquired on a Varian Cary 50 Bio UV-visible spectrophotometer. The concentration of WT Mb
was determined using pyridine hemochromogen method (96) .
2.2.6 Preparation of oxyferrous complexes
The oxyferrous complexes were prepared in a chest freezer at –35 to –45 °C. The
protein sample was taken in 100 mM potassium phosphate buffer (pH 7.0) which contained
65% glycerol (v/v). The ferric protein was first degassed with N2 gas for 2 hr Then it was
reduced to a deoxyferrous species by addition of Na2S2O4 (20 mg/mL stock) under N2
20
atmosphere in a sealed cuvette at 4 °C. Pre-cooled O2 gas was bubbled into the cuvette for 60
s and the UV-visible spectra were recorded (Figure 2.8) as indicated in the previous study (32).
2.2.7 Effect of H2O2
The effect of H2O2 on ferric proteins was observed in the presence of 5 mM H 2O2. The
protein concentration was 5 μM and heme bleaching of proteins was observed at 20 °C (Figures
2.6 and 2.7).
2.2.8 Catalytic sulfoxidation of thioanisole and oxidation of benzaldehyde
In the total volume of 500 μL, 100 mM potassium phosphate buffer pH 7.0, the 5 μM
protein (WT Mb or H64NH2Tyr mutant Mb) was added. To that 1 mM substrate, either
thioanisole or benzaldehyde was added. To initiate the reaction, 5 mM H 2O2 was added to a
final volume of 500 µL. The reaction mixture was incubated for 1 hr After incubation the organic
products were extracted in dichloromethane. The solvent was reduced and the organic products
were analyzed by gas chromatography-mass spectrometry (GC-MS) (Figure 2.5).
2.2.9 Electrochemical instrumentation
Differential pulse voltammetry (DPV) was carried out using a single-compartment,
three-electrode cell at 22 °C on a CHI720C electrochemical analyzer (CH Instruments, Austin,
TX). The working electrode was polycrystalline gold, the counter electrode was a platinum wire,
and the reference electrode was Ag/AgCl/3.5 M KCl (BAS, West Lafayette, IN). All potential
values below are reported with respect to the Ag/AgCl reference electrode. The working
electrode surface was first polished on microcloth with alumina slurry suspension (0.05 μm),
then sonicated in ethanol, and finally rinsed thoroughly with Milli-Q water. The electrolyte
solution was 0.1 M K2HPO4/KH2PO4 buffer at a pH of 7.0. The solutions were deoxygenated by
bubbling nitrogen prior to each experiment. The concentration of H 2O2 used was 5 mM. The
21
DPV measurements were recorded with pulse amplitude of 30 mV, step size set at 4 mV, pulse
width at 0.05 sec, and pulse period at 0.20 sec (Figure 2.9).
22
23
24
Figure 2.5 GC-MS analysis of oxidation products of thioanisole ans benzaldehyde. Conversion of thioanisole (a) to its sulfoxidation
product (b) by 5 mM H2O2. (A), GC data of WT Mb; (B), GC data of H64NH 2Tyr mutant Mb, (C), mass spectrum of thioanisole sulfoxide
product, (D) mass spectrum of benzoic acid product, (E) and (F) show GC and MS data of oxidation of benzaldehyde (a) to benzoic acid
(b) by 5 µM Mb and H2O2 for WT Mb and H64 H64NH2Tyr respectively. The reaction was carried out by adding 5 mM H 2O2 to 5 µM WT
or mutant Mb in 0.5 mL of 100 mM potassium phosphate buffer (pH 7.0) with 1 mM thioanisole or benzaldehyde at 25 °C.
2.3 Results and Discussion
2.3.1 Genetic design rationale and characterization
In the heme-containing peroxidases, the generation of active Cpd I species depends on
precise delivery of two protons at the distal oxygen of the peroxo-ferric intermediate (4). Another
essential feature is the presence of the distal His and the Arg in the active site pocket of the
peroxidases (8, 21, 97). In HRP, the distal Arg influences the distal His to react with H2O2 and
form the active Cpd I through an acid-base mechanism. But in WT Mb, the distal His is not
affected by any other distal amino acid and cannot induce the formation of Cpd I efficiently.
Therefore, I decided to incorporate a redox-sensitive NH2Tyr amino acid in place of the distal
His. In the presence of an oxidant (e.g., H2O2), the redox-active NH2Tyr amino acid should be
able to facilitate an oxidation reaction which releases two electrons and two protons (Figure 2.2)
as well as to induce the formation of Cpd I. Therefore, I expected the H64NH2Tyr mutant Mb to
behave as a considerably more potent peroxygenase catalyst due to the increased oxygenase
activity around the heme environment.
To facilitate the de novo design of Mb with unique peroxidase functionality, I have
genetically encoded NH2Tyr into Mb, replacing His 64 with the amber nonsense codon TAG into
a pBAD expression vector, using a previously reported technique. The method includes the
genetic
incorporation
of
non-canonical
amino
acids
into
proteins
using
orthogonal
tRNACUA/aminoacyl-tRNA synthetase pairs in bacteria. The expressed H64NH 2Tyr Mb showed
were analyzed by SDS- PAGE (Figure 2.3). Mass spectral analysis (MALDI-TOF) showed a
parent ion mass of 18397 Da as expected for the H64NH2Tyr Mb mutant (Figure 2.4).
The electronic absorption spectra of purified H64NH 2Tyr mutant displayed a sharp
Soret absorption peak at 410 nm (visible peaks around 539 nm, 579 nm and 624 nm),
consistent with the aquo-ferric complex. Surprisingly, addition of 5 mM H2O2 did not show
significant Soret shift (λmax = 410 nm) or heme-bleaching for the NH2Tyr mutant Mb (Figure 2.6).
25
Figure 2.6 Electronic absorption spectra of the WT and mutant Mb. H64NH2Tyr Mb in the
presence of 5 mM H2O2 (black solid line), in absence of H2O2 (blue dashed dot dot line) and WT
Mb in the presence of 5 mM H2O2 (green dot line) and in absence of H2O2 (red dashed line). ε =
Molar extinction coefficient.
However, there were isosbestic points in the spectra between the peaks in the
presence and absence of H2O2 with the mutant to indicate that a novel catalytic species had
been generated (Figure 2.6). As expected, the WT protein formed Cpd II (the Soret around 418
nm) with 5 mM H2O2 with significant heme loss within the first 15 min of the reaction (Figure 2.7)
(98).
26
Figure 2.7 Effect of H2O2 on WT and mutant Mb. Absorbance changes monitored over a 30 min
period at λmax = 418 nm for WT Mb (blue dashed dot dot line) and λmax = 410 nm for H64NH2Tyr
mutant Mb (black solid line) in the presence of 5 mM H2O2 and control (red dashed line). The
spectra were measured in 100 mM potassium phosphate buffer, pH 7.0 at 20 °C with 10 μM
protein.
In addition, the oxyferrous and deoxyferrous complexes of the H64NH 2Tyr mutant and
the WT Mb were prepared at –35 to –45 °C. The UV-visible spectra were recorded (Figure 2.8),
which exhibited that the inherent characteristics of the native heme were intact in both the WT
and the H64NH2Tyr mutant Mb.
27
Figure 2.8 Deoxyferrous and oxyferrous spectra of WT and mutant Mb. The spectra of WT Mb
deoxyferrous (blue dashed line), H64NH2Tyr Mb deoxyferrous (red dashed line), WT Mb
oxyferrous (green dotted line) and H64NH2Tyr Mb oxyferrous (black solid line) species. The
spectra were taken at –35 to –45 °C and the samples were examined in 60% glycerol, 100mM
phosphate buffer at pH 7.0.
2.3.2 Catalytic activity
A number of substrates including thioanisole and benzaldehyde were successfully
oxidized to their products by the mutant. Sulfoxidation of thioanisole (Reaction 2) and oxidation
of benzaldehyde to benzoic acid (Reaction 3) by 5 mM H2O2 for H64NH2Tyr mutant Mb showed
–1
2.4 min
(9-fold higher activity compared to WT Mb) and 4.05 min
–1
(81-fold higher activity than
WT Mb) turnover rates, respectively (Table 2.1). However, under the same conditions WT Mb
shows only 0.25 min
–1
for sulfoxidation and 0.05 min
28
–1
for the oxidation of benzaldehyde.
Table 2.1 Rate of sulfoxidation of thioanisole (Reaction 2) and benzaldehyde oxidation
(Reaction 3) by WT Mb and mutant H64NH2Tyr Mb. Kinetic values are based on the average of
at least 2 determinations and the unit for rate is turnover per min.
–1
Protein
Km (µM)
Vmax (µM min )
WT Mb
19.3
25.6
H64NH2Tyr Mb
50.25
69.93
[a]
[a]
This value is in agreement with data previously reported (26)
2.3.3 ABTS peroxidase assay
2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) or ABTS has often been used
to estimate the reaction kinetics of enzymes like peroxidases. In the presence of H2O2 and
peroxidase.enzyme, it is converted to its radical cation. This blue colored radical cation is
utilized to indirectly identify the formation of the ferryl radical species in peroxidases (26, 99). In
order to detect the development of the reactive species in the WT and mutant H64NH 2Tyr Mb,
the ABTS peroxidases assay was performed. The results are contained in Table 2.2. The
29
reaction mixture contained 1 µM protein, 5 mM H2O2 and ABTS. The concentration of ABTS
was varied from 0.02 mM to 2 mM (97, 100, 101).
Table 2.2 Peroxidase assay of WT and mutant Mb with ABTS in presence of H 2O2. ABTS
oxidation reaction assays of Mb and its mutant in the presence of 5 mM H2O2. Kinetic values
are based on the average of at least 2 determinations and the unit for rate is turnover per min.
Reaction has been done at pH 7.0 and at 20 °C.
Protein
Km (µM)
WT Mb
19.3
H64NH2Tyr Mb
[a]
[a]
50.25
–1
Vmax (µM min )
25.6
[a]
69.93
This value is in consistent with data previously reported (99).
The ABTS radical cation formation was monitored at 730 nm. The Km and Vmax values
were calculated from Linewearver-Burk plots. The detection of the ABTS radical cation, in turn,
corroborated the peroxidase activity of the WT and mutant H64NH2Tyr Mb (101).
2.3.4 Electrochemical characterization
To understand the effect of the distal proton and electron delivery network via NH2Tyr
residue in Mb, we measured the reduction potentials (E°) for Mb and its mutant in the presence
of H2O2 at pH 7 as well as at pH 10 (Table 2.3) using differential pulse voltammetry (DPV). The
E° values confirmed that the primary amine and hydroxyl groups in NH 2Tyr amino acid influence
the electronic nature of the iron at the heme active site. In the presence of H2O2 at pH 7.0, the
mutant (–360 mV) exhibited a 40 mV shift from the WT Mb (–320 mV), which is more than the
difference observed (27 mV) at pH 10.0 (Figure 2.9 and Table 2.3). Since a single hydrogen
bond results in a 25-60 mV change in the E° values (102, 103), the observed potential
30
difference between the mutant and the WT Mb is consistent with the presence of an additional
hydrogen bond for H64NH2Tyr mutant than the His-64 in WT Mb.
31
32
Figure 2.9 DPV of WT and mutant Mb in presence and absence of H2O2. The DPV response of 5 µM WT and mutant H64NH2Tyr Mb in
nitrogen saturated 100 mM phosphate buffer and 10 mM KCl solutions (A and C: at pH 7.0; B and D: at pH 10.0) in absence (A and B)
and in presence (C and D) of H2O2 at room temperature conditions. Pulse amplitude = 30 mV, step size = 4 mV, pulse duration = 0.05
sec and pulse period = 0.20 sec.
Table 2.3 Reduction potential of WT and mutant Mb. Reduction potentials were measured at pH
7.0 and 10.0 in the absence and presence of 5 mM H2O2. All reduction potentials are reported
with respect to Ag/AgCl (3.5 M KCl) reference electrode.
Reduction Potential, E (V)
Protein
Without H2O2
With 5 mM H2O2
pH 7.0
pH 10.0
pH 7.0
pH 10.0
WT Mb
–0.232
–0.256
–0.260
–0.292
H64NH2Tyr Mb
–0.244
–0.256
–0.352
–0.320
A possible reason for the 27 mV shift with H2O2 at pH 10 (than the 40 mV at pH 7) is
that the redox-sensitive amino acid (NH2Tyr) can be more easily oxidized (pKa is ~10 for
hydroxyl group in NH2Tyr) to o-imino-quinone by releasing two electrons and two protons, which
would preferentially promote the formation of the heme high valent (ferryl) species in the Mb
mutant.
2.3.5 Ligand binding study of WT and H64NH2Tyr Mb mutant adducts
The replacement of distal His with redox residue like NH2Tyr gave us an important
insight of the nature of electronic absorption spectra. From Figure 3.6, I observed that after
addition of neutral ligands such as CO, NO and O 2 to WT and mutant Mb, the Soret became
sharper at respective places. The CO adduct is linear and both of its π orbitals are empty,
which allows dπ-π orbitals to overlap in a perpendicular direction resulting in greater
overlapping. It provides the perfect condition for π backbonding, while under similar conditions
NO and O2 adducts have less π backbonding because they have one or two electrons in their
orbitals (104). Since mutant Mb form an extra hydrogen bond with the ligands, the adduct with
33
mutant Mb were stronger. The bigger size residue like NH2Tyr, if present near the active site,
also prohibits the release of ligands from the heme center and hence increases the stability.
–
–
CN and N3 , which are rich in electrons, also show the similar effect with lesser intensity as
compared to neutral ligands (Figure 2.10).
34
35
Figure 2.10 Characterization of WT and H64NH2 Mb ligand complexes. Electronic absorption spectra of (A) CO (B) NO, (C) cyanoferric
–
and (D) ferric N3 complexes of WT Mb (red solid line) and H64NH 2Tyr (blue dashed line) Mb. The spectra were taken at 4 °C and the
samples were examined in 100 mM phosphate buffer at pH 7.0.
2.3.6 Proposed mechanical feature
The above spectroscopic and electrochemical characterizations demonstrate the role of
the redox-sensitive NH2Tyr in the introduction of peroxidase activity in Mb (Figure 2.11). When
H2O2 is present in the active site of the mutant Mb, the amine functional group of the NH 2Tyr
can abstract a proton from the H2O2, thereby forming the Fe-O-OH moiety (Cpd 0). This in turn
loses one molecule of H2O to form the active ferryl radical cation (Cpd I). The presence of the
hydroxyl and the amine groups on the NH2Tyr is absolutely essential for this step as it involves
an acid-base catalysis mechanism. The distal “pull” effect via the hydrogen bonding network
facilitates heterolytic cleavage of the hydroperoxide-ferric O-O bond in Cpd 0. The substrate can
IV
now approach the ferryl (Fe ) radical species become oxidized, thereby regenerating the
resting ferric state in the mutant Mb.
This indicates that the redox-properties of NH2Tyr can be exploited to generate the
elusive ferryl radical cation (Cpd I) in the mutant Mb. Hence, H64NH 2Tyr Mb peroxygenase
activities as well as isolation of catalytically active species in this mutant will significantly
increase our understanding of how nature utilizes the heme iron cofactor and the protein
scaffold around the active site in essential biological transformations involving peroxides and
O2.
36
Figure 2.11 Mechanism of NH2Tyr which involves an acid-base catalysis mechanism in
presence of an oxidant [O], such as H2O2.
2.4 Conclusions
In this chapter, I report the creation of a genetically engineered Mb mutant by
incorporating a non-canonical redox-active NH2Tyr into its active site. The replacement of His in
the distal His 64 with NH2Tyr mutant Mb provided an insight into the role of the distal active site
residues in acid-base catalysis and distal charge relay “pull” effect in peroxide activation and
formation of ROS in heme proteins. The H64NH2Tyr mutant Mb showed high turnover rates for
thioanisole oxidation (9 times) and benzaldehyde oxidation (81 times) when compared with the
WT Mb, in the presence of H2O2. The ligand binding studies also explored the importance of
redox residues (e.g., NH2Tyr) near the heme center.
37
CHAPTER 3
INTRODUCTION OF PEROXIDASE ACTIVITY IN MYOGLOBIN BY INCORPORATING
UNNATURAL AMINO ACIDS AT THE DISTAL HISTIDINE POSITION
3.1 Introduction
Among the various heme containing metalloproteins, Mb is found in the muscle tissue
of most vertebrates and all mammals (2). It is an oxygen binding single chain globular protein
with 153 amino acids (sperm whale Mb) (1, 2, 5). The active site of Mb contains iron-heme as
the prosthetic group (105). Since Mb has been extensively characterized, it is frequently used
as a model to study the structure and function of the heme proteins. X-ray crystallography,
NMR, and EPR, have been successfully used to study the structure and function of the Mb
protein (106-108). The Mb gene of many organisms, including human and sperm whale, have
been cloned, and heterologous expression of recombinant protein in E. coli has been carried
out effectively (10, 109-112).
While studying other heme-proteins, like horseradish peroxidase (HRP) and
cytochrome c peroxidase (CcP), it has been revealed that the presence of distal His is very
critical for the formation of Cpd I and peroxidase activity (Figure 3.1) (106-108, 113-115). The
successful oxidation of thioanisole and benzaldehyde by HRP and CcP has demonstrated their
peroxidase property. In contrast, the rate of formation of Cpd I in Mb is quite slow and the
myoglobin Mb Cpd I is also very unstable, hence the WT Mb cannot catalyze sulfoxidation of
thioanisole or benzaldehyde oxidation significantly. To carry out these reactions, it is essential
to increase the rate of Cpd I formation and provide a suitable environment at the active site to
prohibit its decay to Cpd II. The Cpd II is a catalytically inactive and stable iron-oxygen species,
which effectively stores the oxygen in the tissues.
38
A Active site of WT Mb
B Active site of HRP
Figure 3.1 Schematic representation of the formation of Cpd I and Cpd II in Mb and HRP. (A)
Formation of Cpd II by homolytic cleavage of O-O bond in Mb; (B) Cpd I formation in HRP by
heterolytic cleavage.
In order to increase the rate of formation of Cpd I and to stabilize it, we have
incorporated an unnatural amino acid, DOPA, replacing the distal His in the active site of Mb.
This H64DOPA Mb mutant was constructed on the basis of accommodating a redox-sensitive
amino acid in the active site of Mb. Oxidation of thioanisole and benzaldehyde were carried out
in the presence of WT and mutant Mb to monitor their peroxidase property. Herein, we report
39
that the H64DOPA Mb mutant exhibited significantly high peroxidase activity as compared to
the WT Mb.
Furthermore, I have studied the electron rich and neutral ligand adducts of mutant and
WT Mb to understand the effect of redox amino acids such as DOPA, which is capable of giving
electrons to heme center and to ligand when present near the active site. It also was interesting
to explore the role played by hydrogen bonding favoring residues on the stability of ligand
adducts.
3.2 Experimental procedures
3.2.1 Chemicals
The gasses (O2, NO, CO and N2) were purchased from Air Liquide while DOPA,
Na2S2O4, sodium azide, potassium cyanide, thioanisole, benzaldehyde, and 30% H2O2 were
purchased from Sigma-Aldrich USA, and have been used without any further purification. All
chemicals were of analytical grade.
3.2.2 Preparation of WT Mb and H64DOPA Mb mutant constructs
The WT Mb and H64DOPA Mb expression constructs were made by the following
previous method (44). The H64DOPA Mb expression vector and tRNA synthetase plasmid have
been co-transformed in DH10B E.coli. The double antibiotic resistant colonies were picked,
grown, and the cell stocks were stored at –80 °C prior to protein purification.
3.2.3 WT Mb and H64DOPA Mb protein purification and analysis
The H64DOPA Mb was expressed in E. coli that was grown in GMML containing 25%
LB media and suitable antibiotics (ampicillin and tetracycline). Both the WT and mutated
(H64DOPA) Mb proteins were expressed, purified and analyzed as described in chapter 2
(Figure 3.2).
40
Figure 3.2 Analysis of WT and H64DOPA mutant Mb by SDS-PAGE and MALDI-TOF. (A) Lane
1, expressed WT Mb; Lane 2, expressed H64DOPA Mb; Lane 3, Molecular weight standards as
indicated in kDa (B) MALDI-TOF of the H64DOPA mutant Mb protein. A mass of 18397 Da
matches the calculated mass for the Mb mutant. No WT protein (predicted mass, 18356 Da)
was observed.
41
3.2.4 Spectroscopy
UV-visible electronic absorption spectra of the WT Mb and mutant Mb proteins were
taken using Varian Cary 50 Bio UV-visible spectrophotometer. The concentration of WT and
mutant Mb was determined by heme chromatogen method (96).
3.2.5 Effect of H2O2
The effect of H2O2 on ferric proteins has been observed in the presence of 5 mM H2O2.
The protein concentration was 5 μM and heme bleaching of proteins was observed at 20 °C.
3.2.6 Preparation of oxyferrous complex
The oxyferrous complexes were prepared in a chest freezer at –45 to –55 °C. The
protein sample was taken in 100 mM potassium phosphate buffer (pH 7.0) which containing
65% glycerol (v/v). The ferric protein was first degassed with N 2 gas for 2 hr Then, it was
reduced to deoxyferrous by addition of Na2S2O4 (20 mg/mL stock) under N2 atmosphere in a
sealed cuvette at 4 °C. Pre-cooled O2 gas was bubbled into the cuvette for 60 sec and the UVvisible spectra were recorded.
–
–
3.2.7 Preparation of CO, NO, CN and N3 adduct samples
The ferrous-CO adducts were generated by gentle bubbling of CO in deoxyferrous
enzymes for 30 sec at 4 °C in 0.1 M potassium phosphate buffer pH 7.0. To prepare the
ferrous-NO adducts, minimal amount of buffer saturated with NO gas was added to the
deoxyferrous enzymes under N2 at 4 °C in 0.1 M potassium phosphate buffer pH 7.0. The ferric
–
N3 and cyanoferric adducts were formed by addition of minimal amounts of NaN3 and KCN
from their respective stocks (40 mM NaN3 and 1 M KCN) to the degassed ferric proteins. The
ferric proteins were in 100 mM potassium phosphate buffer pH 7.0 and the temperature was 4
42
°C. The deoxyferric enzymes were prepared by bubbling of N2 gas in the ferric enzymes for at
least 30 min.
3.2.8 Catalytic sulfoxidation of thioanisole and oxidation of benzaldehyde
The reactions were performed and the products were analyzed as described in chapter
2 and the data were shown in figure 3.8 and table 3.2.
3.2.9 Electrochemical instrumentation
DPV was carried out following the method as described in chapter 2 and the data were
shown in figure 3.7.
3.3 Results and discussion
The inherent property of Mb is to store and transport oxygen. The WT Mb forms a
stable and catalytically inert oxy-complex. It has been reported that the His 64 stabilizes the
sixth ligand water molecule through hydrogen bonding (110, 116, 117).
3.3.1 UV-visible spectroscopic characterization of WT and H64DOPA mutant Mb
The mutant protein and WT Mb exhibited similar electronic spectra as observed for the
ferric heme species. The maximum absorbance in Soret band was found to be 409 and 411 nm
for the WT and mutant Mb, respectively (Figure 3.3). In the case of WT Mb, small peaks were
observed at 632 nm, 580 nm, 544 nm, and 507 nm, while for H64DOPA Mb, only two peaks
were observed at 632 nm and 530 nm in addition to the 411 nm peak. His 64 has been replaced
by DOPA and the unnatural amino acid was expected to stabilize it further by forming an extra
hydrogen bond. Therefore, I have genetically encoded DOPA into Mb, replacing His 64 with the
amber nonsense codon TAG into a pBAD expression vector, using a previously reported
technique (44, 45). The method includes the genetic incorporation of non-canonical amino acids
43
into proteins using orthogonal tRNACUA/aminoacyl-tRNA synthetase pairs in bacteria. Mass
spectral analysis (MALDI-TOF) showed a parent ion mass of 18597 Da as expected for the
H64DOPA Mb mutant (Figure 3.2 and Figure 3.3).
Furthermore, the H64DOPA Mb mutant showed remarkable tolerance to H2O2. Even at
25 mM concentration of H2O2, heme bleaching was not observed to a significant extent (Figure
3.4). The UV-visible absorption spectrum of H64DOPA Mb mutant in presence of H 2O2 also
exhibited a Soret band at 411 nm similar to that of ferric resting species. However, the
isosbestic points present in the spectra indicate that an active catalytic species had been
formed (Figure 3.3). Under similar conditions, when H2O2 was added to the WT Mb, hemin was
bleached out and it formed an inactive Cpd II species as expected. This fact was verified by the
presence of a Soret at 418 nm (Figure 3.4) in the absorption spectrum of the WT Mb.
Figure 3.3 High-valent heme complexes of WT and H64DOPA Mb. Electronic absorption
spectra of the H64DOPA Mb in the presence (black solid line), in the absence (red dashed line)
and WT Mb in the presence (green dotted line) and in absence (blue dashed dot line) of 5 mM
H2O2.
44
Figure 3.4 Stability of WT and H64DOPA Mb in presence of H2O2. Absorbance changes
monitored over a 30 min period at λmax = 418 nm for WT Mb (blue dashed), λmax = 410 nm for
H64DOPA mutant Mb (black dash dot line) in the presence of 5 mM H 2O2.and control protein
without H2O2 (red solid line) The spectra were measured in 100 mM potassium phosphate
buffer, pH 7.0 at 20 °C with 10 μM protein.
45
3.3.2 Deoxyferrous and oxyferrous species of WT Mb and H64DOPA Mb
The substrate free ferric (low spin) of WT Mb and H64DOPA Mb have been reduced to
ferrous (high spin) with the Na2S2O4 in 65% (v/v) glycerol under N2 at 4 °C. The deoxyferrous
protein shows a shift in the Soret from 409 to 430 nm and from 411 to 432 nm in WT Mb and
mutant Mb respectively (Figure 3.5). In the visible region, a peak at 558 nm was observed in the
deoxyferrous WT Mb, while in mutant H64DOPA Mb, peaks were observed at 560 nm and 515
nm (118).
Figure 3.5 Deoxyferrous and oxyferrous spectra of WT and H64DOPA mutant Mb. The
absorption spectra of WT Mb deoxyferrous (blue dashed dot line), H64DOPA Mb deoxyferrous
(red dashed line), WT Mb oxyferrous (green dotted line) and H64DOPA Mb oxyferrous (black
solid line) species. The spectra were taken at –35 to –45 °C and the samples were examined in
65% glycerol, 100 mM phosphate buffer at pH 7.0.
In the oxyferous WT Mb and mutant H64DOPA Mb, Soret bands at 416 nm and 418 nm
were observed, respectively. In the visible region, peaks at 581 nm and 540 nm for the WT Mb
and at 580 nm and 536 nm for the mutant H64DOPA have been recorded. Since DOPA can
46
give electrons to the heme and can also participate in hydrogen bonding, the Soret (λmax = 418
nm) has been observed to be sharper in the mutant protein as compared to WT mb. (Figure 3.5)
–
–
3.3.3 CO, NO, CN and N3 adducts of WT and H64DOPA Mb
Figure 3.6 clearly indicates that when CO is bound to the enzyme, the Soret (λmax =
422) became sharper for the ferrous enzyme bonded with CO. As Fe-CO is a linear adduct and
both the π orbitals are empty, it allows dπ-π orbitals to overlap in a perpendicular direction,
making the sharper Soret (104). Under similar conditions addition of NO and O2, (because of
having one or two electrons in their orbitals) reduce the chances of backbonding. These
adducts were stronger in the case of mutant protein because of the formation of extra hydrogen
–
–
bonding. The electron rich ligands such as CN and N3 have similar effects with less intensity
as π backbonding was observed less in these cases (32, 119).
47
48
Figure
3.6
–
Study of WT and H64DOPA Mb ligand complexes. The electronic absorption spectra of (A) CO (B) NO, (C) cyanoferric and (D) N3
complexes of WT Mb and H64DOPA Mb. The spectra were taken at 4 °C and the samples were examined in 100 mM phosphate buffer
at pH 7.0.
3.3.4. Redox potentials of WT and mutant Mb
Both the WT and the mutant H64DOPA Mb were further analyzed to obtain their redox
potential. The electrochemistry of the protein solutions were probed by differential pulse
voltammetry. The redox potential of WT and mutant H64DOPA Mb protein in the absence of
H2O2 WT were found to be the same, i.e., –232 mV, while in the presence of H2O2 it was
observed to be –260 mV and –300 mV, respectively (Figure 3.7 and Table 3.1). This
observation was expected as H2O2 was bonded to the heme center (120). The binding of H2O2
to the heme center replaced the H2O molecule, which was the sixth ligand, and a high spin
ferric state was formed. The difference in the reduction potential also removed the energy
barrier of electron transfer to the heme center (79, 120). Upon addition of H2O2 in the mutant
H64DOPA Mb protein, the reduction potential had decreased, which clearly indicates that an
extra hydrogen bond is formed at the active site of the mutant protein when compared to the
WT Mb active site (121).
Table 3.1 Reduction potential of WT Mb and H64DOPA Mb mutant at pH 7.0 and pH 10.0 in the
presence and absence of H2O2 as obtained from DPV. All protein concentrations were 5 µM
and the potentials were recorded vs. Ag/AgCl (3.5 M KCl).
Reduction Potential, E (V)
Without H2O2
Protein
With 5mM H2O2
pH 7
pH 10
pH 7
pH 10
WT Mb
–0.232
–0.256
–0.260
–0.292
H64DOPA Mb
–0.232
–0.244
–0.300
–0.268
49
50
Figure 3.7 Reduction potential of WT and H64DOPA Mb in presence and absence of H2O2 at pH 7.0 and at pH 10.0. The DPV response
of 5 µM WT and mutant H64DOPA Mb in nitrogen saturated 100 mM phosphate buffer and 10 mM KCl solutions (A and C: at pH 7.0; B
and D: at pH 10.0) in presence (A and B) and absence (C and D) of H2O2 at room temperature conditions. Pulse amplitude = 30 mV,
step size = 4 mV, pulse duration = 0.05 sec and pulse period = 0.20 sec.
3.3.5 Thioanisole sulfoxidation and benzaldehyde oxidation
IV
As reported earlier (98, 122), WT Mb reacts with H2O2 and forms ferryl heme (Fe =O)
equivalent to Cpd II. Cpd I has never been isolated, as it is highly unstable, which makes
peroxidase activity of the WT Mb very low. It was observed that, in the presence of H2O2, WT
Mb converts only 14% (Figure 3.8) thioanisole to sulfoxide with the turnover rate of 0.25 min
–1
which matches with the literature reports (Table 3.2). But under identical conditions, H64DOPA
–1
Mb converts 97% (Figure 3.8) thioanisole to its sulfoxide with a turnover rate of 2.5 min . Thus
the turnover rate of sulfoxidation is 10 times higher in the case of the mutant H64DOPA Mb than
that of WT Mb.
51
52
Figure 3.8 GC analysis of thioanisol and benzaldehyde oxidation. Conversion of thioanisole (a) to its sulfoxidation product (b), (A) WT
Mb, (B) H64DOPA Mb. (C) and (D) show oxidation of benzaldehyde (a) to benzoic acid (b) by WT Mb and H64DOPA Mb respectively.
The reaction mixture contained 5 µM Mb and 5 mM H2O2.
Furthermore, H64DOPA Mb converts 80% (Figure 3.8) benzaldehyde to benzoic acid
with 2.5 min
–1
turnover rate (Table 3.2) while under similar conditions WT Mb converts 2%
(Figure 3.8) benzaldehyde to benzoic acid with 0.01 min
–1
turnover rate (Table 3.2). The
turnover rate of benzaldehyde to benzoic acid was 54 times higher by mutant H64DOPA Mb as
compared to the WT Mb.
Table 3.2 Rate of sulfoxidation of thioanisole reaction (Rxn 1) and benzaldehyde oxidation
reaction (Rxn 2) by WT and H64DOPA mutant Mb.
–1
Protein
Rates (min )
Rxn 1
Rxn 2
WT Mb
0.25
0.05
H64DOPA Mb
2.5
2.67
In addition to the above experiments, ABTS oxidation reaction assays of Mb and its
H64DOPA mutant Mb in the presence of 5 mM H2O2 were also carried out at pH 7.0 and at 4
°C. The kinetic values and turnover factor for the WT and the mutant H64DOPA Mb were
calculated based on the data obtained. The H64DOPA mutant Mb exhibited a Km of 0.67 ×10
3
µM, while that for the WT Mb was 19.3 µM, which corresponded to the literature value. The Vmax
–1
for the WT and the mutant was observed to be 25.6 and 15.2 µM min , respectively (Table 3.3)
(100).
53
Table 3.3 Peroxidase assay of WT and H64DOPA mutant Mb with ABTS in presence of H 2O2.
ABTS oxidation reaction with WT Mb and its H64DOPA mutant Mb in the presence of 5 mM
H2O2. Kinetic values are based on the average of at least 2 determinations and the unit for rate
is turnover per min. Reaction has been done at pH 7.0 and at 4 °C.
Protein
WT Mb
19.3
H64DOPA Mb
[a]
–1
Km (µM)
Vmax (µM min )
[a]
25.6
3
0.67 × 10
[a]
15.2
This value is in agreement with data previously reported
Significantly enough, under similar conditions, epoxidation of styrene did not form
substantial amounts of the epoxide product (result not shown). In order to explain the observed
catalytic reactions and the non-formation of epoxides, we propose that the formation of Cpd I
was achieved through general acid base catalysis. The hydroxyl groups of the DOPA facilitate
the mechanism (Figure 3.9) as they are closer to the heme center and can form a hydrogen
bond with H2O2. The extra hydrogen bond is instrumental in stabilizing the Cpd I species in the
mutant H64DOPA Mb, which further generates the peroxidase activity in the mutant Mb.
54
Figure 3.9 Proposed general acid-base catalytic mechanism for Cpd I formation in H64DOPA
mutant Mb in presence of H2O2.
3.4 Conclusion
In this chapter we report the incorporation of an unnatural amino acid to a specific place
in the active site of Mb. The mutant H64DOPA Mb is an active and stable protein. It could carry
out the oxidation of two substrates, namely, thioanisole and benzaldehyde with 20 to 40 fold
higher catalytic rate in mutant Mb as compared to WT Mb. In addition, our observations support
the idea that the protonation of hydroperoxo species is important for the formation of Cpd I. In
order to exhibit peroxygenase activity in Mb, the mutation of the distal His with an unnatural
amino acid assisted in substrate binding at the active site and enhanced the Cpd I formation. In
addition, the ligand binding studies demonstrate electron deficient ligands exhibit sharper Soret
than the electron rich ones for the mutant protein.
55
CHAPTER 4
FAVOURABLE ACTIVE SITE ORIENTATION IN MYOGLOBIN FOR DIRECT ELECTRON
TRANSFER, ELECTROCATALYSIS AND MICROARRAY INVESTIGATION
4.1. Introduction
Much attention has been focused in recent years on the immobilization of
proteins on electrode surfaces (123-126). Such studies are driven by possible applications in a
broad range of chemical, biological, and medical technologies spanning bioelectrocatalysis,
biomaterials, and biosensors (127-129). Further, immobilized protein assemblies provide a
versatile platform for designing mechanistic studies on heterogeneous electron transfer
between a cofactor and an active center of a protein. However, direct electron transfer (DET)
between proteins and the electrode surface, which can provide new mechanistic insights, is
difficult to achieve, mainly due to the lack of good immobilization techniques. Most of the current
immobilization techniques show denaturation of the protein on adsorption on the electrode
surface. Unfavorable active site orientation of the protein during random adsorption process on
the electrode also hinders DET. Strategies to circumvent these difficulties include the use of
organic mediators (e.g., dye molecules, conducting polymers, etc) and protein films which again
can lead to denaturing of the proteins (130-133). In the past, various immobilization methods
have been employed to immobilize proteins on the electrode surface. For example, noncovalent attachment of proteins (134, 135), entrapment in porous
matrix (136-138),
nanoparticles (139, 140), and sol gels (141-143) have been extensively studied. There are also
some reports of covalent immobilization of proteins on the surface of the electrodes, where
either chemically modified protein (which often leads to protein denaturation due to harsh
chemical treatment) or the functional groups (such as Cys, Arg, Lys, Asp, Glu, etc.) available on
the surface of the proteins have been utilized to covalently attach the protein onto the suitably
modified electrode surface (123, 144-146).
56
Mb is a water soluble heme containing protein, which is present in all mammals and
vertebrates, and has been used extensively for investigating electron transfer and
electrocatalysis with the protein immobilized on an electrode surface. Its main function is to
store oxygen and enhance diffusion of oxygen in the muscle. This globular protein has a single
153 or 154 amino acid chain as in sperm whale, with a molecular weight of ~17 kDa (147). It is
a versatile protein with high tolerance for chemical and mechanical environments and can be
easily expressed and purified in large quantities in E. coli. Another important feature is that it
can accommodate various mutations without any adverse effects on its conformational and
functional properties (19, 24, 148). Mb contains a single iron-porphyrin center (heme b) that can
accommodate ferrous, ferric or ferryl oxidation states within this heme active site moiety. For
these reasons Mb has become an accepted natural model for exploring redox functional
properties and electron transfer reactions in electrochemistry (149-152). For example, coadsorption of Mb with surfactants like sodium dodecyl sulfate or cetyltrimethylammonium
bromide on various electrodes such as glassy carbon, pyrolytic graphite, and platinum was
reported (150). These protein films were utilized for the electrocatalytic reduction of 1,2dibromocyclohexane and trichloroacetic acid (150). In the case of immobilization of Mb, a selfassembled monolayer using L-cysteine (L-Cys) on the modified electrode surface exhibited DET
between the protein active site and the electrode as well as good electrocatalytic activity toward
ascorbic acid oxidation (151).
Even though a wide range of reports are available for immobilization of proteins for
carrying out DET and electrocatalysis with electrodes, a direct comparison involving active site
orientation of monolayer and multilayer covalent attachment of proteins is absent. This is mainly
due to the difficulty to achieve a site-directed covalent tagging of the native conformation of Mb
on the surface with the full control over active site orientation. Furthermore, formation of both a
monolayer as well as a multilayer on the electrode surface is a challenging task. Herein we use
a unique biochemical technique based on the incorporation of an unnatural amino acid in vivo to
57
generate a native Mb protein with additional functional groups (92, 153). NH2Tyr was genetically
encoded with the amber nonsense codon, TAG, by means of a previously evolved orthogonal
tRNACUA/aminoacyl-tRNA synthetase pair that is specific for NH2Tyr (154). The S3NH2Tyr
mutant Mb was immobilized by forming a benzoxazine ring through a Diels-Alder reaction in
buffer (155). This provides an effective strategy for covalent immobilization of a single
monolayer of Mb mutant on a gold electrode surface without impairing its electroactivity (Figure
4.1A). The random multiple covalent attachment of the WT Mb was achieved through 1-ethyl-3(dimethylamino-propyl) carbodiimide (EDC)-catalyzed bioconjugation reaction. The DET
characteristics and electrocatalytic behavior of such random multilayered assembly of
covalently-bound WT Mb counterpart (Figure 4.1B) are compared in this study with a sitedirected monolayer protein on the gold surface.
Due to its ability to perform total genomic analysis, DNA microarray has revolutionized
the genomic biotechnology. Likewise, protein microarray has the potential to generate advanced
bioinformatics platforms for proteomics and it could transform the field of drug discovery,
medical diagnostics, and biochemical analysis. Development of functional protein microarray is
more difficult than DNA, peptide, and antibody microarrays. Strong attachment of proteins on
surface, native protein conformation, homogeneous protein monolayer, control over active site
orientation, and retention of protein activity have been the major problem while dealing with
protein microarray.
In order to understand the role of the amino acid residues which are located far from the
active site such Ser 3 in ligand adduct formation, we have extensively formed and studied
–
–
ligand adducts with various ligands such as CO, NO, O 2, CN , and N3 in this chapter.
58
Figure 4.1 Graphical representation of WT and S3NH2Tyr mutant Mb attachment on modified
gold electrodes. (A) Ser 3 replaced with NH2Tyr Mb, with its heme active site, which leads to
site-directed covalent attachment, with full control over active site orientation of S3NH 2Tyr Mb
mutant on the modified gold electrode; (B) WT Mb (PDB: 1MBO) with all surface-exposed
amino acids are shown, aspartate and glutamate in red color (with acidic side-chains) and
arginine and lysine in blue color (with basic side-chains) as points of immobilization for the
bioconjugated crosslinking of the WT Mb proteins. Orange ellipses represent random active site
orientation (with limited substrate accessibility) of bioconjugated protein cluster.
59
4.2 Experimental procedures
4.2.1 Chemicals
L-Cys, tetraethyleneglycol diacrylate (TEGDA), sodium periodate (NaIO 4) and
N,N-di-iso-propylethyl amine (DIPEA) were obtained from Sigma-Aldrich USA and were used
without further purification. All chemicals were of analytical grade.
4.2.2 Preparation of WT Mb and S3NH2Tyr mutant Mb constructs
The WT Mb expression construct (pBAD with ampicillin resistant marker), S3NH 2Tyr Mb
gene expression vector (pBAD with kanamycin resistant marker) and aminoacyl tRNA
synthetase (aatRNA S) with tetracycline resistant marker vector (PAC) were a gift from Dr.
Peter Schultz (The Scripps Research Institute, La Jolla, CA USA). The S3NH2Tyr Mb
expression vector and tRNA synthetase plasmid were co-transformed in DH10B E. coli cells.
The double antibiotic resistant colonies were picked, grown, and the cell stocks were stored at –
80 °C prior to protein expression.
4.2.3 WT Mb and S3NH2Tyr Mb protein purification and analysis
The S3NH2Tyr Mb was expressed in DH10B and was grown in GMML containing
ampicillin and tetracycline antibiotics and the unnatural amino acid, NH2Tyr. The WT and
S3NH2Tyr Mb mutated proteins were expressed, purified, and analyzed as described in chapter
2 (Figure 4.2). The mass of mutant S3NH2Tyr Mb protein mass was analyzed by MALDI-TOF
and was observed to be 18446 Da (Figure 4.3), which matched with the theoretically calculated
mass.
60
1
2
3
150 kDa
20 kDa
15 kDa
Figure 4.2 Coomassie-stained SDS-PAGE analysis of S3NH2Tyr Mb and WT Mb proteins.
Lane 1, expressed WT Mb; Lane 2, expressed S3NH2Tyr Mb mutant in presence of 1 mM
NH2Tyr; Lane 3, molecular weight standards as indicated in kDa unit.
61
62
Figure 4.3 Electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) analysis of the incorporation of S3NH2Tyr into S3
(TAG) Mb mutant showing a mass of 18446 Da (calculated mass, 18446 Da) for S3NH 2Tyr Mb.
4.2.4 UV-vis spectroscopy
UV-visible spectra were acquired on a Cary 50 Bio UV-visible spectrophotometer. The
concentrations of the WT and mutant Mb were determined from the absorption spectrum of the
protein, using an extinction coefficient of 170 mM
–1
cm
–1
at 410 nm.
4.2.5 AFM measurements
The experiments were performed with a Veeco MultiMode V SPM instrument. The
cantilevers used in this experiment were made of P-doped n-type Si. The cantilever was
oscillated in the constant excitation mode. Tapping mode Atomic force microscopy (AFM)
images were acquired in the constant frequency shift mode using frequency modulation (FM)
detection. Data analysis was done with the Nanoscope software 7.0.
63
4.2.6 Ligands adduct formation
The protein samples containing 65% glycerol (v/v) were taken in airtight cuvettes
containing 0.1 M potassium phosphate buffer pH 7.0 and were degassed with the N 2 gas for ~2
hr at 4 °C and then reduced with minimal amount of Na2S2O4 (in µL) from the stock of 20
mg/mL. In a chest freezer at –35 to –45 °C containing the above sample, pre-cooled O2 gas
was bubbled for 60 s and the UV-visible spectra were recorded. The ferrous-CO adducts were
generated by gentle bubbling of CO in the deoxyferrous enzymes under N 2 for 30 s at 4 °C in
0.1 M potassium phosphate buffer pH 7.0 while ferrous-NO adducts were generated by µL
addition of buffer saturated with NO gas. Ferric azide and cyanoferric adducts were prepared by
addition of minimal amount of NaN3 and potassium cyanide solution from their respective stocks
4.2.7 Preparation of microarray
Clean glass slides were silanized, extended, and acryloyl functionalized. To obtain a
pegylated linker, extended acryloyl glass slides were obtained from amine-coated glass slide by
treating with 1 mM diisopropylethylamine followed by reaction with 1 mM tetra (ethylene glycol)
diacrylate in dry dimethylformamide (DMF). In the presence of 100 µM NaIO4, at room
temperature, the mutant protein (3NH2Tyr incorporated) was attached to the pegylated acryloyl
glass slide, for 2 hr The slides were washed in 100 mM potassium phosphate and water for 2
hr. At room temperature, carbonic anhydrase (CA) catalytic assay was carried out using pnitrophenyl acetate (0.1 mM) in 15 mM Tris buffer (pH 7.6). Using Veeco Multimode V SPM
instrument, AFM measurements were recorded for glass chip-bound proteins. To prepare
microarrays, proteins were mixed with 0.1 mM NaIO4 for 10 min prior to printing in a solution of
100 mM potassium phosphate buffer (pH 6.0). BioRobotics MicroGrid II 600 with an internal
chamber humidity of 70% was used for printing. After printing, before being transferred to
phosphate buffered saline (PBS) buffer (pH 7.5) for 48 hr of washing, proteins were allowed to
bind for 4 hr at room temperature. With a 1:500 dilution in PBS buffer containing 1% bovine
64
serum albumin (BSA) and 0.1% Tween 20 for 1 hr, binding of HiLyte 647 fluorophoretagged
anti-6xHis tag rabbit Immunoglobulin G (IgG) was performed, followed by washing in PBS buffer
containing 0.1% Tween 20 for 5 min twice. Using Coomassie bromophenol blue staining
solution, Coomassie staining was done for 10 min, followed by 24 hr of washing in water.
4.2.8 Electrochemical instrumentation and procedures
Cyclic voltammetry (CV) was carried out using a three-electrode cell at 22 °C on a
CHI720C electrochemical analyzer (CH Instruments, Austin, TX). The working electrode was
2
polycrystalline gold (1 × 1 cm in area, Alfa Aesar), the counter electrode was a platinum wire,
and the reference electrode was Ag/AgCl (3.5 M KCl). The counter electrode and reference
electrode were obtained from CH Instruments. All potential values below are reported with
respect to the Ag/AgCl (3.5 M KCl) reference electrode. The working electrode surface was first
polished on microcloth (Buehler No. 40-7212) with alumina slurry suspension (0.05 μm), then
sonicated in ethanol, and finally rinsed thoroughly with Milli-Q water. The electrolyte solution
was 0.1 M potassium phosphate buffer with 10 mM KCl solutions at pH 7.0. The solutions were
deoxygenated by bubbling nitrogen prior to each experiment. The CV measurements were
recorded at potential scan rates ranging from 0.05 Vsec
–1
–1
to 0.5 Vsec .
4.2.9 Electrode modification procédures
4.2.9.1 Au/L-Cys/WT Mb
The freshly polished and clean gold electrode was immersed in a 2 mM ethanol
solution of L-Cys for 2 hr. It was then washed thoroughly and immersed in the 5 µM WT Mb
solution in MES buffer at pH 5.0. To this solution, a freshly prepared aqueous solution of EDC
was added and the electrode was incubated in this protein medium (2 mL) overnight at 4 °C.
The electrode was then removed, rinsed thoroughly with Milli-Q water, and allowed to dry under
nitrogen flow before use.
65
4.2.9.2 Au/L-Cys/TEGDA/ S3NH2Tyr Mb
The freshly polished and clean gold electrode was immediately immersed in a 2 mM
ethanolic solution of L-Cys for 2 hr. It was then washed thoroughly and immersed in 20 mM
N,N-di-iso-propyl-ethylamine in dimethylformamide (2 mL). To this solution, 10 mM
tetraethylene glycol diacrylate was added and the electrode was kept in it for 3 hr. The
S3NH2Tyr Mb was added in phosphate buffer solution and an aqueous solution of 100 µM
NaIO4 was added to it. The chemically-modified gold electrode was washed thoroughly to
remove any traces of unreacted molecules and incubated in the mutant protein solution
overnight at 4 °C. The electrode was then removed, rinsed with Milli-Q water thoroughly for 1 hr,
and allowed to dry under nitrogen flow before use.
4.2.9.3 Electrocatalysis
Electrocatalysis was carried out in 8 mL of potassium phosphate buffer (100 mM at pH
7.0) containing 5 mM thioanisole dissolved in 30% t-butanol. O2 was bubbled into the solution
throughout the reaction time. Bulk electrolysis was then performed for 2 hr at a constant
potential of –0.50 V. On completion of the catalytic cycle, the organic products were extracted in
dichloromethane (3 × 2 mL). The solution was concentrated, and the products were analyzed by
GC.
4.3. Results and discussion
4.3.1 Monolayer vs. multilayer covalent immobilization of Mb on Au
A central challenge in designing an Mb array for DET is that the electro-active heme
centers are randomly oriented within the protein matrix. Further, conversion of heme protein into
a protein film on the electrode surface may also lead to heme bleaching and protein denaturing
(156-158). To address these challenges, a mild and native immobilization strategy was
66
developed. In this study, site-directed mutagenesis was used to incorporate an unnatural amino
acid (Scheme 4.1) to immobilize Mb on the gold electrode as a monolayer using a unique DielsAlder reaction in water or buffer at room temperature or 4 °C (155). The Ser 3 group replaced
with an NH2Tyr of the Mb provides an ideal basis for this reaction as it is solvent exposed at that
position as well as it orients the active site away from the surface.
67
68
Scheme 4.1 Schematic representation of Au electrode modification for monolayered- immobilization of S3NH2Tyr Mb mutant on the Au
surface; see text for details.
Incorporation of NH2Tyr into the protein was carried out by using orthogonal
tRNACUA/aminoacyl-tRNA synthetase pairs in bacteria. The mutant Mb was analyzed by SDSPAGE (Figure 4.2) and MALDI-TOF (Figure 4.3). Surface modification of the gold electrode was
initiated by modifying it first with an amine-functionalized thiol surface (Scheme 4.1). This
chemically-modified surface was reacted with tetraethyleneglycol diacrylate to obtain the
acroylyl moiety on the surface. Note that the tetraethyleneglycol portion serves to also provide a
lipid-like environment to the covalently attached protein, thus protecting its native conformation
on the surface. In the presence of mild oxidant NaIO4, the NH2Tyr on the surface of the
S3NH2Tyr Mb mutant was easily oxidized to form the o-iminoquinone. This intermediate, in turn,
underwent a Diels-Alder cyclo-addition reaction with suitable alkenes to form a benzoxazine
moiety. The entire process of the covalent attachment of mutant S3NH2Tyr Mb onto the acryloyl
derivatized gold electrode surface was carried out under ambient or 4 °C temperature. The
mutation at Ser 3 with the unnatural amino acid, NH2Tyr, enables full control of the orientation of
the active site of the protein, when covalently immobilized onto the modified gold electrode
(Figure 4.1A). Thus the catalytic and electro-activity of the heme can be easily achieved even
when the mutant protein is covalently attached to the surface of the electrode.
To immobilize the WT Mb as a multilayer, a different strategy had to be employed
(Figure 4.1B). The presence of carboxylic acid and amine groups on the protein surface can be
utilized to form covalent cross linking bonds between these groups and also with suitably
modified, i.e., amine-functionalized, gold surfaces. This bioconjugation reaction is facilitated by
the amino acids with carboxylate groups, such as aspartate (20, 27, 44, 122, 126, and 141
positions), glutamate (4, 6, 18, 38, 41, 52, 54, 59, 83, 136, and 148), and amine groups, such as
arginine (31, 45, 118, and 139) and lysine (16, 34, 42, 47, 50, 56, 62, 63, 77, 78, 79, 96, 98,
102, 133, 140, 145, and 147 positions). Compounds such as 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide possessing the carbodiimide functional group can be successfully used for
crosslinking these amino acids to form amide linkages (159, 160). The EDC reaction chemistry
69
(Scheme 4.2) was exploited in this study to covalently bind the WT Mb to the aminefunctionalized gold surface, as well as to bioconjugate the Mb as a polymer to generate Mb
multilayer with disoriented active sites. The random orientation of the active sites hinders
exposure of some of them to the solvent phase for catalytic or electrochemical activity.
Scheme 4.2 Schematic representation of multilayered WT Mb immobilization on the modified
gold electrode surface using EDC catalyzed bioconjugation reaction. Inset: Graphical
representation of WT Mb (PDB: 1MBO) with all surface-exposed amino acids. The aspartate
and glutamate are represented in red color and arginine and lysine residues in blue color.
70
The surface topography of the covalently attached Mb assemblies were probed
by AFM. Figure 2 compares the AFM images for a chemically-modified amine-functionalized Au
surface before (Figure 4.2a) and after S3NH2Tyr Mb mutant (Figure 4.2b) or WT Mb (Figure
4.2c) covalent attachment. While the pristine surface before protein attachment was featureless
(Figure 4.2 a), an average feature height of ~5 nm was observed in the case of the mutant
protein monolayer, which correlated to the actual size of the protein based on the X-ray
crystallographic structure of Mb (Figure 4.2b). On the other hand, covalently-attached WT Mb
was noticeably rougher, with an average feature height of ~175 nm (Figure 4.2c). As a control
experiment, the WT Mb was also immobilized on the acryolyl-derivatized gold electrode surface
using NaIO4 under the same reaction conditions as the S3NH2Tyr Mb mutant. However, as
expected by the absence of the o-iminoquinone moiety, the WT Mb did not form a monolayer on
the modified surface (Figure 4.3) compared to the genetically modified S3NH2Tyr Mb mutant
(Figure 4.2b). Since the covalent linkage is formed only between the o-iminoquinone moiety of
the protein and the acryloyl group on the modified electrode surface by the Diels-Alder reaction,
the WT Mb does not get covalently immobilized on the electrode surface.
71
72
Figure 4.4 Tapping mode AFM topographic images for (a) chemically modified Au surface, (b) with immobilized S3NH2Tyr Mb mutant
and (c) and with WT Mb on the surface.
4.3.2 Electrochemical characterization
Cyclic voltammetry (CV) was used to study the DET electrochemistry of immobilized Mb
on the Au electrodes. In this study, though L-Cys and cysteamine were used to modify the gold
electrode surface (Figure 4.1 and Table 4.1), cysteamine-bound Mb failed to display reversible
redox properties in CV measurements (data not shown). Both WT Mb and the S3NH2Tyr Mb
mutant were immobilized on L-Cys functionalized surface to obtain the CV data. The results
show well defined redox electrochemistry when examined by CV from scan rates 0.05 Vsec
73
0.5 Vsec
–1
(Figure 4.4).
–1
to
Immobilized Mb on modified Au electrode (CV)
-6e-5
Au-LCys
Au-LCys-WT Mb
Au-LCys-TEGDA-S3NH2Tyr Mb
Current /A
-4e-5
-2e-5
0
2e-5
4e-5
0.4
0.2
0.0
-0.2
-0.4
Potential /V vs. Ag/AgCl
Figure 4.5 CV response of L-Cys-modified, WT Mb and S3NH2Tyr Mb mutant immobilized gold
electrode in nitrogen-purged 100 mM phosphate buffer with 10 mM KCl (pH 7.0) at room
–1
temperature at potential scan rate = 0.1 Vsec .
73
74
74
75
Figure 4.6 CV and peak current response of WT and mutant Mb. Mutant (A) S3NH2Tyr Mb and
(C) WT Mb immobilized gold electrode in nitrogen-purged 100 mM phosphate buffer with 10 mM
KCl (pH 7.0) at room temperature. Potential scan rate ranges from 0.05 to 0.5 V/sec. Plot of
peak current vs scan rate for mutant S3NH2Tyr Mb (B) and WT Mb (D) immobilized on L-Cys
modified gold electrode in nitrogen-purged 100 mM phosphate buffer with 10 mM KCl (pH 7.0)
–1
at room temperature. Potential scan rate ranges from 0.05 to 0.50 Vsec
75
It was observed that the peak currents were directly proportional to the scan rates,
which signals that the proteins were immobilized to the electrode surface (Figure 4.4). The peak
current, Ip for a surface confined reactant is given by Eq. 1 (161).
Ip 
n2 F 2
A
4 RT
(Eq. 1)
where, n = number of electrons transferred, F = Faraday constant, R = universal gas
constant, T = absolute temperature in Kelvin (T = 295 K), A = surface area of the electrode, Γ =
surface coverage or the concentration of the redox-sensitive covalently immobilized Mb protein
76
2
2 2
in mol/cm , υ = scan rate. From equation 1, the slopes of the plots were given by (n F /4RT) AΓ
and substituting the known values, such as the geometric area of the electrode surface (A = 2.0
2
cm ) and the other constants, the estimated values of the number of molecules of the enzyme
associated with the electrode surface (Γ) were 1.54 × 10
–10
mol cm
–2
for the S3NH2Tyr Mb
mutant protein immobilized on the modified gold electrode. Similarly, for the WT Mb protein, Γ
was 1.29 × 10
–10
mol cm
–2
on the modified gold electrode through the bioconjugation reaction. A
comparison of the surface coverage of the monolayered S3NH2Tyr Mb mutant and the
multilayered WT Mb indicated that random orientation of the active sites in the multilayered WT
Mb leads to lesser number of redox-sensitive proteins on the surface. In contrast, the
S3NH2Tyr Mb mutant allows proper orientation of the active site, thereby exhibiting
homogenously distributed redox-sensitive proteins on the electrode surface.
To investigate the influence of pH on the electron transfer between the active site of the
immobilized protein and the gold electrode, CV was carried out in solutions of different pH
ranging from 5 to 9. It was observed that with increasing pH, the reduction potential of the Fecenter exhibited a linear negative shift (Figure 4.5). These data support the notion that
denaturation of the Mb protein did not occur within the pH range of 5 to 9.
76
Table 4.1 Comparison of heterogeneous electron transfer rates of Mb on gold electrodes.
Surface modification used
Approximate
Heterogeneous
theoretical length of
electron
anchoring tether (Å);
Reference
transfer
–1
rate, ks (s )
molecule used
77
5.35
Homocysteine
4.75
L-Cys
4.75
L-Cys
0.93
(162)
1.66
(151)
2.26
This work
1.25
This work
26.66
L-Cys-linked with
tetraethylglycol
diacrylate
77
Influence of pH on immobilized WT Mb and S3NH2Tyr Mb
0.20
Potential/ V vs Ag/AgCl
WT Mb
S3NH2Tyr Mb
0.15
0.10
0.05
78
0.00
5
6
7
8
9
pH
Figure 4.7 Plot of peak potentials vs pH for WT Mb and S3NH2Tyr Mb. Proteins are immobilized
on L-Cys-modified gold electrode in nitrogen purged 100 mM phosphate buffer with 10 mM KCl
solutions (pH 7.0) at room temperature.
A wide variety of electrode systems have been used to explore the electrochemistry of
Mb. A comparison of some of these systems is tabulated below (Table 4.2). Similar cathodic
and anodic peak separation due to slow DET has also been reported in earlier literature. In this
study, when employing L-Cys for the gold electrode surface modification, the electrochemical
behaviour of the WT Mb and the S3NH2Tyr Mb mutant was significantly different. Since the
electrochemical reaction was quasi-reversible in nature, the formal reduction potential E°′ was
estimated from the midpoint potential E1/2 [obtained from (Epc + Epa)/2]. The WT Mb and the
immobilized S3NH2Tyr Mb mutant gave values of E1/2 between 0.080 V and 0.103 V with
respect to Ag/AgCl (3.5 M KCl) reference, respectively. These midpoint potentials of the heme
center of the WT Mb are in accord with reported values in the literature.(151, 162) The rather
large separation of the cathodic and anodic peaks indicated that DET to the protein was quite
slow.
78
Table 4.2 Comparison of direct electrochemistry of Mb on various electrode systems.
Electrode
E1/2 values
(V)
Gold/L-Cys-linked with
[a]
∆Ep
Reference
(V)
0.103
~0.150
This work
Gold/L-Cys-WT Mb
0.080
~0.091
This work
Gold/L-Cys-WT Mb
0.086
Gold/Homocysteine-WT Mb
-0.012
~0.246
(162)
Gold nanopyramids-WT Mb
0.210
~0.060
(163)
Gold/azide-terminated alkane thiols-
-0.335
tetraethylglycol diacrylate and
S3NH2Tyr Mb mutant
(151)
(123)
79
WT Mb
NAF/Mb/IL/PtNPs/MWCNTs/GCE
[b]
[c]
~0.140
(134)
Mb-CA/GCE
-0.281
~0.102
(164)
[d]
Mb/DDAB–HIMIMPF6
-0.261
~0.044
(165)
[a]
All literature values for E1/2 have been converted to Ag/AgCl (3.5 M KCl) reference electrode,
[b]
for clarity; NAF/Mb/IL/PtNPs/MWCNTs/GCE = Glassy carbon electrode modified with multiwalled carbon nanotubes (MWCNTs), followed by platinum nanoparticles (PtNPs), ionic liquids
[c]
(IL) and Nafion (NAF) with WT Mb; Mb-CA/GCE = Glassy carbon electrode modified with
[d]
calcium alginate and WT Mb; Mb/DDAB–HIMIMPF6 = Glassy carbon electrode modified with
didodecyldimethylammonium
bromide
(DDAB),
1-hexyl-3-methylimidazolium
hexafluorophosphate (HIMIMPF6) and WT Mb.
The rate of heterogeneous electron transfer between the electrode and the immobilized
protein was calculated using Laviron’s method (161). The value of the transfer coefficient ()
was determined using Eq. 2:
E pc  E 0 
E pa
 nF
RT
ln 
nF  RTk s
  0
 nF
RT
   E 
ln 
nF  RTk s
 
 (1   )nF
RT
E 
ln 
(1   )nF 
RTk s
0

RT
 
ln  
 nF
  0
 (1   )nF
RT
   E 
ln 
(
1


)
nF
 
 RTk s

RT
 
ln  
(
1

 )nF

(Eq. 2)
where Eº is the standard potential, ks is the heterogeneous electron transfer rate
constant, and n = 1. The potentials for the cathodic (Epc) and anodic (Epa) peaks were plotted
against ln (v) and on analyzing the ratio of the slopes of the plots, an average value of the
79
transfer coefficient, , was found to be 0.54 for the S3NH2Tyr Mb mutant, and 0.51 for the WT
Mb immobilized electrode. The heterogeneous electron transfer rate constants for immobilized
WT Mb and the S3NH2Tyr Mb mutant were determined from the following equation (Eq. 3) at
different scan rates:
 RT   (1   )nFE p
log k s   log(1   )  (1   ) log   log

2.3RT
 nF 
2.3RT 
2.3RT
 RT  
E p 
log
  log(1   )  (1   ) log   log k s  log
  
nF (1   ) 
 nF   nF (1   )
(Eq. 3)
80
where ΔEp is the peak potential separation. Using the respective values of α and from
the plot of ΔEp vs log v (see Supporting Information Figure SI-6), ks was estimated to be 1.3
sec
–1
and 2.3 sec
–1
respectively for the immobilized S3NH2Tyr Mb mutant and WT Mb on the
chemically modified gold electrode. The S3NH2Tyr Mb mutant monolayer has slightly slower
electron transfer kinetics than the multilayered WT Mb presumably due to the varying length of
the anchoring tethers in the two cases (see Table 4.1).
Table 4.1 compares surface modification chemistries based on anchoring molecules
used for the immobilization of Mb on gold electrode surfaces and their associated ks values
reported in two previous studies (151, 162) with the results from this study. It is observed that
random immobilization of WT Mb leads to a decrease in the DET from 2.26 sec
–1
to 0.93 sec
–1
as the linker molecule length is increased from L-Cys (~4.75 Å) to homocysteine (~5.35 Å).
Though the theoretical length of the tether is longer (~26 Å) in the case of S3NH 2Tyr Mb mutant,
it facilitates faster electron transfer compared to homocysteine. This could be due to the
tetraethylene glycol-linker providing a through-bond electron transfer environment from the
electrode to the redox-sensitive heme center of the Mb (164, 166).
80
-2e-4
Au-LCys
Au-LCys-WT Mb
Au-LCys-TEGDA-S3NH2Tyr Mb
Current /A
-2e-4
Au-Lcys-Hemin
Hemin (5M) in solution
-1e-4
-5e-5
81
0
5e-5
0.4
0.2
0.0
-0.2
-0.4
Potential /V vs. Ag/AgCl
Figure 4.8 Comparision of immobilized Mb with hemin on modified gold electrode. CV response
of hemin in solution, L-Cys modified, hemin immobilized, WT Mb and S3NH2Tyr Mb mutant
immobilized gold electrode in nitrogen-purged 100 mM phosphate buffer with 10 mM KCl (pH
–1
7.0) at room temperature and at a potential scan rate of 0.1 Vsec .
An important aspect of the heme bleaching from the protein immobilized electrode
suface was carefully investigated for both the S3NH2Tyr Mb mutant and the WT Mb protein. The
redox responses of the free hemin (5 µM) in solution and when immobilized on the aminefunctionalized gold electrode surface were observed to be quite different from those of the
S3NH2Tyr Mb and the WT Mb-bound surface (Figure 4.6). From these data, it can be inferred
that heme bleaching was absent from both the S3NH2Tyr Mb mutant and the WT Mb
immobilized electrodes under the above mentioned experimental conditions.
81
4.3.3 Catalytic studies
Mb has often been utilized to explore the monooxygenase activity of heme proteins in
presence of H2O2, in order to investigate the P450 catalytic shunt-pathway. A common
substrate used in such studies is thianisole, which can be catalytically oxidized to its sulfoxide
by high valent ferryl-porphyrin-π-cation radical species (also known as Cpd I) of Mb. Hence,
electrocatalytic conversion of thioanisole to its oxidized form provides an ideal comparison
platform for the active site orientation in these two modes of immobilization. Dioxygen was
bubbled into the solution and bulk electrolysis was carried out for 2 hr with the covalently
immobilized S3NH2Tyr Mb mutant and the WT Mb (Table 4.3).
82
Table 4.3 Electrocatalytic conversion of thioanisole to its oxidized form and turnover frequency
(TOF) using bare, multilayered WT Mb-immobilized and monolayered S3NH2Tyr Mbimmobilized Au electrodes.
Unreacted
Thioanisole
thioanisole (%)
sulfoxide
(%)
TOF for the oxidized
–1
products (sec )
Bare electrode
86
14
WT Mb
13
87
1.9 × 10
3
S3NH2Tyr Mb
19
81
1.5 × 10
3
The GC results demonstrated that 87% conversion of the thioanisole to its oxidized
form had taken place for the WT Mb immobilized electrode, while for the S3NH 2Tyr Mb mutant,
81% conversion was obtained. A reduced reaction time (1 hr) did not yield sufficient product
formation in comparison to that of 2 hr catalytic reaction. A control experiment was also carried
out for the electrocatalytic oxidation of thioanisole in the presence of bare Au. Only 14%
conversion of the thioanisole to its sulfoxide was observed in this case after 2 hr. Considering
82
the amount of electro-active protein covalently attached onto the surface from Eq. 1, the
3
–1
turnover frequency (TOF) for the WT Mb was 1.9 × 10 sec . Similarly, the TOF for the
3
–1
S3NH2Tyr Mb mutant was 1.5 × 10 sec . Significantly, the comparable catalytic activity
observed for the multilayered WT Mb and the monolayered S3NH 2Tyr Mb mutant reiterates the
importance of orientation of the active sites of both the proteins. The random orientation of
active site in the multilayered protein leads to loss of catalytic activity (considering the greater
number of proteins on the surface), than the monolayered mutant S3NH2Tyr Mb. Proper
orientation of the heme active site in the monolayered S3NH 2Tyr Mb mutant assisted the
catalytic and redox-activity of the protein, when covalently immobilized onto the modified gold
83
electrode surface.
4.3.4 Microarray studies
Functionalization of glass surface with polyethylene glycol (PEG) was done by using the
methods as previously described (167). To attach S3NH2Tyr Mb on PEG linker, Diels–Alder
reaction was used in mild conditions, such as in water (168). Since the protein will bind only
through its unnatural amino acid, we have control of the active site in mutants. The immobilized
protein on glass surface was analyzed by AFM, in which monomeric S3NH2Tyr Mb showed
expected dimensions of height and width (Figure 4.3) based on crystal structure.
4.3.4.1 Preparation of functional protein microarrays
A library of five proteins was printed on a single slide, to determine if our technique can
be used for microarray studies (Figure 4.7). In order to understand orientation of the protein
arrays and selective protein-protein interaction, we allowed HiLyte 647 fluorophore-conjugated
anti-6×His tag rabbit IgG to bind with the proteins that were spotted on the surface. S3NH2Tyr
Mb and T365 (3NH2Tyr P450 BM3) (Figure 4.7) were the only two proteins that show surfaceexposed 6×His-tag chains when we observed the site of immobilization. Thus these were the
83
two proteins that showed the direct and efficient binding with IgG. However, all other proteins
except the control were attached to the surface after the tagging reaction, as they were visible in
coomassie blue staining. This experiment confirmed the interaction between IgG and the protein
could be generated in a specific manner as well as control over the orientation of the protein.
84
84
85
Figure 4.9 A microarray of five proteins on a single slide displays. (a) T365 (3NH2Tyr) P450 BM3. (b) P450 BM3 control without NaIO4
(control shows that printed proteins were detached from the surface after washing). (c) T73 (3NH2Tyr) CA. (d) D125 (3NH2Tyr) CA. (e)
D133 (3NH2Tyr) EGFP. (f) S3 (3NH2Tyr) Mb. Column 2 displays an image of excitation at 649 nm and emission at 666 nm.
86
87
Figure 4.10 Spectroscopic characterization of high spin, low spin and ligand complexes of WT and mutant Mb. Electronic absorption
–
spectra of (A) ferric and ferrous, (B) CO, (C) NO, (D) oxyferrous, (E) cyanoferric and (F) N3 complexes of WT Mb and S3NH2Tyr Mb.
The spectra were taken at 4 °C and the samples were examined in 100mM phosphate buffer at pH 7.0.
4.3.5 Ligand binding studies
To understand the effect of different ligands upon replacement of residues which are
away from the active heme center of the protein, we have replaced the serine 3 (S3) with
3NH2Tyr, a redox amino acid. Figure 4.8 clearly indicates that S3NH2Tyr Mb protein behaves
more or less similar to that of WT Mb with respect to ligand-binding studies. Since position 3 is
quite distant from the heme center, it has almost no role in influencing the electronic density of
the Asheme center. As position 3 is quite away, it did not play a major role in determining the
ligand-metal charge transfer, metal-ligand charge transfer, and in π backbonding (104, 119).
We did not observe significant difference in the electron absorption spectra of mutant protein as
compared to WT Mb (15, 31).
4.4. Conclusion
Though, previous research groups have observed facile electron transfer using specific
organic molecules as promoters, no prior studies were reported using mutant protein that
utilizes its unnatural amino acid moiety to specifically immobilize onto the electrode surface. In
this chapter, I report monolayer and multilayer immobilization of Mb on to chemically-modified
gold electrode surfaces using two unique immobilization techniques as described above.
Further, electrocatalysis of thioanisole oxidation was carried out successfully with both the
protein immobilized electrode cases, albeit at variant facility. These results underline that the
S3NH2Tyr Mb mutant monolayer exhibits well-defined catalytic and electro-activity because of
proper orientation of its heme active site, in comparison to the random multilayered WT Mb
case.
To investigate protein-protein interactions, five separate proteins P450 BM3, Mb,
enhanced green fluorescent protein (EGFP), HRP, and CA have been covalently attached onto
a solid support and analyzed. Functional protein microarray concept development was
confirmed by a catalytic activity designed assay using spectroscopic characterization. This
88
concept developed microarray will bring novelty in the field of drug discovery and biomedical
and proteomic research. The ligand binding studies showed that the S3 position being quite
away from the active heme center, does not affect the metal-ligand binding to a greater extent.
89
Chapter 5
ELECTRONIC NATURE INVESTIGATION OF HEME CENTER BY MODIFYING PROXIMAL
AND DISTAL ACTIVE SITE ENVIRONMENT OF SPERM WHALE MYOGLOBIN WITH
ELECTRON RICH RESIDUES
5.1 Introduction
The heme group is the most common prosthetic group in the metalloproteins (1). The
iron center, porphyrin, and axial ligand constitute the three covalently linked integral
components of a heme protein. In the heme proteins, a wide variety of porphyrines are present
such as heme a, heme b, heme c, heme d, heme d1, heme o, heme P460, chloroheme, and
siroheme. Among the globins and cytochrome, heme b and heme c are the most common
(Figure 5.1). A widely studied example is Mb (sperm whale), a small protein with 153 amino
acids and molecular weight of 17.5 kD, with oxygen storage as its main function in vertebrates
(2, 14, 169, 170). The presence of different varieties of amino acids around the active site has
always been a matter of interest for the understanding of heme ligand interaction and its
catalytic activity (169, 170). With the tool of site directed mutagenesis divers, residues can be
introduced to the proteins, and complexes formed with different ligands can be investigated.
Among the heme protein, various axial ligands such as His, Cys, Met, Tyr, and Lys are found
and they play a crucial role in their properties (14, 22). His present at 64 position plays a major
role in stabilizing the ligand adduct by forming bonds to the ligands attached to the heme
moiety. It has been proposed that with open and closed confirmation distal His can act like a
gate for ligand adducts (1, 15, 29, 171, 172).
Figure 5.2 B shows the details of amino acid residues around the heme center in sperm
whale Mb. It is also believed that the size of certain residues also contributes significantly to
determining the heme ligand complex stability. But the role played by the electronic nature
around heme center is not clearly understood. The introduction of noncannonical amino acids
with electron rich functional groups such as pNO2Phe, will allow us to investigate the electronic
–
–
nature of heme center, and its effect on ligand (CO, NO, O 2, CN , and N3 ) binding to a greater
90
–
−
extent. For our further understanding of interaction of CO, NO, O2, N3 , and CN with Mb as a
model for heme protein, I have created mutations at its distal and proximal sites (15). By site
directed mutagenesis we have replaced the His with pNO2Phe at His 64 and His 93 positions
and compared the interaction of different ligands with these mutants, as well as to WT Mb (15,
88).
Figure 5.1 Structures of heme b and heme c.
91
Figure 5.2 Schematic representations of WT and mutants Mb. (A) Overall structure of WT Mb.
(B) enlarged active site of WT Mb, (C) structure of H64pNO2Phe and (D) structure of
H93pNO2Phe mutants.
5.2 Experimental procedure
5.2.1 Chemicals
The gasses (O2, CO, and N2) have been purchased from Air Liquide, USA while
pNO2Phe, sodium azide, potassium cyanide, and Na2S2O4 were purchased from Sigma-Aldrich
92
USA. All chemicals were of analytical grade and have been used without any further purification
(173).
5.2.2 Preparation of WT, H64pNO2Phe and H93pNO2Phe Mb mutant constructs
The WT Mb expression constructs H64pNO2Phe (TAG) and H93pNO2Phe (TAG) Mb
gene expression vector were prepared by Dr. Roshan Perera and aminoacyl tRNA synthetase
(aatRNA S) plasmids were donated by Dr. Peter Schultz (The Scripps Research Institute, La
Jolla, CA USA). The H64pNO2Phe and H93pNO2Phe Mb expression vector were cotransformed with tRNA synthetase separately in DH10B E.coli. The bacterial colonies which are
double antibiotic (ampicillin and tetracycline) resistant were picked, grown, and the cell stocks
were stored at –80 °C prior to the start of bacterial cell culture for protein purification (15).
5.2.3 Purification and analysis of WT Mb, H64pNO2Phe and H93pNO2Phe Mb
The H64pNO2Phe Mb and H93pNO2Phe Mb were expressed in E. coli bacteria which
were grown in GMML containing 5% LB media and suitable antibiotics (ampicillin and
tetracycline). Both the WT and mutated (H64pNO2Phe and H93pNO2Phe) Mb proteins were
induced, expressed, purified, and analyzed as described in chapter 2.
93
Figure 5.3 Coomassie-stained SDS-PAGE analysis of expression of WT, H64pNO2Phe and
H93pNO2Phe mutants Mb. Lane 1 expressed WT Mb, Lane 2 H64pNO2Phe mutant Mb, Lane 3
Molecular weight standards and Lane 4 H93pNO2Phe mutant Mb.
5.2.4 Electronic absorption spectroscopy
UV-visible electronic absorption spectra of WT Mb proteins and the mutant H64pNO2
and H93pNO2 Mb proteins were taken using Varian Cary 50 Bio UV-visible spectrophotometer.
The concentrations of proteins were determined from the absorption spectrum of the protein,
using the molar extinction coefficient determined by the heme chromatogen method (96).
94
5.2.5 Preparation of deoxyferrous Mb
The WT Mb and mutants Mb proteins were taken in their respective airtight cuvettes
containing 0.1 M potassium phosphate buffer, pH 7.0. The N2 gas was passed for ~2 hr at 4 °C
to degas the sample. The minimal amount of Na2S2O4 (in µL) was added from the stock of 20
mg/mL and the electronic absorption spectra were recorded by Varian Cary 50 Bio UV-visible
spectrophotometer (15).
5.2.6 Preparation of oxyferrous complex
The oxyferrous complexes were prepared in a chest freezer at −35 to −45 °C. The
protein sample was placed in a 100 mM potassium phosphate buffer (pH 7.0), which contained
65% glycerol (v/v). The ferric protein was first degassed with N 2 gas for 2 hrs. Then it was
reduced to deoxyferrous by addition of Na2S2O4 (20 mg/mL stock) under N2 atmosphere in a
sealed cuvette at 4 °C. Pre-cooled O2 gas was bubbled into the cuvette for 60 sec and the UVvisible spectra were recorded (15, 31, 40).
5.2.7 CO and NO complex preparation
The ferrous-CO adducts were generated by gentle bubbling of CO in deoxyferrous
enzymes for 30 sec at 4°C in 0.1 M potassium phosphate buffer pH 7.0. The ferrous-NO
complexes were generated by micoliter addition of buffer saturated with NO gas in deoxyferrous
enzymes under N2 at 4 °C in 0.1 M potassium phosphate buffer pH 7.0. The saturated buffer
was prepared by gentle bubbling of NO gas in a sealed cuvette containing 0.1 M potassium
phosphate buffer pH 7.0 for 30 min at room temperature (15, 31, 32, 40).
95
–
–
5.2.8 N3 and CN complex preparation
–
WT Mb and mutants Mb N3 complexes were formed by µL addition of NaN3 solution
from 40 mM stock solution. The NaN3 stock solution and protein were in 100 mM potassium
phosphate buffer pH 7.0 and the temperature was kept at 4 °C. The cyanoferric adducts were
prepared from the deoxyferric enzymes by addition of minimal volumes (µL) of a 1 M KCN stock
solution, prepared in 100 mM potassium phosphate buffer pH 7.0. The deoxyferric enzymes
were prepared by bubbling of N2 gas in the ferric enzymes for at least 30 min.
5.3 Results and discussion
Here we studied the ferrous and ferric electronic absorption spectra of Mb and its
mutants (H64pNO2Phe Mb and H93pNO2Phe) in the absence and the presence of exogenous
–
–
ligands such as CO, NO, O2, N3 , and CN using UV-vis spectroscopy. The electronic absorption
spectra of the ferric form of the enzyme showed that there are significant differences in their
character, particularly at 550-650 nm (Figure 5.4). The peaks around 539, 578, and 628 nm in
ferric and around 662 nm in ferrous enzyme were not prominent in mutants when compared to
the WT Mb. Due to the presence of the electron rich functional group (pNO2Phe), the Soret
peaks around 409 nm in ferric and around 430 nm in ferrous were a little sharper in case of
mutant enzymes (Figures 5.4 and 5.5).
96
Figure 5.4 Characterization of ferric WT and mutants Mb. Electronic absorption spectra of the
H64pNO2Phe (blue dashed-dot), H93pNO2Phe (black solid line) and WT (red dashed line) of
ferric Mb enzymes. The spectra were recorded in 100 mM potassium phosphate buffer, pH 7.0
at 4 °C with 5 μM protein.
97
Figure 5.5 Deoxyferrous electronic absorption spectra of WT, H64pNO2Phe and H93pNO2Phe
mutant Mb. WT Mb (red dashed line), H64pNO2Phe (blue dashed-dot) and H93pNO2Phe Mb
mutant (black solid line). The spectra were taken at 4 °C and the samples were examined in
100mM phosphate buffer at pH 7.0.
5.3.1 Ferrous-CO, ferrous-NO and ferrous O2 complexes of WT, H64pNO2Phe and
H93pNO2Phe Mb
To understand the effect of different ligands upon replacement of distal and proximal
His and electronic nature of the heme center, I have investigated the ferrous-XO (X= C, N, O)
complexes of Mb and its mutants. As XO molecules have vacant π orbitals which closely
matched with filled dπ orbitals of the Fe (II) ion of porphyrin, it makes the heme group
electronically more favorable to bind to CO, NO and O 2. Furthermore, this kind of arrangement
was found to be optimal for the Fe-XO π-backbonding. The backbonding was responsible for
shortening (strengthening) the Fe-X bond and lengthening (weakening) of the X-O bond
(Figures 5.6 and 5.7) (40, 174-176).
98
Figure 5.6 The heme carbonyl complex showing dπ-pπ* backbonding.
−
Figure 5.7 Showing presence of electron rich functional group (NO 2 ) at distal position.
99
−
−
Table 5.1 The electronic absorption spectral features of the oxy, carbonyl, NO, CN , and N3 complexes with H64pNO2Phe Mb,
H93pNO2Phe Mb mutants and WT Mb.
H64pNO2Phe
Soret
(nm)
Ferric
408.9
Deoxyerrous
430
558
Visible
(nm)
100
H93pNO2Phe
Soret
(nm)
Visible (nm)
WT Mb
Soret
(nm)
Visible (nm)
(References)
Ferric
408.9
635
Ferric
409
Ferrous
433
635
Ferrous
431
505; 539;
578; 628; 675
(175)
556; 662
(175)
3+
–
422.9
537; 576
423
536.9
420
Fe - N3
3+
–
422
541; 576
421
536; 571
416
Fe -CO
2+
424
539; 573
422.9
542; 577
422
2+
419.9
542; 580
416.9
416
2+
419
545, 577
419
541.5;
579.6
546, 580
Fe - CN
Fe -O2
Fe -NO
420
542; 642.2
(175)
534; 583;
636; 661
540; 570;
649; 611 (10)
540; 581; 668
(15)
547, 581, (40)
Ferrous-CO complexes of the H64 and H93 pNO2Phe have shown higher intensity of
absorbance compared to ferrous-NO and ferrous–O2 complexes (Figure 5.8). This may be due
to Fe-CO being linear adduct and both of its π orbitals have empty shells. It allows dπ-π
orbitals to overlap in a perpendicular direction, which results in a prominent overlapping, and
more backbonding giving rise to sharper Soret (119). However under the similar conditions, NO
and O2, occupies one and two π electrons, respectively in their π orbitals. In order to
accommodate any π-antibonding interaction, the NO and O2 adducts have to bend, which
further lowers the possibility of overlapping and backbonding. This leads to less prominent Soret
in NO and O2 as compare to CO (Figure 5.8, 5.9 and 5.10). It is noteworthy that there were
significant differences within the XO adducts of different proteins’ electronic spectra (Table 5.1).
When axial His H93 attached to heme center, was replaced by pNO2Phe (an electron rich
functional group), the sharper Soret was recorded due to stretching one dπ-π backbonding
electron transition via push effect The pNO2Phe increases the electron density near the Fe (II)
center, which enhances the metal-ligand charge transfer and backbonding. Similar results were
not observed when the distal His 64 was replaced by pNO2Phe. The distal pNO2Phe probably
increases the electron density around the CO orbitals which favors ligand-metal charge transfer
and lowers the backbonding. Since O2 has two π electrons, reverse characteristics have been
observed in the oxyferrous complex (Figure 5.10). The different enzymes show different peaks
in 550-700 nm regions (Table 5.1), which explains that they also have diverse, features in
visible region (3, 176).
101
Figure 5.8 Comparative studies of carbonyl complexes of WT, H64pNO2Phe, and H93pNO2Phe.
Electronic absorption spectra of CO complex of WT Mb (red dashed line), H64pNO2Phe (blue
dashed-dot) and H93pNO2Phe Mb mutant (black solid line). The spectra were taken at 4 °C and
the samples were examined in 100 mM phosphate buffer at pH 7.0.
102
Figure 5.9 Comparative studies of NO complexes of WT, H64pNO 2Phe, and H93pNO2Phe.
Electronic absorption spectra of NO complex of WT Mb (red dashed line), H64pNO2Phe (blue
dashed-dot) and H93pNO2Phe Mb mutant (black solid line). The spectra were taken at 4 °C and
the samples were examined in 100 mM phosphate buffer at pH 7.0.
103
Figure 5.10 Characterization of oxyferrous complexes of WT, H64pNO2Phe, and H93pNO2Phe
Mb. Electronic absorption spectra of WT Mb oxyferrous (red dashed line), H64pNO2Phe mutant
Mb oxyferrous (blue dashed-dot) and H93pNO2Phe mutant Mb oxyferrous (black solid line). The
spectra were taken at −35 to −45 °C and the samples were examined in 60% glycerol, 100 mM
phosphate buffer at pH 7.0.
The stability of XO adducts are dictated by hydrogen bonding between the XO and
distal protein moiety (figure 5.11). In WT Mb at the 64 position, His is present, which is primarily
responsible for the H-bonding, hence the stability of XO adducts (176). When I replaced the His
with pNO2Phe, the possibility of H-bonding at distal pocket was reduced, which lowers stability
of complex. Furthermore, the electron rich oxygen allowed more electronic distribution, which
led to prominent ligand to metal charge transfer as seen in uv-vis spectrum. The replacement of
the proximal His 93 with pNO2Phe led to increased electron density, and backbonding was
more prominent around XO orbital; it helped in forming the H-bond with distal His. Thus I
104
conclude that when we replaced the axial His with the pNO2Phe, the XO adducts were more
stable than in other cases.
Figure 5.11 Proposed structure of (A) Fe (II) O2, (B) Fe (II) CO and (C) Fe (II) NO.
5.3.2 Ferric-cyanide and ferric-azide complexes of WT and H64pNO2Phe and H93pNO2Phe
mutants of Mb
–
–
Characterization of anionic ligand adducts such as CN and N3 to ferric WT Mb and
mutant Mb were important to understand how the electronic nature affects heme center. It gives
us the idea of the impact of protein environment on heme center. Ligand to metal charge
3+
transfer are ideal for metals in high oxidation states such as Fe that are bound to electron rich,
–
–
low electronegativity ligands such as N3 and CN (15, 175-177). The charge transfer from
ligand to metal will cause the increase in Soret, but due to lack of backbonding, it was not as
105
prominent as in the case of the CO adduct. The charge transfer from ligand to heme center
made it a low spin complex; the red shift was observed to high energy (Figures 5.12 and 5.13).
–
As CN is a stronger field ligand, the Soret was less sharp in the ferrycyanide complex .
The introduction of electron rich oxygen in pNO2Phe at axial and distal positions has a
significant impact on the electronic density of the heme center. When we replaced distal His
with pNO2Phe in electron density around CN
–
–
and N3
ligands, orbitals increased.
Consequently, there was more ligand-metal charge transfer, resulting in an increase in Soret
and red shift. While we replaced axial His with pNO2Phe, the Fe center was more electron rich,
which caused the red shift (λmax 423, 436.9, 437.4, and 576). There was less possibility of metal
to ligand charge transfer and backbonding, as the ligand was electron rich and its orbitals are
filled, and therefore no significant increase in Soret (119). The adducts also showed quite a
distinct feature in their 550-700 nm region (Table 5.1), which supports that the heme
environment has a significant role on heme proteins in the determination of their catalytic and
ligand binding properties.
106
–
Figure 5.12 Comparative studies of CN complexes of WT, H64pNO2Phe, and H93pNO2Phe
–
Mb. Electronic absorption spectra of ferric CN complex of WT Mb (red dashed line),
H64pNO2Phe (blue dashed-dot) and H93pNO2Phe Mb mutant (black solid line). The spectra
were taken at 4 °C and the samples were examined in 100 mM phosphate buffer at pH 7.0.
107
–
Figure 5.13 Characterization of N3 complexes of WT, H64pNO2Phe, and H93pNO2Phe Mb.
–
Electronic absorption spectra of ferric N3 complex of WT Mb (red solid line), H64pNO2Phe
(blue dashed-dot) and H93pNO2Phe Mb mutant (black solid line). The spectra were taken at 4
°C and the samples were examined in 100mM phosphate buffer at pH 7.0.
–
–
The H-bonding also plays a significant role in the stability of CN and N3 adducts
(Figures 5.14 and 5.15). Any residue that strengthens the H-bonding will make a stronger
adduct and vice versa. In this case, as pNO2Phe reduces the possibility of H-bonding at the
distal pocket, it leads to weaker adducts. But the size of the residues also plays a significant
role in holding the ligands by not allowing them to leave the active site environment.
108
–
Figure 5.14 Two canonical forms of N3 bound to heme iron of WT Mb and H64pNO2Phe Mb
mutant.
–
Figure 5.15 Two canonical forms of CN bound to heme iron of WT Mb and H64pNO2Phe Mb
mutant.
109
5.3 Conclusion
To summarize, the importance and significance of electron rich functional groups on the
electronic nature of heme center had been explored. It has been found that the axial His
attached to the heme center plays a crucial role in dictating the electron cloud near the heme
center. When the axial heme is replaced by an electron rich residue like pNO2Phe, the electron
density was higher near heme center, which contributes to higher energy and red shift of the
spectra. This study also concludes that the oxidation states of metal centers and the natures of
ligand (electron rich) play an important role in determining the backbonding and direction of
charge transfer (from metal to ligand or ligand to metal). The redox nature and size of the distal
residue also has a high impact on determining the stability of the ligand adducts.
110
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BIOGRAPHICAL INFORMATION
Subhash Chand the elder son of Mr. Keshaw Prasad Yadav and Mrs. Urmila Devi
Yadav has obtained his high school education at Shri Shanker Ji Intermediate College,
Pushpanagar, Azamgarh. Ever since the initial years of education, his interests were focused on
subjects like Science and Mathematics. In his primary school days, he has been academically
oriented, always stood out for his scholastic performance and earned his way into the top rank
in his class. He received B.Sc. (Biology) degree from the Ewing Christian College, Allahabad,
India and then M.Sc. (Botany) degree from University of Allahabad, Allahabad, India. His
respectable educational credentials landed him in the Indian Institute of Technology, Kharagpur
(IIT Kharagpur), the prestigious science and technology institute of India, to pursue Master of
Technology in Applied Botany (Biotechnology). After receiving his Masters degree he joined
Meerut Institute of Engineering and Technology and served as a Lecturer in the Department of
Biotechnology and Applied Sciences. He soon realized that higher education plays an important
role in advancing one’s professional life and moved to the USA to join University of Texas at
Arlington to carry out doctoral research. He joined Dr. Roshan Perera’s group to obtain his
Doctoral degree in Biochemistry. In the Perera lab he conducted stimulating research to
incorporate unnatural amino acids in genetically modified myoglobin mutants and mimic the
catalytic activities of various heme proteins.
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