. 9.“;
.. Hp! ,

.1 I}...

It. .
a II"...

; ah.
1. {131.9}.
15“. 15
1.1.,

5.13.3
a. t.
. 1; , Vaughn... ,
. art. 2.1 =an
K. . it, W—ié
5:35..
“ﬁrs... L

niob-

I
.33..
.I- I w.

 

i
If. 1 not?
.{E.§!.XJ.I‘

 

 

 

’Z-

10“” LIBRARY
Michigan State
University

 

 

 

 

This is to certify that the
dissertation entitled

DEVELOPMENT OF A TRANSMISSION
SPECTROELECTROCHEMICAL METHOD WITH
OPTICALLY TRANSPARENT DIAMOND ELECTRODE FOR
THE STUDY OF REDOX PROTEINS

presented by

Yingrui Dai

has been accepted towards fulﬁllment
of the requirements for the

Ph. D. degree in Chemistry

 

 

 

 

Major Professor’s Signature

M10; 1900?

Date

 

 

MSU is an afﬁnnative-action, equal-opportunity employer

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K1IProlecc8PrelelRC/DateDue indd

DEVELOPMENT OF A TRANSMISSION SPECTROELECTROCHEMICAL
METHOD WITH OPTICALLY TRANSPARENT DIAMOND ELECTRODE FOR
THE STUDY OF REDOX PROTEINS
By

Yingrui Dai

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Chemistry
2009

ABSTRACT
DEVELOPMENT OF A TRANSMISSION SPECTROELECTROCHEMICAL
METHOD WITH OPTICALLY TRANSPARENT DIAMOND ELECTRODE FOR
THE STUDY OF REDOX PROTEINS
By
Yingrui Dai

The goal of the research was to develop a transmission
Spectroelectrochemical method with an optically transparent boron-doped
diamond electrode that is useful for studying structure-function relationships of
redox proteins.

First, the electrochemical behavior of the cytochrome c was investigated
at boron-doped ultrananocrystalline diamond (UNCD) and microcrystalline
diamond (MCD) electrodes. Quasi-reversible, diffusion-controlled electron
transfer was observed at oxygen-terminated, but not hydrogen-tenninated
electrodes. UNCD electrodes exhibited a similar electrochemical response as the
MOD electrodes but showed higher peak currents due to a larger electrochemical
active area. pH-dependent studies of the voltammetric response revealed that
the largest peak current and smallest peak separation were observed between
pH 7 and 7.5.

Next, a custom-designed thin-layer transmission spectroelectrochemical
cell was constructed and characterized. Electrochemical characterization of the
thin-layer cell showed Gaussian-shaped thin-layer voltammetric i-E curves with a

linear relationship between the peak current and the scan rate. Time-dependent

measurements indicated rapid and complete electrolysis in the cell, and good

agreement between electrochemical and spectral data. The
spectroelectrochemical studies of cytochrome 0 revealed an advantage of
diamond OTE in terms of its fouling resistance. The spectral characterization of
the cytochrome c—cyanide complex was also presented for the ﬁrst time. The
reversible spectroelectrochemical response of myoglobin demonstrated the
applicability of the cell under anaerobic condition.

An analysis method was developed for studying a multicomponent redox
protein, Na*-translocating NADH:quinone oxidoreductase (Na"-NQR). The UV-
Vis difference spectra of this protein at diamond OTE without mediators indicated
that the reversible reaction and complete electrolysis occurred in the thin-layer
cell. The spectra were modeled with global regression analysis to resolve the

redox centers in Na*-NQR and determine the corresponding formal potentials.

DEDICATION

To my husband

ACKNOWLEDGEMENTS

There are many people I want to thank during my time at Michigan State
University. The ﬁrst person I want to thank is my advisor, Dr. Greg M. Swain.
Thanks for his guidance over the last ﬁve years. Thanks to Dr. Denis A.
Proshlyakov for the helpful discussion and comments. Thanks to my committee
members, Dr. Gary J. Blanchard, Dr. David P. Weliky and Dr. Shelagh Ferguson-
Miller for the comments about my work. I also want to thank Dr. Denise A. Mills
and Yi Zheng for the help with the protein work.

I have been fortunate to work with people in the Swain Group. Words
alone can not express my gratefulness to my friends in chemistry department. It
is nice to have you in my life that saw me through the process.

Finally, and most importantly, I would like to say thank you to my parents
and husband. Without your love and support, I would not be here today. Thank
you for always being there with me.

Grants from the National Science Foundation (CHE-0616730) and

National Institutes of Health (GM 070544) are gratefully acknowledged.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ VIII
LIST OF FIGURES .............................................................................................. IX
CHAPTER 1 INTRODUCTION ......................................................................... 1
1.1 Spectroelectrochemistry .......................................................................... 1
1.2 Transmission Spectroelectrochemistry .................................................... 5
1.2.1 Optically Transparent Electrodes (OTE) .......................................... 6
1.2.2 Development of Optically Transparent Boron-Doped Diamond
Electrodes (Diamond OTE) ............................................................. 8
1.2.2.1 Boron-Doped Diamond Electrodes .............................................. 8
1.2.2.2 Optically Transparent Boron-Doped Diamond Electrodes ......... 11
1.3 The Importance of Studying Structure-Function Relationships of
Metalloproteins ..................................................................................... 13
1.4 Na*-Translocating NADHzQuinone Oxidoreductase .............................. 15
1.5 Speciﬁc Aims ......................................................................................... 17
1.6 Organization of the Dissertation ............................................................ 18
1.7 References ............................................................................................ 19
CHAPTER 2 EXPERIMENTAL METHODS .................................................... 32
2.1 Electrode Materials ................................................................................ 32
2.1.1 Boron-Doped Diamond .................................................................. 32
2.1.1.1 Ultrananocrystalline and Microcrystalline Boron-Doped Diamond
Electrodes (UNCD and MCD) .................................................... 34
2.1.1.2 UV-Vis Transparent Boron-Doped Diamond Electrode
(Diamond/Quartz OTE) .............................................................. 36
2.1.2 Glassy Carbon ............................................................................... 37
2.1.3 Gold Minigrid ................................................................................. 37
2.2 Cyclic Voltammetric Experiments .......................................................... 38
2.3 Electrochemical Kinetics ........................................................................ 39
2.4 X-ray Photoelectron Spectroscopy (XPS) .............................................. 40
2.5 Spectroelectrochemical Measurements ................................................. 41
2.5.1 Thin-Layer Spectroelectrochemical Cell ........................................ 41
2.5.2 Potential Step Method ................................................................... 42
2.5.3 UV-Vis Spectroelectrochemical Measurements ............................ 45
2.6 Global Regression Analysis ................................................................... 46
2.7 Chemicals .............................................................................................. 46
2.8 References ............................................................................................ 49

vi

CHAPTER 3 DIRECT ELECTROCHEMISTRY OF CYTOCHROME 0 AT

BORON-DOPED DIAMOND ELECTRODES ............................. 50

3.1 Introduction ............................................................................................ 50
3.1.1 Electrochemical Studies of Cytochrome c ..................................... 50
3.1.2 Oxygen-Terminated Boron-Doped Diamond Electrodes ............... 54
3.2 Results .................................................................................................. 57
3.2.1 XPS Measurements ....................................................................... 57
3.2.2 Cyclic Voltammetric Studies .......................................................... 61
3.3 Discussion ............................................................................................. 68
3.4 Conclusions ........................................................................................... 73
3.5 References ............................................................................................ 74

CHAPTER 4 SPECTROELECTROCHEMICAL BEHAVIOR OF REDOX

PROTEINS IN A THIN-LAYER TRANSMISSION CELL ............ 78

4.1 Introduction ............................................................................................ 78
4.1.1 Ideal Thin-Layer Behavior ............................................................. 79
4.1.2 Ideal Thin-Layer Transmission Cell ............................................... 81
4.1.3 Electrochemical Studies of Myoglobin ........................................... 81
4.2 Results .................................................................................................. 86
4.2.1 Electrochemical Performance of the Cell ....................................... 86
4.2.2 Spectroelectrochemcial Kinetics .................................................... 91
4.2.3 Spectroelectrochemistry of Cytochrome c — Cyanide Complex ..... 96
4.2.4 Spectroelectrochemistry of Myoglobin ......................................... 100
4.3 Discussion ........................................................................................... 104
4.4 Conclusions ......................................................................................... 107
4.5 References .......................................................................................... 109

CHAPTER 5 SPECTROELECTROCHEMICAL STUDIES OF Na“-
TRANSLOCATING NADHIQUINONE OXIDOREDUCTASE

FROM VIBRIO CHOLERAE .................................................... 112

5.1 Introduction .......................................................................................... 1 12
5.2 Results ................................................................................................ 115
5.3 Discussion ........................................................................................... 125
5.4 Conclusions ......................................................................................... 127
5.5 References .......................................................................................... 129
CHAPTER 6 CONCLUSIONS AND OUTLOOK ............................................ 130

vii

Table 2.1

Table 2.2

Table 3.1

Table 3.2

Table 3.3

Table 4.1
Table 5.1

Table 5.2

LIST OF TABLES

Growth conditions for UNCD and MCD thin ﬁlms deposited on p-
type Si ........................................................................................... 35

Growth conditions for UV-Vis optically transparent diamond
electrode ........................................................................................ 37

Summary of the surface composition on various diamond electrode
surfaces ......................................................................................... 60

Apparent heterogeneous electron-transfer rate constant, kgpp,
calculated from cyclic voltammetric data ....................................... 67

pH dependence of the midpoint potential Em of cytochrome c at
boron-doped diamond electrodes. Scan rate=20 mV/s. Electrode

area=0.2 cm2 ................................................................................. 68
Extinction coefﬁcients for horse myoglobin at pH 7, ~20 °C .......... 83
Determined redox potentials for Na"-NQR .................................. 125

Redox transitions for known cofactors in Na"-NQR determined by
Bogachev .................................................................................... 126

viii

Figure 1.1

Figure 1.2

Figure 1.3

Flgure 1.4

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

LIST OF FIGURES

Different types of spectroelectrochemical techniques ..................... 2

General design of an OTE and a thin-layer electrochemical cell for
transmission spectroelectrochemical measurement ........................ 6

Two conventional OTEs for transmission measurements: (A) a
metal minigrid and (B) a thin ﬁlm supported on an optically
transparent substrate ....................................................................... 7

Schematic bonding arrangement of spa-bonded diamond ............... 9

A diagram of a microwave plasma-assisted CVD reactor used to
grow boron-doped diamond thin-ﬁlm electrodes ............................ 32

Design of the single—compartment, three-electrode electrochemical
cell with (a) the diamond ﬁlm working electrode, (b) o-ring seal, (c)
rubber spacer (and nickel foil), (d) reference electrode inside a
glass capillary tube with a cracked tip, (e) carbon rod counter
electrode, and (f) input for nitrogen purge gas ............................... 38

Diagram of the thin-layer transmission spectroelectrochemical cell.
A thin layer (TL) is formed between the working electrode (WE) and
the transmission window (not shown) and its thickness is
determined by the spacer (SP). The thin layer is connected to the
Pt counter electrode (CE) via electrolyte capillary (EC) between the
transmission window and outer cell body. A miniature Ag/AgCl
reference electrode (RE) is also connected to the electrolyte
capillary via a cracked glass junction (GJ). Reference electrode
capillary is also used for loading sample (SM), which is exhausted
via a similar capillary on the opposite side (not shown) ................. 41

Scheme for measuring the steady-state absorbance of a redox
analyte as a function of the potential in a spectroelectrochemical
measurement. An absorbance spectrum was recorded at each
potential, as indicated by the arrows ............................................. 43

Principle of difference absorbance spectroscopy. The difference
spectrum B-A is generated by subtracting spectrum of initial state A
from spectrum of product state B ................................................... 44

Figure 2.6

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Potential-time proﬁle for potential step studies of Na”-NQR.
Absorbance spectra at the reference potential (dashed arrow) were
recorded before and after each potential step (solid arrow). The
difference absorbance spectrum was generated by subtracting the
average of the absorbance spectra at reference potential from
absorbance spectrum at each potential (e.g., AA=A2-(A1+A3)/2) ...45

Heme c structure of cytochrome c. (A) Planar porphyrin ring system;
(B) heme Fe is also bonded to histidine (His) and methionine (Met)
axial ligands ................................................................................... 51

Terminal atomic structure for hydrogen- and oxygen-terminated
diamond (100) and (111) surfaces ................................................. 55

XPS spectra for (A) as-deposited, (B) oxygen-terminated, and (C)
hydrogen-terminated UNCD electrode surfaces. ........................... 58

XPS spectra for UNCD electrode surfaces after potential cycling in
cytochrome 0 solution. (A) Hydrogen-terminated surface; (B)
oxygen-terminated surface. ........................................................... 60

Cyclic voltammetric i—E curve of 0.1 mM cytochrome c in 0.1 M
NaCl, 10 mM Tris-HCI (pH 7) solution at a hydrogen-terminated
UNCD electrode. Scan rate=20 mV/s. ........................................... 61

Cyclic voltammetric i-E curves for 0.1 mM cytochrome c in 0.1 M
NaCl, 10 mM Tris-HCI buffer (pH 7) at oxygen-terminated (A)
UNCD and (B) MCD electrodes. Curves are shown for different
scan numbers. Scan rate=20 mV/s ............................................... 62

Cyclic voltammetric background i-E curves for an oxygen-
terminated UNCD electrode. Cyclic voltammograms were
measured in 0.1 M NaCl and 10 mM Tris-HCI (pH 7) before (solid
line), after (dashed dot line) cytochrome 0 potential cycling and
after purging with N2 (dashed line) following cytochrome c
measurements. Scan rate=20 mV/s .............................................. 63

Cyclic voltammetric i-E curves for 0.1 mM cytochrome c in 0.1 M
phosphate buffer (pH 7.5) at UNCD electrodes at different scan
rates. Electrode area=0.2 cm2 ....................................................... 65

Cyclic voltammetric data for cytochrome c at UNCD electrodes.
Electrode area=0.2 cmz. (A) Plot of reduction peak current for 0.1
mM cytochrome 0 solution versus the square root of the scan rate;
(B) Plot of reduction peak current versus cytochrome c

Figure 3.10

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

concentration from 0.025 to 0.2 mM in 0.1 M phosphate buffer (pH
7.5) at 20 mV/s .............................................................................. 66

Effect of the solution pH on the cytochrome 0 reduction peak
current at boron-doped diamond electrodes. The solutions were 0.1
mM cytochrome c in 0.1 M phosphate buffer at different pH. Scan
rate=20 mV/s. Electrode area=0.2 cm2 .......................................... 68

A theoretical cyclic voltammetric i-E curve for a reversible, one-
electron redox reaction in a thin-layer cell ..................................... 79

Heme b structure of myoglobin. (A) Planar porphyrin ring system
and (B) heme Fe is axially bound to a histidine molecule with the
sixth coordination site remaining unoccupied or weakly ligated by a
solvent molecule ............................................................................ 82

UV-Vis spectra for horse myoglobin in different oxidation states with
different bound ligands. Metmyoglobin (meth): MbFe(lll)H20,
deoxymyoglobin (deoxyMb): MbFe(lI), oxymyoglobin (Mb02)2
MbFe(ll)Oz and carboxymyoglobin (MbCO): MbFe(l|)CO ............. 83

Background-corrected cyclic voltammetric i-E curves for Fe(CN)6-4
in 1 M KCI in the thin-layer cell with a4 glassy carbon electrode. (A)
Effect of scan rate for 1 mM Fe(CN)6 using the 75 pm spacer; (B)

.4
effect of Fe(CN)6 concentration using the 75 pm spacer at 1 mV/s;

(C) effect Of layer thickness for 1 mM Fe(CN)6'4 at 1 mV/s; and (D)
peak separation as a function of layer thickness ........................... 87

Plots of (A) oxidation peak current and (B) faradaic charge vs. scan
rate for different concentrations Of Fe(CN)6 .The spacer thickness
was 75 pm and the supporting electrolyte was 1 M KCI. ............... 90

Time depenfence of the current and optical absorbance at 220 nm
for Fe(CN)6 at a gold minigrid electrode in the thin-layer cell during

a potential step from -0.1 to 0.6 V. Data are shown for supporting
electrolyte concentration of (A) 100 mM KCI and (B) 1 M KCl. Lines
are for the current on the left and markers are for the simultaneous
optical changes on the right. The spacer thickness was 25 pm ..... 92

Cyclic voltammetric i-E curve for cytochrome c at a gold minigrid

electrode in a thin-layer cell with a 75 pm spacer. The analyte
solution was 0.1 mM cytochorme c in 10 mM phosphate buffer (pH
7) and 100 mM KCI. Scan rate=10 mV/s ....................................... 93

xi

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12
Figure 5.1

Figure 5.2

Figure 5.3

Difference absorbance spectra of cytochrome c at a gold minigrid
electrode in a thin-layer cell with a 75 pm spacer. The Red-Ox
difference spectra were generated during a potential step from 0.5
to -0.3 V. The analyte solution was 0.1 mM cytochorme c in 10 mM
phosphate buffer (pH 7) and 100 mM KCI. The spacer thickness
was 75 pm ..................................................................................... 94

Absorbance change of cytochrome c on (A) a gold minigrid
electrode and (B) a boron-doped diamond thin ﬁlm at 418 nm. The
analyte solution was 0.1 mM cytochorme c in 10 mM phosphate
buffer (pH 7) and 100 mM KCI. The spacer thickness was 75 pm.
Repetitive potential steps between 0.5 and -0.3 V were applied, as
indicated in the ﬁgure .................................................................... 95

Spectroelectrochemical measurements of cytochrome c—cyanide
complex on a boron-doped diamond electrode. (A) Absorption
changes at characteristic wavelengths; (B-D) difference
absorbance spectra between key states as designated by letters a
to g; and (E) thermodynamic cycle of cytochrome c—cyanide
complex. The analyte solution was 0.1 mM cytochorme c in 0.1 M
KCN, Tris buffer (pH 7) and 100 mM KCI. Potentials were stepped
between +0.3, -0.3 and -0.7 V as indicated in the ﬁgure ............... 98

Absorbance-time proﬁles and absorption spectra of myoglobin
without (A, B) and with (C, D) far-UV light exposure. Spectra were
recorded in the thin-layer transmission cell with a gold minigrid
electrode. The spacer thickness was 75 pm. The analyte solution
was 50 IIM myoglobin in 10 mM sodium phosphate buffer (pH 7)
and 100 mM KCI .......................................................................... 102

Proposed mechanism for photoreduction of myoglobin ............... 107
Chemical structures of riboﬂavin, FMN, FAD and ubiquinone-8..113

(A) UV-Vis absorption spectra for air-oxidized (solid line) and
sodium dithionite reduced (dashed line) Na*-NQR puriﬁed from
Vibrio cholerae. The solution was 0.01 mM Na*-NQR in 50 mM
sodium phosphate buffer (pH 8), 100 mM NaCl, 1 mM EDTA, 5%
glycerol and 0.05% dodecylmaltoside; (B) difference absorbance
spectrum (Red-Ox) for Na”-NQR ................................................. 1 16

Background-corrected cyclic voltammetric i-E curves for Na*-NQR
at (A) a freshly polished glassy carbon electrode and (B) a
diamond/quartz electrode with a 75 pm spacer. The analyte
solution was 0.1 mM Na”-NQR in 50 mM sodium phosphate buffer

xii

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

(pH 8), 500 mM NaCl, 1 mM EDTA, 5% glycerol and 0.05%
dodecylmaltoside. The scan rate was 0.5 mV/s .......................... 118

Absorbance change of Na"-NQR (460nm) at a diamond/quartz OTE
in a transmission cell with a 75 pm spacer. Potential steps to 0.2
and -0.7 V were applied as indicated in the ﬁgure. The analyte
solution was 0.1 mM Na*-NQR in 50 mM sodium phosphate buffer
(pH 8), 500 mM NaCl, 1 mM EDTA and 0.05% dodecylmaltoside
.................................................................................................... 119

UV-Vis difference absorbance spectra measured for Na"-NQR at a
diamond/quartz OTE in a transmission cell with a 75 pm spacer.
Ox-Red and Red-Ox difference spectra were generated from the
oxidized and reduced spectra collected at 0.2 and -0.7 V. The
analyte solution was 0.1 mM Na+-NQR in 50 mM sodium phosphate
buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside ......................................................................... 120

Difference absorbance-potential curve (460 nm) for Na*-NQR at a
diamond/quartz OTE in a transmission cell with a 75 pm spacer.
The analyte solution was 0.1 mM Na+-NQR in 50 mM sodium
phosphate buffer (pH 8), 500 mM NaCI, 1 mM EDTA and 0.05%
dodecylmaltoside. The data were collected at a scan rate of 2 mV/s
.................................................................................................... 121

UV-Vis difference absorbance spectra of Na"-NQR during cathodic
and anodic potential steps at a diamond/quartz OTE in a
transmission cell with a 75 pm spacer. The difference spectra were
generated from the oxidized spectra collected at reference potential
0.2 V for reduction and oxidation potential steps every 100 mV and
20 min. The analyte solution was 0.1 mM Na+-NQR in 50 mM
sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM EDTA and
0.05% dodecylmaltoside .............................................................. 122

Absorbance changes of Na*-NQR upon reduction (vs. -0.1 V) and
oxidation (vs. -O.6 V) potential steps every 10 mV and 20 min.
Markers: experimental data, solid line: global ﬁtting results. The
analyte solution was 0.1 mM Na+-NQR in 50 mM sodium phosphate
buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside ......................................................................... 124

Resolved spectra of redox transitions in Na+-NQR ...................... 124

xiii

CHAPTER 1

INTRODUCTION

1.1 Spectroelectrochemistry

Spectroelectrochemistry is a hybrid of two techniques: reaction-oriented
electrochemistry and species—focused spectroscopy. The technique can be used
to record the change in spectroscopic signature of an analyte associated with a
change in redox state caused by electron transfer at an electrode. It provides
more information about a redox reaction mechanism than can be acquired using

traditional electrochemical methods alone.

Considerable development in spectroelectrochemistry has taken place
since its inception in 1964 when Kuwana introduced this hybrid analytical
method.1 Over the years, spectroelectrochemistry has been used with
electromagnetic radiation from multiple regions of the electromagnetic spectrum
in the study of inorganic, organic, and biological redox systems?5 Some
commonly used spectroscopic methods that have been coupled with

electrochemistry are listed in Figure 1.1.

 

  
   

  

ABSORPTION
SPECTROSCOPY LUM'NESSENCE
SCATTERING
uv, VISIBLE, IR SPECTROSCOPIES

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

TRANSMISSION REF ANGIE
H 0.2 mm Raman scatterlng
' l \EK excitation
A- Thin-layer 00" C. Specular reflectance E. Raman

 

 

 

MEIR. R m
D f /

B, Conventional cell D. Internal reﬂectance F. Fluorescence

\/

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.1 Different types of Spectroelectrochemical techniques.2

Absorption spectroscopy using ultraviolet, visible, or infrared radiation is
most often employed. In the transmission mode (Figure 1.1, A and B), the optical
beam is directed orthogonally through an optically transparent electrode (OTE)
and the adjacent solution layer. Specular reﬂectance Spectroscopy (Figure 1.1, C)
involves passing light through a solution layer and measurement of the light
reﬂected from the electrode surface. In the internal reﬂection mode (Figure 1.1,
D), the light beam is introduced through the back Side of an OTE at an angle
greater than the critical angle so that the beam is totally internally reﬂected. The
light interacts with species present adjacent to the electrode surface. The
sensitivity can be increased by introducing multiple reﬂections. In normal and

resonance Raman Spectroscopy (Figure 1.1, E), a laser beam is directed through

the solution at an electrode and the Raman back-scattered light is detected. A
beam of excitation light is passed through an electrochemical cell and the
resulting luminescence from electrogenerated species is recorded (Figure 1.1, F).
Many other techniques, such as surface plasmon resonance (SPR), mass
Spectrometry (MS), electron paramagnetic resonance (EPR), nuclear magnetic
resonance (NMR) and X-ray diffraction (XRD), have also been coupled with
electrochemistry. In summary, the transmission mode provides information about
the electrochemical reaction in the solution; the reﬂectance mode is more
focused on the electrode/electrolyte interface; and the scattering mode is useful
for the investigation of electrogenerated species adsorbed on the electrode

surface.

The combination of spectroscopic and electrochemical techniques has
enabled new and powerful combinations to be applied to complex redox systems.
A recent book reviewed UV-Vis/lR/Raman/NMR/EPR spectroelectrochemistry
and their application in analysis of redox reactions through identiﬁcation of the
intermediates and products to study iron-containing proteins, metalloporphyrins,
mixed-valence complexes and organometallics.6 A series of papers by Heineman
et al. reported a spectroelectrochemical sensor that is based on an attenuated
total reﬂection measurement at an indium tin oxide (ITO) optically transparent
electrode coated with a thin (20-500 nm thick) ion-exchange ﬁlm.7'29 This type of
sensor has been employed as a small portable unit for the detection of

ferrocyanide in tank waste.“ ‘6 Saavedra et al. used spectroelectrochemical

analysis to study surface-conﬁned redox couples and characterize adsorbed
polymer ﬁlms on lTO electrodes with attenuated total reﬂectance spectroscopy.”
36 Lopez-Palacios et al. focused on using a reﬂection-transmission bidimensional
spectroelectrochemistry cell to analyze the ion exchange processes during
doping/dedoping of conducting polymers.”44 The Porter and Barbara groups
demonstrated that Single-molecule ﬂuorescence spectroelectrochemistry was a
powerful approach for the study of electron transfer process at highly
heterogeneous interfaces using carbon OTE and ITO separately.45 This
technique was later employed by the Ackerman group to investigate the electron
transfer dynamics of single redox molecules in aqueous solution using cresyl
violet as a model ﬂuorescent redox molecular probe.46 Mantele and coworkers
have used electrochemically-induced UV-ViS/IR transmission spectroscopy
(spectroelectrochemistry) over the last two decades to study redox protein
structure-function relationship.”76 The informative mid-IR difference spectra
revealed information about complex molecular mechanisms, such as the
conformational and hydrogen-bonding changes in the backbone and the side
chains, protonation and deprotonation reactions Of amino acid residues, and
interactions between prosthetic groups and the protein.58 Berthomieu and
coworkers extended this method to the far infrared (50 cm“) domain for the
analysis of metal-ligand interactions in redox proteins with diamond windows.77
The far-IR difference spectra can extend the analysis of vibrational properties to
metal sites or redox states that are not accessible to resonance Raman

spectroscopy.

1.2 Transmission Spectroelectrochemistry

The transmission spectroelectrochemical measurement mode is most
Often used with a thin-layer cell and an optically transparent electrode (OTE). The
general design of an OTE and the thin-layer electrochemical cell is shown in
Figure 1.2.78 A thin layer solution containing a redox analyte is conﬁned between
an optically transparent window and the working electrode. The OTE acts as the
working electrode as well as the optically transparent window. The reference and
counter electrodes are in contact with the thin layer solution but outside the
optical path. Potential step and potential sweep methods are usually employed to
drive the redox reaction inside the thin volume of solution. As the electromagnetic
radiation with intensity, Io, passes through the cell, changes in the spectroscopic
signature for the redox analyte are recorded as a function of the applied potential
(i.e., change in oxidation state). One of the main advantages of the thin-layer cell
design is that the electroactive analyte present can be exhaustively electrolyzed
in a very short time (typically 20-120 3 depending on the cell thickness). In the
design, there is ﬁnite diffusion, which means that the mathematical description of

the electrochemical process can be simpliﬁed.

Opﬁcal

 

 

 

Thin solution layer window
As,
I" \ /
0x
III I
# w —>
by
Red

1Tansparent Carbon
substrate ﬁlm

Figure 1.2 General design of an OTE and a thin-laayer electrochemical cell for
transmission Spectroelectrochemical measurement.7

1.2.1 Optically Transparent Electrodes (OTE)

Transmission spectroelectrochemical measurements require an electrode
that is transparent to the electromagnetic radiation. The electrode transparency
enables potential-dependent spectra of electrogenerated Species to be recorded
in the thin layer solution. An ideal OTE should possess good electrical

conductivity, high optical transparency, robustness and chemical inertness.78

A. Minigrid OTE B. Film-Based OTE

 

 

 

. OTE ,.
Substrate

 

 

 

 

:[25 pm

4 F-
9W"

Figure 1.3 Two conventional OTEs for transmission measurements: (A) a
metal minigrid and (B) a thin ﬁlm supported on an optically transparent substrate.

One electrode architecture that would work for such measurements is a
porous electrode. Examples are a minigrid electrode (e.g., Au, Ni, Ag or Cu),7M1
which is simply a metal mesh (Figure 1.3 A) and porous reticulated vitreous
carbon.82' 83 Light is transmitted through the electrode openings, allowing one to
probe changes in electronic properties or structural features of a molecule,
brought about by a change in redox state. A gold minigrid is the most commonly
used electrode material due to its accessibility and transmission over the entire
electromagnetic spectrum.“ 48 The disadvantages of this electrode type are the
narrow potential window, the large background charging current and temporal
instability due to protein denaturation even after surface modiﬁcation. Due to their
geometry, they are not suitable for optical probing of surface localized processes,
such as adsorption. Also, their fragility limits the lifetime of the cell using such

materials.

The other design is a thin conductive ﬁlm (Figure 1.3 B) supported on an

optically transparent substrate. Depending on the spectral region of interest,

84, 85 78, 83, 86-89

conductive thin ﬁlms, such as metals, and metal

carbon materials,

oxides,”92

can be employed. Usually, a compromise between electrical
conductivity and transparency has to be reached as these two parameters are
inversely related to one another, and both of them are controlled by ﬁlm
thickness.93 The transparent substrate improves the mechanical stability of the
thin OTE. Currently used OTE materials are indium—doped tin oxide (ITO) and
boron-doped diamond. Both materials can be deposited as a thin ﬁlm on
transparent substrates, such as glass (visible), quartz (UV-Visible), germanium or

silicon (infrared). The development and application of carbon OTEs were recently

reviewed.78

1.2.2 Development of Optically Transparent Boron-Doped

Diamond Electrodes (Diamond OTE)

1.2.2.1 Boron-Doped Diamond Electrodes

Diamond is the hardest natural material, consisting of a crystal lattice of
tetrahedrally-arranged sp3-bonded carbon atoms. This arrangement leads to six-
membered rings in a chair conformation, as shown in Figure 1.4.94 It can be
produced synthetically by two methods: (1) high—pressure-high-temperature
(HPHT) growth (>1400 °C, >50,000 bar) in the presence of a catalyst; and (2) low

pressure, low temperature (<1000 °C, 0.01-1 bar) chemical vapor deposition

(CVD) from a carbon-containing source gas mixture.95 CVD diamond is usually
polycrystalline with randomly-oriented crystallites and grain boundaries between
the crystallites. These grain boundaries consist of a mixture of sp2- and spa-

bonded carbon.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

V

Figure 1.4 Schematic bonding arrangement of sp3- bonded diamond.

Diamond is naturally an insulating material, so in order for it to be
electrically conducting, it must be doped. Boron dopant atoms substitute for
carbon atoms in the growing crystal lattice, which produces p-type
semiconductivity with an acceptor level of 0.37 eV above the valence band.96
Resistivities as low as 0.01 D-cm have been reported for degenerately boron-
doped ﬁlms, making them useful as electrochemical electrodes.96109 The surface
carbon atoms are stabilized by strong covalent bonds with hydrogen for ﬁlms
deposited from hydrogen-rich source gas mixtures, as illustrated in Figure 1.4.
Diamond can also be made n-type by dOping with nitrogen110 or phosphorusm'
"3. The deposition and properties of diamond with different dopants, such as

SU'fUF. boron, nitrogen and phosphorus, have been studied by Haubner.114

The use of electrically conductive boron-doped diamond (BDD) as an
electrode material is a ﬁeld of research that had its beginnings back in the early
19905.115117 There are three types of polycrystalline BDD: microcrystalline,
nanocrystalline and ultrananocrystalline. Butler and Sumant recently reviewed
the growth, Characteristics, and applications of chemical vapor deposited
ultrananocrystalline and nanocrystalline diamond ﬁlms.118 Angus et al. also
reported on the growth, electrochemical properties and applications of diamond
ﬁlms.‘“"127 Their recent ﬁnding regarding the surface conductivity of hydrogen-
terminated diamond may enable a new class of sensorsm"130 Compton and
coworkers also worked on the fabrication, characterization and application (e.g.,
metal ion analysis) of boron-doped diamond electrodes.‘31‘135 Moreover, Swain
and Fujishima groups have been reporting on the deposition and application of
boron-doped diamond electrodes in electroanalysis in the last two decades.96' 99'
108'109'136‘16‘ Much Of the work, so far, has involved the characterization of the
physical and electrochemical properties of boron-doped diamond and its
electrochemical response toward a variety of aqueous and nonaqueous redox
systems. From these results, it can be concluded that high-quality diamond,
which means minimal nondiamond carbon impurity content, low secondary
nucleation density, and hydrogen surface termination, offers signiﬁcant
advantages over other carbon and metal electrodes in terms of a wide potential
window, lower and stable background current, fouling resistance, low limits of

detection, and better robustness. 95'109-139'155'162

10

1.2.2.2 Optically Transparent Boron-Doped Diamond Electrodes

Another unique property of diamond is its optical transparency. Diamond
(undoped) has one of the widest optical windows of any material, extending from
the band gap absorption edge (~225 nm) deep into the far infrared (~30 cm").163
Factors that inﬂuence the optical throughput of diamond include the defect
density, dopant type and doping level, ﬁlm thickness and grain Size (roughness).
Adding boron dopant to improve the electrical conductivity reduces the optical
throughput in isolated regions of the spectrum. It is, therefore, essential to
establish a balance between the electrical and optical properties of boron-doped
diamond in order to make the material a useful OTE. By adjusting the CVD

growth conditions, it is possible to manipulate and optimize this balance.

The versatility of boron-doped diamond, both as an electrode and an
Optically transparent material, makes it an obvious choice for
spectroelectrochemical measurements.164 In general, diamond offers several
advantages over other commonly used OTES, including (1) a wide optical
window extending from near UV into far-IR regions of the electromagnetic
spectrum, (2) a wide working potential window and low background current, (3)
stability during both cathodic and anodic polarization even at high current density
and potential, (4) reproducibility of the electrical, optical and electrochemical
properties from batch-to-batch, and (5) resistance to fouling because of a

nonpolar, hydrogen-terminated surface.164

11

Diamond OTES have been employed by several groups in the
spectroelectrochemical measurements. Martin and Morrison used internal
reﬂection IR spectroelectrochemical measurements to study the types of carbon-
oxygen functional groups formed on the diamond surface during anodic
polarization.165' ‘66 Zhang et al. reported the use of a boron-doped diamond-
coated platinum mesh electrode in the spectroelectrochemical measurement of
high-potential redox species in aqueous solutions.167 Our group has been
developing optically transparent diamond electrodes for applications in the
Spectroelectrochemical measurements for some time now.‘64' 16“" Both free-
standing diamond disks and diamond thin ﬁlm deposited on quartz or undoped Si
have been prepared, characterized and utilized in transmission
Spectroelectrochemical measurements. Zak et al. characterized a free-standing
diamond disk in a thin-layer spectroelectrochemical cell for UV-Vis transmission

measurements of Fe(CN)6'3’4

and methyl viologen.168 Haymond et al. applied the
free-standing diamond disk to study ferrocene in acetonitrile.170 Their work
demonstrated that the boron-doped diamond is a practical OTE material for
reproducible spectroelectrochemical measurements both in aqueous and
nonaqueous media. However, due to the large thickness (380 pm), the
transparency was limited. In general, the low transparency, the long deposition
time and laborious mechanical polishing needed to smooth the surface are
limitations of this OTE architecture. Stotter et al. reported on the preparation and

characterization of a thin ﬁlm of optically transparent boron-doped diamond

deposited on quartz for UV-Vis Spectroelectrochemical measurements.169

12

Absorption Spectra of chlorpromazine and Fe(CN)I.;.'3’4 were recorded at different
potentials, and the spectral changes were correlated with alterations in redox
state of the molecules. Stotter et al. also compared the electrical, optical, and
electrochemical properties of two optically transparent electrodes: diamond and
ITO.164 Boron-doped diamond has also been deposited on undoped silicon and
been used in preliminary mid-IR spectroelectrochemical studies of cytochrome 0.
Such measurements allow one to probe the amino acid Side chain and peptide
backbone vibrations as a function of the redox state of the protein.171 The
preparation and application of a diamond/Si OTE in the mid- and far-IR
transmission spectroelectrochemical measurements was recently reportedm'172
The results represent a ﬁrst-step toward the ultimate goal of applying the
spectroelectrochemical method to study metal-ligand vibrations in

metalloproteins.

1.3 The Importance of Studying Structure-Function

Relationships of Metalloproteins

Metalloproteins, about one-third of all proteins, are biomolecules that
contain metal cofactors. A metal cofactor is either loosely or tightly bound to a
protein and can be essential for the protein’s biological functions. Metalloproteins
are generally composed of protein atoms (carbon, nitrogen, oxygen, hydrogen,
sulfur) and metal ions, such as iron, copper, calcium, zinc, magnesium, and so
on. Iron and copper metalloproteins are usually referred to electron-transfer

redox proteins, such as mononuclear copper proteins (cupredoxins),

13

mononuclear iron proteins (rubredoxin and desulforedoxin), iron-sulfur proteins
(ferredoxins), and heme proteins (cytochromes).173 Hemoglobin, for example,

which carries oxygen in the bloodstream, is an iron-containing metalloprotein.

Metalloproteins are involved in all fundamental biological processes
including respiration, photosynthesis, regulation of transcription and translation,
and metabolism.”3 Thus, metalloproteins make life on Earth possible and the
ability to understand and ultimately control the binding and activity of protein
metal sites is of great biological and medical importance. For example,
myoglobin functions to transport and store oxygen and it has been used as a
model system for the study of heme proteins. This protein has been studied
extensively by a variety of techniques, such as spectroscopy, crystallography,

and computation."“’183

However, the structure and its relationship to the function of many
metalloproteins is still unclear. Due to their biological importance, structure-
function relationships of metalloproteins are of considerable interest?“186 The
metal ions in metalloproteins are critical to the protein's function, structure and
stability. In fact, numerous essential biological functions require metal ions, and
most of the metal ion functions involve metalloproteins. Metal-substituted heme
proteins, which are created by replacing the heme iron with copper, cobalt or
manganese, have been studied to elucidate the functional role of the heme iron

in biological processes.""'190 Biological ligands that bind to metalloproteins, such

14

as NO, 02, CO and H2, are very important in order for the metalloproteins to
function properly in respiration, catalysis and signal transduction.182 One of the
major structural determinants of heme protein functional properties is the identity
of the axial ligands of the protein which form the coordination environment of the
heme iron.191 The ligand dissociation pathways and protein structural change are
key issues in understanding protein function.192 It is almost certain that there are
intermediate states along the pathway. Site-directed mutagenesis or semi-
synthesis have been employed to change the axial ligands of heme proteins in

order to study their functions.193'201

1.4 Na*-Translocating NADH:Quinone Oxidoreductase

Vibrio cholerae, a motile bacterium with a single polar ﬂagellum, is a
causative agent of the potentially lethal disease cholera.202 The ability of the
bacterium to use sodium in bioenergetic processes plays a key role in both
adaptation to different environments and pathogenic function.202 This halophilic
organism has a strict requirement of Na” for its growth, with an optimal pH
ranging from 7 to 9 depending on the salinity.203 The enzyme that transports Na+
across the cell membrane is essential for providing energy to the cell and for

maintaining a constant intracellular environment.

Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) acts as a
redox-driven sodium pump in prokaryotes, such as marine bacteria (e.g., Vibn'o

alginolyticus) and human pathogenic bacteria (e.g., Vibrio cholerae).204 This

15

membrane-bound enzyme couples the electron transfer from NADH to
ubiquinone with Na+ translocation across the membrane. It catalyzes the same
redox reaction as the H+-pumping NADH:quinone oxidoreductase (complex I).
However, the two enzymes Share no homology.205 The efﬁciency of Na+
translocation by the Na"-NQR has been shown to be 1 Na“/ 1 e'.206

NADH + H”in+ Q + 2 Naiin —> NAD"+ 0H2 + 2 Na‘out
The electrochemical sodium gradient generated by Na*-NQR provides the
energy for cellular metabolic function such as driving bacterial ﬂagellum and

transporting nutrients.2°7' 208

The mechanism of energy transformation between the redox transitions
and sodium translocation across the membrane of Na*-NQR is still unknown.
Na+-NQR is a favorable system to study the mechanism of a redox-driven ion
pump. For example, as the redox transitions often involve H+ uptake and release,
the concentration of Na+ can be changed over a wide range without changing the
pH or causing a deleterious effect, allowing the variables to be examined
independently. We would like to use the transmission spectroelectrochemical
method together with global nonlinear regression analysis, to characterize the
redox potentials of the cofactors in Na+-NQR and to determine their Na+
sensitivity. The spectra with the global ﬁtting can also resolve redox centers with

similar thermodynamic parameters.

16

1.5 Speciﬁc Aims

The long-term goal of this project is to develop a transmission
spectroelectrochemical method using a diamond OTE to study structure-function
relationships of redox-linked metalloproteins. We desire to use the technique to
elucidate structural changes in redox systems as a function of the oxidation state.

The speciﬁc aims of the research are listed below:

1. To investigate the electrochemical response of cytochrome c at boron-
doped diamond electrodes as a function of surface chemistry, ﬁlm morphology
and solution pH. This aim will test the hypothesis that boron-doped diamond
electrodes with different surface terminations should exhibit different
performance in electrochemistry of cytochrome c;

2. To characterize a thin—layer transmission cell and study
spectroelectrochemical behavior of model redox proteins in the cell;

3. To apply the spectroelectrochemical method together with global
regression analysis to study a multicomponent redox protein, Na"-translocating
NADH:quinone oxidoreductase. This aim will test the hypothesis that the
spectroelectrochemical titrations can be used to determine redox potentials for

the protein with multiple cofactors.

17

1.6 Organization of the Dissertation

Chapter 1 gives background information about spectroelectrochemical
methods, describes development of optically transparent boron-doped diamond
electrodes and introduces the targeted redox protein. Chapter 2 details the
experimental parameters and procedures used in the work, including the

methods, instrumentation and materials.

Chapter 3 presents the direct electrochemistry of horse heart cytochrome
c recorded with UNCD and MCD electrodes. A key ﬁnding was that well-defined
electrochemical behavior was observed at oxidized boron-doped diamond
electrode surfaces, but not at hydrogen-terminated electrodes. In Chapter 4, a
specially designed thin-layer transmission cell is reported. It was
electrochemically characterized with a glassy carbon electrode and employed in
the spectroelectrochemical measurements of several model redox proteins. In
Chapter 5, the spectroelectrochemical method and global nonlinear regression
analysis were employed to study a redox protein (Na"-NQR) with several

cofactors.

Finally, Chapter 6 summarizes the key ﬁndings from this work and

provides some future perspectives.

18

1.7

(1)

(2)

(3)

(4)

(5)

(6)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

References

Kuwana, T.; Darlington, K. R.; Leedy, D. W. Anal. Chem. 1964, 36, 2023-
2025.

Heineman, W. R. Anal. Chem. 1978, 50, 390A-402A.
Heineman, W. R. J. Chem. Educ. 1983, 60, 305-308.

Heineman, W. R.; Hawkridge, F. M.; Blount, H. N. In Electroanalytical
Chemistry, Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 1-
113.

Bard, A. J.; Faulkner, L. R. In Electrochemical Methods: Fundamentals
and Applications, 2 ed.; John Wiley & Sons: New York, 2001, pp 680-735.

Kaim, W.; Klein, A. Spectroelectrochemistry, Royal Society of Chemistry
2008.

Wansapura, C. M.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2007, 79,
5594-5600.

Conklin, S. D.; Heineman, W. R.; Seliskar, C. J. Electroanalysis 2007, 19,
523-529.

Heineman, W. R.; Seliskar, C. J.; Morris, L. K.; Andria, S. E.; Wansapura,
C.; Shtoyko, T. ECS Trans. 2006, 3, 5-12.

Shtoyko, T.; Richardson, J. N.; Seliskar, C. J.; Heineman, W. R.
Electrochim. Acta 2005, 50, 31 91 -31 99.

Conklin, S. D.; Heineman, W. R.; Seliskar, C. J. Electroanalysis 2005, 17,
1433-1440.

Andria, S. E.; Richardson, J. N.; Kaval, N.; Zudans, l.; Seliskar, C. J.;
Heineman, W. R. Anal. Chem. 2004, 76, 3139-3144.

Stegemiller, M. L.; Heineman, W. R.; Seliskar, C. J.; Ridgway, T. H.; Bryan,
S. A.; Hubler, T.; Sell, R. L. Environ. Sci. Technol. 2003, 37, 123-130.

Monk, D. J.; Ridgway, T. H.; Heineman, W. R.; Seliskar, C. J.
Electroanalysis 2003, 15, 1198-1203.

Kaval, N.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2003, 75, 6334-
6340.

19

(15)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(25)

(27)

(28)

(29)

(30)

(31)

Heineman, W. R.; Seliskar, C. J.; Richardson, J. N. Aust. J. Chem. 2003,
56, 93-102.

Richardson, J. N.; Dyer, A. L.; Stegemiller, M. L.; Zudans, l.; Seliskar, C.
J.; Heineman, W. R. Anal. Chem. 2002, 74, 3330-3335.

Maizels, M.; Seliskar, C. J.; Heineman, W. R.; Bryan, S. A. Electroanalysis
2002, 14, 1345-1352.

Ross, S. E.; Seliskar, C. J.; Heineman, W. R.; Aryol, S.; Nevin, J. H. Proc.
- Electrochem. Soc. 2001, 2001-18, 499-510.

630, L.; Seliskar, C. J.; Heineman, W. R. Electroanalysis 2001, 13, 613-
620.

Slaterbeck, A. F.; Stegemiller, M. L.; Seliskar, C. J.; Ridgway, T. H.;
Heineman, W. R. Anal. Chem. 2000, 72, 5567-5575.

Ross, S. E.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2000, 72, 5549-
5555.

Maizels, M.; Seliskar, C. J.; Heineman, W. R. Electroanalysis 2000, 12,
1356-1362.

DiVirgilio-Thomas, J. M.; Heineman, W. R.; Seliskar, C. J. Anal. Chem.
2000, 72, 3461-3467.

Slaterbeck, A. F.; Ridgway, T. H.; Seliskar, C. J.; Heineman, W. R. Anal.
Chem. 1999, 71, 1196-1203.

Gao, L.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1999, 71, 4061-
4068.

Slaterbeck, A. F.; Shi, Y.; Seliskar, C. J.; Ridgway, T. H.; Heineman, W. R.
Proc. - Electrochem. Soc. 1997, 97-19, 50-60.

Shi, Y.; Slaterbeck, A. F.; Seliskar, C. J.; Heineman, W. R. Anal. Chem.
1997, 69, 3679-3686.

Shi, Y.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 4819-
4827.

Doherty, W. J., lll; Wysocki, R. J., Jr.; Armstrong, N. R.; Saavedra, S. S. J.
Phys. Chem. B 2006, 110, 4900-4907.

Doherty, W. J., Ill; Wysocki, R. J.; Armstrong, N. R.; Saavedra, S. S.
Macromolecules 2006, 39, 4418-4424.

20

(32)
(33)

(34)
(35)

<36)
<37)
(38)
(39)
(40)
(41)
(42)
(43)
(44>
<45)

(46)
(47)

(48)

Araci, Z. O.; Runge, A. F.; Doherty, W. J., lll; Saavedra, S. S. Isr. J. Chem.

2006, 46, 249-255.

Ge, C.; Doherty, W. J.; Mendes, S. 8.; Armstrong, N. R.; Saavedra, S. S.
Talanta 2005, 65, 1126-1 131.

Runge, A. F.; Saavedra, S. S. Langmuir 2003, 19, 9418-9424.

Doherty, W. J., Ill; Donley, C. L.; Armstrong, N. R.; Saavedra, S. S. Appl.
Spectrosc. 2002, 56, 920-927.

Dunphy, D. R.; Mendes, S. B.; Saavedra, S. S.; Armstrong, N. R. Anal.
Chem. 1997, 69, 3086-3094.

Lopez-Palacios, J.; Colina, A.; Heras, A.; Ruiz, V.; Fuente, L. Anal. Chem.
2001, 73, 2883-2889.

Ruiz, V.; Colina, A.; Heras, A.; Lopez-Palacios, J.; Seeber, R. Helv. Chim.
Acta 2001, 84, 3628-3642.

Colina, A.; Lopez-Palacios, J.; Heras, A.; Ruiz, V.; Fuente, L. J.
Electroanal. Chem. 2003, 553, 87-95.

Lopez-Palacios, J.; Heras, A.; Colina, A.; Ruiz, V. Electrochim. Acta 2004,
49, 1027-1033.

Ruiz, V.; Colina, A.; Heras, A.; Lopez-Palacios, J. Electrochim. Acta 2004,
50, 59—67.

Lopez-Palacios, J.; Munoz, E.; Heras, M. A.; Colina, A.; Ruiz, V.
Electrochim. Acta 2006, 52, 234-239.

Orcajo, 0.; Ventosa, E.; Martinez, A.; Colina, A.; Heras, A.; Ruiz, V.;
Lopez-Palacios, J. J. Electroanal. Chem. 2006, 596, 95-100.

Ruiz, V.; Colina, A.; Heras, A.; Lopez-Palacios, J. Polym. Degrad. Stab.
2006, 91, 3117-3123.

Palacios, R. E.; Fan, F.-R. F.; Bard, A. J.; Barbara, P. F. J. Am. Chem.
Soc. 2006, 128, 9028-9029.

Lei, C.; Hu, D.; Ackerman, E. J. Chem. Commun. 2008, 5490-5492.

Moss, 0.; Nabedryk, E.; Breton, J.; Mantele, W. Eur. J. Biochem. 1990,
187, 565-572.

Schlereth, D. D.; Mantele, W. Biochemistry 1992, 31, 7494-7502.

21

(49)

(50)
(51)
(52)

(53)
(54)

(55)

(55)

(57)

(58)

(59)

(50)

(51)

(52)

(53)

(54)

(55)

Brischwein, M.; Scharf, B.; Engelhard, M.; Mantele, W. Biochemistry 1993,
32, 13710-13717.

Leonhard, M.; Mantele, W. Biochemistry 1993, 32, 4532—4538.
Mantele, W. Trends Biochem. Sci. 1993, 18, 197-202.

Schlereth, D. D.; Fernandez, V. M.; Mantele, W. Biochemistry 1993, 32,
9199-9208.

Schlereth, D. D.; Mantele, W. Biochemistry 1993, 32, 1118-1126.

Hellwig, P.; Rost, 8.; Kaiser, U.; Ostermeier, C.; Michel, H.; Mantele, W.
FEBS Lett. 1996, 385, 53-57.

Behr, J.; Hellwig, P.; Mantele, W.; Michel, H. Biochemistry 1998, 37, 7400-
7406.

Hellwig, P.; Behr, J.; Ostermeier, C.; Richter, O. M.; Pﬁtzner, U.;
Odenwald, A.; Ludwig, 8.; Michel, H.; Mantele, W. Biochemistry 1998, 37,
7390-7399.

Hellwig, P.; Ostermeier, C.; Michel, H.; Ludwig, B.; Mantele, W. Biochim.
Biophys. Acta, Bioenerg. 1998, 1409, 107-112.

Baymann, F.; Robertson, D. E.; Dutton, P. L.; Mantele, W. Biochemistry
1999, 38, 13188-13199.

Hellwig, P.; Grzybek, S.; Behr, J.; Ludwig, 8.; Michel, H.; Mantele, W.
Biochemistry 1999, 38, 1685-1694.

Hellwig, P.; Mogi, T.; Tomson, F. L.; Gennis, R. B.; lwata, J.; Miyoshi, H.;
Mantele, W. Biochemistry 1999, 38, 14683-14689.

Hellwig, P.; Soulimane, T.; Buse, G.; Mantele, W. FEBS Lett. 1999, 458,
83-86.

Hellwig, P.; Soulimane, T.; Buse, G.; Mantele, W. Biochemistry 1999, 38,
9648-9658. .

Kuglstatter, A.; Hellwig, P.; Fritzsch, G.; Wachtveitl, J.; Oesterhelt, D.;
Mantele, W.; Michel, H. FEBS Lett. 1999, 463, 169-174.

Taneva, S. G.; Kaiser, U.; Donchev, A. A.; Dimitrov, M. l.; Mantele, W.;
Muga, A. Biochemistry 1999, 38, 9640-9647.

Friedrich, T.; Brors, B.; Hellwig, P.; Kintscher, L.; Rasmussen, T.; Scheide,

D.; Schulte, U.; Mantele, W.; Weiss, H. Biochim. Biophys. Acta, Bioenerg.
2000, 1459, 305-309.

22

(55)

(57)

(53)

(59)

(70)

(71)

(72)

(73)

(74)

(75)
(75)

(77)

(78)
(79)

(30)
(81)
(82)
(83)

Hellwig, P.; Scheide, D.; Bungert, S.; Mantele, W.; Friedrich, T.
Biochemistry 2000, 39, 10884-10891 .

Hellwig, P.; Rost, B.; Mantele, W. Spectrochim. Acta, Part A 2001, 57A,
1123-1131.

Hellwig, P.; Pﬁtzner, U.; Behr, J.; Rost, B.; Pesavento, R. P.; Donk, W. v.;
Gennis, R. 8.; Michel, H.; Ludwig, B.; Mantele, W. Biochemistry 2002, 41,
9116-9125.

Hellwig, P.; Soulimane, T.; Mantele, W. Eur. J. Biochem. 2002, 269, 4830-
4838.

Ritter, M.; Anderka, O.; Ludwig, B.; Mantele, W.; Hellwig, P. Biochemistry
2003, 42, 12391-12399.

Wille, G.; Ritter, M.; Friedemann, R.; Mantele, W.; Hubner, G.
Biochemistry 2003, 42, 14814-14821 .

Ritter, M.; Palsdottir, H.; Abe, M.; Mantele, W.; Hunte, C.; Miyoshi, H.;
Hellwig, P. Biochemistry 2004, 43, 8439-8446.

Haas Alexander, H.; Sauer Ursula, 8.; Gross, R.; Simon, J.; Mantele, W.;
Lancaster, C. R. D. Biochemistry 2005, 44, 13949-13961.

Mileni, M.; Haas, A. H.; Mantele, W.; Simon, J.; Lancaster, C. R. D.
Biochemistry 2005, 44, 16718-16728.

Bernad, S.; Mantele, W. Anal. Biochem. 2006, 351, 214-218.

Hellwig, P.; Bohm, A.; Pﬁtzner, U.; Mantele, W.; Ludwig, B. Biochim.
Biophys. Acta 2008, 1777, 220-226.

Berthomieu, C.; Marboutin, L.; Dupeyrat, F.; Bouyer, P. Biopolymers 2006,
82, 363-367.

Dai, Y.; Swain, G.; Porter, M. D.; Zak, J. Anal. Chem. 2008, 80, 14-22.

Murray, R. W.; Heineman, W. R.; O'Dom, G. W. Anal. Chem. 1967, 39,
1666-1668.

Petek, M.; Neal, T. E.; Murray, R. W. Anal. Chem. 1971, 43, 1069-1074.
Blaedel, W. J.; Boyer, S. L. Anal. Chem. 1973, 45, 258-263.

Norvell, V. E.; Mamantov, G. Anal. Chem. 1977, 49, 1470-1472.
Sorrels, J. W.; Dewald, H. D. Anal. Chem. 1990, 62, 1640-1643.

23

(84)
(85)
(85)

(37)
(38)
(89)

(90)

(91)
(92)

(93)
(94)

(95)

(96)

(97)

(98)

(99)

(100)
(101)

(102)

Yildiz, A.; Kissinger, P. T.; Reilley, C. N. Anal. Chem. 1968, 40, 1018-1024.
Benken, W. v.; Kuwana, T. Anal. Chem. 1970, 42, 1114-1116.

DeAngelis, T. P.; Hurst, R. W.; Yacynych, A. M.; Mark, H. B.; Jr.; William
R, H.; Mattson, J. S. Anal. Chem. 1977, 49, 1395-1398.

Zak, J.; Porter, M. D.; Kuwana, T. Anal. Chem. 1983, 55, 2219-2222.
Anjo, D. M.; Brown, S.; Wang, L. Anal. Chem. 1993, 65, 317-319.

Donner, S.; Li, H.-W.; Yeung, E. 3.; Porter, M. D. Anal. Chem. 2006, 78,
2816-2822.

Armstrong, N. R.; Lin, A. W. C.; Fujihira, M.; Kuwana, T. Anal. Chem.
1976, 48, 741-750.

Ginley, D. 8.; Bright, C. MRS Bulletin 2000, 25, 15-18.

Donley, C.; Dunphy, D.; Paine, 0.; Carter, 0.; Nebesny, K.; Lee, P.;
Alloway, D.; Armstrong, N. R. Langmuir 2002, 18, 450-457.

Haacke, G. Ann. Rev. Mater. Sci. 1977, 7, 73-93.

Fischer, A. Ph. D. dissertation, Michigan State University, East Lansing,
MI, 2005.

Pankove, J. |.; Qiu, C. In Synthetic Diamond: Emerging CVD Science and
Technology, Spear, K. E., Dismukes, J. P., Eds.; John Wiley & Sons: New
York, 1994.

Swain, G. M.; Anderson, A. B.; Angus, J. C. MRS Bulletin 1998, 23, 56-59.

Swain, G. M. In Electroanalytical Chemistry, Bard, A. J., Rubinstein, l.,
Eds.; Marcel Dekker, 2004; Vol. 22, pp 182-277.

Swain, G. M. In Thin-Film Diamond ll (Semiconductors and Semimetals);
Nebel, C. E., Ristein, J., Eds.; Elsevier, 2004; Vol. 77, pp 121-148.

Sonthalia, P.; MCGaw, E.; Show, Y.; Swain, G. M. Anal. Chim. Acta 2004,
522, 3544.

Fischer, A. E.; Show, Y.; Swain, G. M. Anal. Chem. 2004, 76, 2553-2560.

Bennett, J. A.; Wang, J.; Show, Y.; Swain, G. M. J. Electrochem. Soc.
2004, 151, E306-E313.

Haymond, S.; Babcock, G. T.; Swain, G. M. Electroanalysis 2003, 15, 249-
253.

24

(103) Show, Y.; Witek, M. A.; Sonthalia, P.; Swain, G. M. Chem. Mater. 2003, 15,
879-888.

(104) Chen, 0.; Gruen, D. M.; Krauss, A. R.; Corrigan, T. D.; Witek, M.; Swain,
G. M. J. Electrochem. Soc. 2001, 148, E44-E51.

(105) Granger, M. C.; Witek, M.; Xu, J.; Wang, J.; Hupert, M.; Hanks, A.;
Koppang, M. 0.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.;
Swain, G. M. Anal. Chem. 2000, 72, 3793-3804.

(106) Koppang, M. D.; Witek, M.; Blau, J.; Swain, G. M. Anal. Chem. 1999, 71,
1188-1195.

(107) Granger, M. C.; Swain, G. M. J. Electroanal. Chem. 1999, 146, 4551-4558.
(108) Xu, J.; Swain, G. M. Anal. Chem. 1998, 70, 1502-1510.

(109) Xu, J.; Granger, M. C.; Chen, Q.; Strojek, J. W.; Lister, T. E.; Swain, G. M.
Anal. Chem. 1997, 69, 591A-597A.

(110) James, 8.; Carlisle, J. A.; Auciello, 0.; Gruen, D. M.; Gibson, J. M. Appl.
Phys. Lett. 2002, 81, 2235-2237.

(111) Okano, K.; Kiyota, H.; lwasaki, T.; Nakamura, Y.; Akiba, Y.; Kurosu, T.;
lida, M.; Nakamura, T. Appl. Phys. A:Mater. 1990, 51, 344-346.

(112) Koizumi, S.; Kamo, M.; Sato, Y.; Ozaki, H.; lnuzuka, T. Appl. Phys. Lett.
1997, 71, 1065-1067.

(113) Koizumi, S. Phys. Status SolidiA 1999, 172, 71-78.

(114) Haubner, R. Diem. Relat. Mater. 2005, 14, 355-363.

(115) Bachmann, P. K.; Leers, D.; Lydtin, H. Diam. Relat. Mater. 1991, 1, 1-12.
(116) Liu, H.; Dandy, D. S. Diam. Relat. Mater. 1995, 4, 1173-1188.

(117) Ashfold, M. N. R.; May, P. W.; Rego, C. A.; Everitt, N. M. Chem. Soc. Rev.
1994, 21-30.

(118) Butler, J. E.; Sumant, A. V. Chemical Vapor Deposition 2008, 14, 145-160.

(119) Martin, H. B.; Eaton, S. C.; Landau, U.; Angus, J. C. Diamond
Electrochem. 2005, 26-50.

(120) Angus, J. C.; Pleskov, Y. V.; Eaton, S. C. Semiconductors and
Semimetals 2004, 77, 97-119.

25

(121) Eaton, S. C.; Anderson, A. B.; Angus, J. C.; Evstefeeva, Y. E.; Pleskov, Y.

V. Diamond Relat. Mater. 2003, 12, 1627-1632.

(122) Albu, T. V.; Anderson, A. B.; Angus, J. C. J. Electrochem. Soc. 2002, 149,

E143-E147.

(123) Angus, J. C.; Martin, H. B.; Landau, U.; Evstefeeva, Y. E.; Miller, B.;

Vinokur, N. New Diamond Front. Carbon Technol. 1999, 9, 175-187.

(124) Martin, H. B.; Smith, B. A.; Angus, J. C.; Landau, U.; Anderson, A. B.

(125)

(126)

(127)

(128)

(129)

(130)

(131)

(132)

(133)

(134)

(135)

Mater. Res. Soc. Symp. Proc. 1999, 555, 217-226.

Angus, J. C.; Evans, E. A. Mater. Res. Soc. Symp. Proc. 1994, 349, 385-
390.

Angus, J. C.; Argoitia, A.; Get, R.; Li, Z.; Sunkara, M.; Wang, L.; Wang, Y.
Thin Film Diamond 1994, 1-14.

Angus, J. C. Synth. Diamond: Emerging CVD Sci. Technol. 1994, 21-39.

Chakrapani, V.; Anderson, A. B.; Angus, J. C. AlChE Annual Meeting,
Conference Proceedings, Cincinnati, OH, United States, Oct. 30-Nov. 4,
2005 2005, 3950/391-3950/310.

Chakrapani, V.; Angus, J. C.; Anderson, A. B.; Sumanasekera, G. U.
Mater. Res. Soc. Symp. Proc. 2007, 956, 89-96.

Chakrapani, V.; Angus, J. C.; Anderson, A. B.; Wolter, S. D.; Stoner, B. R.;
Sumanasekera, G. U.; Landstrass, M. l.; Ravi, K. V.; Maier, F.; Riedel, B.;
Mantel, J.; Ristein, J.; Ley, L. Science (Washington, DC, United States)
2007, 318, 1424-1430.

Toghill, K. E.; Wildgoose, G. G.; Moshar, A.; Mulcahy, C.; Compton, R. G.
Electroanalysis 2008, 20, 1731 -1 737.

Rassaei, L.; Sillanpaa, M.; French, R. W.; Compton, R. G.; Marken, F.
Electroanalysis 2008, 20, 1286-1292.

Omanovic, D.; Kwokal, 2.; Goodwin, A.; Lawrence, A.; Banks, C. E.;
Compton, R. G.; Komorsky-Lovric, S. J. Iran. Chem. Soc. 2006, 3, 128-
139.

Lawrence, N. S.; Pagels, M.; Meredith, A.; Jones, T. G. J.; Hall, C. E.;
Pickles, C. S. J.; Godfried, H. P.; Banks, C. E.; Compton, R. G.; Jiang, L.
Talanta 2006, 69, 829-834.

Ji, X.; Buzzeo, M. 0.; Banks, C. E.; Compton, R. G. Electroanalysis 2006,
18, 44-52.

26

(136) Wang, Y.; Novotny, M.; Quaiserova-Mocko, V.; Swain, G. M.; Wang, D. H.
Am. J. Physiol. 2008, 294, R1517-R1523.

(137) Muna, G. W.; Swope, V. M.; Swain, G. M.; Porter, M. D. J. Chromatogr., A
2008, 1210, 154-159.

(138) Song, Y.; Swain, G. M. Anal. Chem. 2007, 79, 2412-2420.
(139) MCGaw, E. A.; Swain, G. M. Anal. Chim. Acta 2006, 575, 180-189.

(140) Rao, T. N.; Yagi, l.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal. Chem. 1999,
71 , 2506-2511.

(141) Yamada, D.; lvandini, T. A.; Komatsu, M.; Fujishima, A.; Einaga, Y. J.
Electroanal. Chem. 2008, 615, 145-153.

(142) Wei, M.; Zhou, Y.; Zhi, J.; Fu, 0.; Einaga, Y.; Fujishima, A.; Wang, X.; Gu,
Z. Electroanalysis 2008, 20, 137-143.

(143) Wei, M.; Sun, L.-G.; Xie, Z.-Y.; Zhii, J.-F.; Fujishima, A.; Einaga, Y.; Fu,
D.-G.; Wang, X.-M.; Gu, Z.-Z. Adv. Funct. Mater. 2008, 18, 1414-1421.

(144) Preechaworapun, A.; lvandini, T. A.; Suzuki, A.; Fujishima, A.; Chailapakul,
O.; Einaga, Y. Anal. Chem. 2008, 80, 2077-2083.

(145) Kondo, T.; Tamura, A.; Fujishima, A.; Kawai, T. ECS Trans. 2008, 16,
465-473.

(146) Kondo, T.; Niwano, Y.; Tamura, A.; lvandini, T. A.; Einaga, Y.; Tryk, D. A.;
Fujishima, A.; Kawai, T. Electroanalysis 2008, 20, 1556-1564.

(147) Chiku, M.; lvandini, T. A.; Kamiya, A.; Fujishima, A.; Einaga, Y. J.
Electroanal. Chem. 2008, 612, 201 -207.

(148) Tryk, D. A.; Tachibana, H.; lnoue, H.; Fujishima, A. Diam. Relat. Mater.
2007, 16, 881-887.

(149) Terashima, C.; Fujishima, A. Compr. Anal. Chem. 2007, 49, 211-232.
(150) Spataru, T.; Spataru, N.; Fujishima, A. Talanta 2007, 73, 404-406.

(151) lvandini, T. A.; Sato, R.; Makide, Y.; Fujishima, A.; Einaga, Y. Anal. Chem.
2006, 78, 6291-6298.

(152) Dragoe, D.; Spataru, N.; Kawasaki, R.; Manivannan, A.; Spataru, T.; Tryk,
D. A.; Fujishima, A. Electrochim. Acta 2006, 51 , 2437-2441.

(153) Fujishima, A.; Einaga, Y.; Rao, T. N.; Tryk, D. A. Diamond
Electrochemistry, 2005.

27

(154) Rao, T. N.; Sarada, B. V.; Fujishima, A. Advances in Surface Treatment:
Research & Applications (ASTRA), Proceedings of the lntemational
Conference, Hyderabad, India, Nov. 3-6, 2003 2004, 716-722.

(155) Rao, T. N.; Loo, B. H.; Sarada, B. V.; Terashima, C.; Fujishima, A. Anal.
Chem. 2002, 74, 1578-1583.

(156) Yagi, l.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal.
Chem. 1999, 473, 173-178.

(157) Quaiserova-Mocko, V.; Novotny, M.; Schaefer, L. S.; Fink, G. D.; Swain, G.
M. Electrophoresis 2008, 29, 441-447.

(158) Park, J.; Quaiserova-Mocko, V.; Patel, B. A.; Novotny, M.; Liu, A.; Bian, X.;
Galligan, J. J.; Swain, G. M. Analyst (Cambridge, United Kingdom) 2008,
133, 17-24.

(159) Park, J.; Galligan, J. J.; Fink, G. D.; Swain, G. M. J. Physiol. 2007, 584,
819-834.

(160) Novotny, M.; Quaiserova-Mocko, V.; Wehnrvein, E. A.; Kreulen, D. L.;
Swain, G. M. J. Neurosci. Methods 2007, 163, 52-59.

(161) Song, Y.; Swain, G. M. Anal. Chim. Acta 2007, 593, 7-12.

(162) Fujishima, A.; Rao, T. N.; Try, D. A. Proc.-Electrochem. Soc. 2000, 99-32,
383-388.

(163) Zaitsev, A. M. Optical Properties of Diamond; Springer-Verlag: Berlin,
2001.

(164) Stotter, J.; Show, Y.; Wang, S.; Swain, G. Chem. of Mater. 2005, 17,
4880-4888.

(165) Martin, H. B.; Morrison, P. W. Electrochem. and Solid-State Left. 2001, 4,
E17-E20.

(166) Martin, H. B.; Morrison, P. W., Jr. Proc. - Electrochem. Soc. 2002, 2001-
25, 66-73.

(167) Zhang, Y.; Kato, Y.; Yoshihara, S.; Watanabe, T. J. Electroanal. Chem.
2007, 603, 135-141.

(168) Zak, J. K.; Butler, J. E.; Swain, G. M. Anal. Chem. 2001, 73, 908-914.

(169) Stotter, J.; Zak, J.; Behler, Z.; Show, Y.; Swain, G. M. Anal. Chem. 2002,
74, 5924-5930.

28

(170) Haymond, S.; Zak, J. K.; Show, Y.; Butler, J. E.; Babcock, G. T.; Swain, G.
M. Anal. Chim. Acta 2003, 500, 137-144.

(171) Stotter, J.; Haymond, S.; Zak, J. K.; Show, Y.; Cvackova, Z.; Swain, G. M.
Interface 2003, 12, 33-38.

(172) Dai, Y.; Proshlyakov, D. A.; Zak, J. K.; Swain, G. M. Anal. Chem. 2007, 79,
7526-7533.

(173) Degtyarenko, K. In Encyclopedia of Genetics, Genomics, Proteomics and
Bioinfonnatics; Lynn Jorde , P. L., Mike Dunn and Shankar Subramaniam
Ed.; John Wiley & Sons, Inc, 2005.

(174) Austin, R. H.; Beeson, K. W.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, l.
C. Biochemistry 1975, 14, 5355-5373.

(175) Alben, J. O.; Beece, D.; Bowne, S. F.; Doster, W.; Eisenstein, L.;
Frauenfelder, H.; Good, 0.; McDonald, J. D.; Marden, M. C.; et al. Proc.
Natl. Acad. Sci. U. S. A. 1982, 79, 3744-3748.

(176) Tilton, R. F., Jr.; Singh, U. C.; Weiner, S. J.; Connolly, M. L.; Kuntz, l. D.,
Jr.; Kollman, P. A.; Max, N.; Case, D. A. J. Mol. Biol. 1986, 192, 443-456.

(177) Elber, R.; Karplus, M. Science (Washington, D. C., 1883-) 1987, 235, 318-
321.

(178) Powers, L.; Chance, 8.; Chance, M.; Campbell, 8.; Friedman, J.; Khalid,
S.; Kumar, 0.; Naqui, A.; Reddy, K. 8.; Zhou, Y. Biochemistry 1987, 26,
4785-4796.

(179) Carlson, M. L.; Regan, R. M.; Gibson, O. H. Biochemistry 1996, 35, 1125-
1136.

(180) Scott, E. E.; Gibson, O. H. Biochemistry 1997, 36, 11909-11917.

(181) Chu, K.; Berendzen, J.; Sweet, R. M; Schlichting, I. Biophys. J. 1999, 77,
2153-2174.

(182) Chu, K.; Vojtchovsky, J.; McMahon, B. H.; Sweet, R. M.; Berendzen, J.;
Schlichting, I. Nature 2000, 403, 921-923.

(183) Antonini, E.; Brunori, M. Hemoglobin and myoglobin in their reactions with
ligands; North-Holland Pub. 00.: Amsterdam, 1971.

(184) Ortiz de Montellano, P. R. Acc. Chem. Res. 1987, 20, 289-294.
(185) Dawson, J. H. Science 1988, 240, 433-439.

29

(186) Barker, P. D.; Ferrer, J. C.; Mylrajan, M.; Loehr, T. M.; Feng, R.; Konishi,
Y.; Funk, W. D.; MacGillivray, R. T.; Mauk, A. G. Proc. Natl. Acad. Sci. U.
S. A. 1993, 90, 6542-6546.

(187) Fabry, T. L.; Simo, C.; Javaherian, K. Biochim. Biophys. Acta 1968, 160,
118-122.

(188) Zomzely, C. E.; Roberts, 3.; Peache, S. Proc. Natl. Acad. Sci. U. S. A.
1970, 67, 644-651.

(189) Yonetani, T.; Drott, H. R.; Leigh, J. 8., Jr.; Reed, G. H.; Waterman, M. R.;
Asakura, T. J. Biol. Chem. 1970, 245, 2998-3003.

(190) Miller, L. M.; Chance, M. R. Biochemistry 1995, 34, 10170-10179.

(191) Lloyd, E.; Hildebrand, D. P.; Tu, K. M.; Mauk, A. G. J. Am. Chem. Soc.
1995, 117, 6434-6438.

(192) Nishihara, Y.; Sakakura, M.; Kimura, Y.; Terazima, M. J. Am. Chem. Soc.
2004, 126, 11877-11888.

(193) Sorrell, T. N.; Martin, P. K.; Bowden, E. F. J. Am. Chem. Soc. 1989, 111,
766-767.

(194) Carver, T. E.; Rohlfs, R. J.; Olson, J. 8.; Gibson, O. H.; Blackmore, R. S.;
Springer, B. A.; Sligar, S. G. J. Biol. Chem. 1990, 265, 20007-20020.

(195) Pakrasi, H.; De Ciechi, P.; Whitmarsh, J. EMBO J. 1991, 10, 1619-1627.

(196) Adachi, S.; Nagano, S.; Watanabe, Y.; lshimori, K.; Morishima, I. Biochem.
Biophys. Res. Commun. 1991, 180, 138-144.

(197) Van Dyke, B. R.; Bakan, D. A.; Glover, K. A. M.; Hegenauer, J. C.;
Saltman, P.; Springer, B. A.; Sligar, S. G. Proc. Natl. Acad. Sci. U. S. A.
1992, 89, 8016-8019.

(198) Adachi, S.; Nagano, S.; lshimori, K.; Watanabe, Y.; Morishima, |.; Egawa,
T.; Kitagawa, T.; Makino, R. Biochemistry 1993, 32, 241-252.

(199) Smulevich, G.; Hu, 8.; Rodgers, K. R.; Goodin, D. B.; Smith, K. M.; Spiro,
T. G. Biospectroscopy1996, 2, 365-376.

(200) Ozaki, S.-i.; Roach, M. P.; Matsui, T.; Watanabe, Y. Acc. Chem. Res.
2001, 34, 818-825.

(201) Smulevich, G. Physical Chemistry 2006, Proceedings of the lntemational

Conference on Fundamental and Applied Aspects of Physical Chemistry,
8th, Belgrade, Serbia, Sept. 26-29, 2006 2006, 1, 31-37.

30

(202) Hase, C. C.; Barquera, B. Biochim. Biophys. Acta 2001, 1505, 169-178.
(203) Miller, C. J.; Drasar, B. S.; Feachem, R. G. J. Hyg. 1984, 93, 475-495.

(204) Turk, K.; Puhar, A.; Neese, F.; Bill, E.; Fritz, G.; Steuber, J. J. Biol. Chem.
2004, 279, 21349-21355.

(205) Juarez, 0.; Morgan, J. E.; Barquera, B. J. Biol. Chem. 2009, 284, 8963-
8972.

(206) Bogachev, A. V.; Bertsova, Y. V.; Aitio, O.; Permi, P.; Verkhovsky, M. l.
Biochemistry 2007, 46, 10186-10191 .

(207) Barquera, 8.; Zhou, W.; Morgan, J. E.; Gennis, R. B. Proc. Natl. Acad. Sci.
U. S. A. 2002, 99, 10322-10324.

(208) Barquera, B.; Hellwig, P.; Zhou, W.; Morgan, J. E.; Hase, C. C.; Gosink, K.

K.; Nilges, M.; Bruesehoff, P. J.; Roth, A.; Lancaster, C. R. D.; Gennis, R.
B. Biochemistry 2002, 41, 3781-3789.

31

CHAPTER 2

EXPERIMENTAL METHODS

2.1 Electrode Materials

2.1.1 Boron-Doped Diamond

The diamond thin-ﬁlm electrode was deposited by microwave-assisted
chemical vapor deposition (1.5 kW, 2.54 GHz, Astex, Inc., Lowell, MA).1 The

diagram of a typical reactor is shown in Figure 2.1.

m crowave
generator

 

 

 

 

   
 
 

quartz
chamber \ \

...— substrate

 

 

gas inlet ——>‘

rotary pump

Figure 2.1 A diagram of a microwave plasma-assisted CVD reactor used to
grow boron-doped diamond thin-ﬁlm electrodes.

32

All source gases were ultrahigh purity (99.999%) grade (CH4 and H2 from
AGA Specialty Gas, Cleveland, OH; Ar from BOC Group, Inc, Murray Hill, NJ;
0.1% B2H5 diluted in H2 from Matheson Gas Products, Inc.). The gases were
introduced into the chamber at controlled rates regulated by mass ﬂow
controllers. The microwave plasma was then ignited inside a quartz cavity with
the ﬂowing source gas mixture. The plasma contains the reactive species
important for growth. The pressure of the chamber was regulated using a throttle

exhaust valve in series with a rotary pump.

Before growth, the silicon substrate was mechanically scratched for 5 min
on a felt polishing pad using a 100 nm diameter diamond powder (Diamond
Innovations, Worthington, OH) suspended in ultrapure water. The scratched
substrate was then ultrasonically cleaned for 5 min each in ultrapure water, 2-
propanol, acetone, 2-propanol, and ultrapure water to remove polishing debris
from the surface, partially from the scratches or striations. The substrate was
then dried and examined under an Olympus BX60M optical microscope
(Olympus America, Inc.) for Cleanliness. Embedded diamond powder particles as
well as the scratching defects serve as the initial nucleation Sites for diamond
growth. For the quartz substrate, it was scratched similarly as the silicon
substrate using a 100 nm diameter diamond (Diamond Innovations, Worthington,
OH) / water suspension prior to diamond deposition, but on a UltraPrepTM
diamond lapping ﬁlm disc (Buehler, Lake Bluff, IL). The cleaning steps were the

same as those for the silicon substrate.

33

2.1.1.1 Ultrananocrystalline and Microcrystalline Boron-Doped

Diamond Electrodes (UNCD and MCD)

UNCD and MCD thin ﬁlms (not optically transparent) were deposited on
highly conductive p-type Si (100) substrates (~10'3 Q-Cm, 0.05 cm thick x 1 cm2
area, Virginia Semiconductor, Inc., Fredericksburg, VA). The UNCD ﬁlms were
deposited using gas ﬂow rates of 1, 94, 5 sccm for CH4, Ar, H2, respectively.
Deposition was accomplished using a power of 800 W, a system pressure of 140
Torr, a substrate temperature of ~750 °C and a deposition time of 2 h. The MCD
ﬁlms were deposited using a 0.5% CH4IH2 (v/v) source gas mixture, a total gas
ﬂow of 200 sccm, 1 kW of microwave power, a system pressure of 45 Torr, a
substrate temperature of ~750 °C and a growth time of 10 h. Both the UNCD and
MCD ﬁlms were boron-doped during deposition by adding 10 ppm B2H6 to the
source gas mixture. The Speciﬁc deposition conditions used for growth of each
diamond type are listed in Table 2.1. Following the deposition, the CH4 and B2H5
gas ﬂows were stopped, and the films remained exposed to the Ar/H2 (UNCD) or
H2 (MCD) plasma for an additional 10 min. The plasma power and pressure were
Slowly reduced over a 15 min period to cool the samples in the presence of
atomic hydrogen to a temperature below 300 °C. Post growth annealing in atomic
hydrogen is essential for etching away adventitious nondiamond carbon impurity,
minimizing dangling bonds, and ensuring full hydrogen termination. The plasma
power was then turned off and the ﬁlms were cooled to room temperature under

a ﬂow of Ar/H2 (UNCD) or H2 (MCD). Both ﬁlm types had a nominal boron dopant

34

concentration in the high 1019 to low 1020 B/cm3 range, and a ﬁlm resistivity of

~0.01 Q-cm, or less.

Table 2.1 Growth conditions for UNCD and MCD thin ﬁlms deposited on p-

 

 

type Si.
UNCD MCD
Gas MiXtUl‘G Ar/H2/CH4/B2H5 CH4/H2/BzH5
Total Gas Flow 100 sccm 200 sccm
Methane Concentration 1% CH4 0.5% CH4
Dopant 10 ppm B2H5 10 ppm B2H5
Microwave Power 800 W 1000 W
Chamber Pressure 140 Torr 45 Torr
Substrate Temperature ~750°C ~750°C
Growth Time 2 h 10 h

 

After growth, the diamond ﬁlms were chemically cleaned to remove
adventitious metal and sp2-bonded nondiamond carbon impurities from the
surface. A tvvo-step procedure was employed: (i) exposure to warm 3:1
HCI/HN03 (v/v) for 30 min followed by a rinse with ultrapure water and (ii)
exposure to warm 30% hydrogen peroxide (CCI) for 30 min followed by a rinse
with ultrapure water. The acid oxidatively removes metallic impurity while the
hydrogen peroxide effectively removes nondiamond sp2 carbon impurity. These
chemical treatments introduce surface carbon-oxygen functionalities, and such a
surface is referred to as oxygen-terminated. To regenerate the hydrogen-
terminated surface, the diamond ﬁlms were placed back into the CVD reactor for
a 30-min hydrogen-plasma treatment. The conditions were as follows: a

microwave power of 1000 W, a system pressure of 35 torr, and hydrogen gas

35

ﬂow of 200 sccm. The ﬁlms were slowly cooled in atomic hydrogen, as described

above, over a 15-min period.

2.1.1.2 UV-Vis Transparent Boron-Doped Diamond Electrode

(Diamond/Quartz OTE)

The UV-Vis optically transparent diamond thin ﬁlm was deposited on
quartz using a CH4/H2/B2H5 source gas mixture consisting of 0.5% CH4/H2 with
10 ppm B2H6 added for boron doping, a total gas ﬂow of 100 sccm, a power of
600 W, a system pressure of 45 torr, a substrate temperature of ~450 °C (roughly
estimated with an optical pyrometer), and a growth time of 1 h. The growth
conditions are listed in Table 2.2. At the end of the deposition period, the ﬁlm
remained exposed to a hydrogen plasma for an additional 10 min at 600 W and
45 Torr. The plasma power and pressure were then slowly reduced over a 15-
min period to cool the samples in the presence of atomic hydrogen. The 1-h ﬁlm
was estimated to be 500 nm thick with an electrical resistivity of ~0.026 Q-cm
and an optical transparency of 45-60% between 300 and 800 nm.2 The diamond
ﬁlm received no pretreatment prior to use other than a 20 min soak in distilled
isopropanol. The current ﬂow was laterally through the ﬁlm with an ohmic contact
made along the edge of the electrode. To improve the conductivity, a metallic
layer (50A Ti, 200A Au) was deposited on the surface of the ﬁlm outside the

electrochemical cavity.

36

Table 2.2 Growth conditions for UV-Vis optically transparent diamond electrode.

 

 

Diamond/quartz OTE
Gas Mixture CH4/H2/B2H5
Total Gas Flow 100 sccm
Methane Concentration 0.5% CH4
Dopant 10 ppm B2H6
Microwave Power 600 W
Chamber Pressure 45 Torr
Substrate Temperature ~450°C
Growth Time 1 h

 

2.1.2 Glassy Carbon

The glassy carbon electrode (T okai, GC-20) was prepared by polishing to
a mirror-like ﬁnish with successively smaller grades of alumina powder Slurried in
ultrapure water (1.0, 0.3, and 0.05 pm). The polishing was carried out by hand on
individual felt polishing pads. After each polishing step, the electrode was rinsed
thoroughly with ultrapure water and then ultrasonically cleaned in the same

medium for 20 min to remove polishing debris and clean the surface.

2.1.3 Gold Minigrid

The gold minigrid electrode (Precision Eforming LLC, MG44, 55%
transmission) was modiﬁed prior to use by oxidizing in a freshly prepared

concentrated HZSO4z30% H202 (3:1 v/v) solution for 30 min followed by

incubation in 2 mM cysteamine for 2 h.

37

2.2 Cyclic Voltammetric Experiments

All electrochemical measurements were performed at room temperature in
a single compartment glass cell using a computer-controlled potentiostat (Model
900, CH Instruments, Inc., Austin, TX). A standard three-electrode conﬁguration

was employed, as Shown in Figure 2.2.3

 

Figure 2.2 Design of the single-compartment, three-electrode electrochemical
cell with (a) the diamond ﬁlm working electrode, (b) o-ring seal, (c) rubber spacer
(and nickel foil), (d) reference electrode inside a glass capillary tube with a
cracked tip, (e) carbon rod counter electrode, and (f) input for nitrogen purge gas.

The electrochemical cell was housed inside a grounded Faraday cage for
electrical shielding. The working electrode was clamped against a clean Viton® o-

ring (i.d. 0.5 cm) at the bottom of the cell to conﬁne the solution. The o-ring was

sonicated for 10 min in ultrapure water, rinsed with distilled isopropyl alcohol

38

(IPA), and dried under a steam of N2 before use. Electrical contact was made to
the diamond working electrode by scratching the backside of the Si substrate
with a diamond scribe, and then cleaning and coating the area with graphite from
a pencil to ensure good ohmic contact with the copper current-collector plate.
The geometric area of the electrode exposed to the solution was 0.2 cm2. The
electrode was cleaned prior to use by soaking in IPA for 20 min.“ 5 After
decanting the IPA and rinsing thoroughly with distilled water, the cell was ﬁlled

with the electrolyte solution of interest. A silver/silver chloride reference electrode
(4 M KCI saturated with AgCl, E° = -45 mV vs. SCE) was used, which was placed

inside a cracked—glass capillary ﬁlled with 1 M KCI and positioned in close
proximity to the working electrode. A large area carbon rod served as the counter

electrode and was positioned normal to the working electrode.

2.3 Electrochemical Kinetics

The apparent heterogeneous electron-transfer rate constant, kgpp was

determined from the scan rate dependence of AEp using Nicholson’s theoretical
method.6 According to the theory, AEp for an electrochemical reaction is
dependent on the scan rate (v, V/s), transfer coefﬁcient (a) and heterogeneous
electron-transfer rate constant (kgpp, cm/s). The behavior of quasireversible

reactions can be described by the equation:

39

 

Where Do and DR are the diffusion coefﬁcients of the oxidized and reduced
species (cmzls), respectively, f is the Faraday constant divided by the ideal gas

constant and temperature (F/RT), and \P is a dimensionless parameter. From the

relationship between ‘P and AE,,, kgpp can be calculated from the above equation
at different scan rates. In this research, AEp varied over the scan rate range

tested and the apparent heterogeneous electron-transfer rate constant, kgpp,

was statistically determined from data at several scan rates.

2.4 X-ray Photoelectron Spectroscopy (XPS)

XPS was used to determine the oxygen and carbon atomic ratios (O/C) on
diamond surface.7'9 XPS spectra were obtained using a Physical Electronics PHI
5400 XPS spectrometer with 3 Mg X-ray source. A take-off angle of 45 degrees
was used for all measurements at a base pressure less than 5 x 10'9 Torr.
Software provided with the instrument was used to deconvolute the peaks and
integrate peak areas. The relative atomic percentage of each element was
estimated from peak areas using atomic sensitivity factors speciﬁc for the PHI

5400 instrument.

40

2.5 Spectroelectrochemical Measurements

2.5.1 Thin-Layer Spectroelectrochemical Cell

 

Figure 2.3 Diagram of the thin-layer transmission spectroelectrochemical cell.
A thin layer (TL) is formed between the working electrode (WE) and the
transmission window (not shown) and its thickness is determined by the spacer
(SP). The thin layer is connected to the Pt counter electrode (CE) via electrolyte
capillary (EC) between the transmission window and outer cell body. A miniature
Ag/AgCI reference electrode (RE) is also connected to the electrolyte capillary
via a cracked glass junction (GJ). Reference electrode capillary is also used for
loading sample (SM), which is exhausted via a Similar capillary on the opposite
side (not Shown).

The new transmission thin-layer cell design (Figure 2.3) builds upon an
original design (Mantelle et al.”) with major modiﬁcations to meet desired criteria
for thin-layer cells. The electrochemical volume of the cell is deﬁned by the
narrow space between two planes — typically a working electrode (front) and a
transmission window (back; 9 mm dia. x 5 mm, CaF2 used in this work)
separated by a Kapton® polyimide ﬁlm spacer of the desired thickness (7.5 to 75
pm). A platinum wire ring outside the sealed cavity was used to establish electric

contact with the working electrode surface. The cell was equipped with a

41

miniature Ag/AgCl reference electrode for aqueous solution measurements. The
reference electrode was connected to the side of the back window between the
working and counter electrodes via a cracked glass junction and a 1 mm dia.
capillary. Another Pt wire was employed as the counter electrode. It was either
located in a 1x1 mm groove encircling the back transmission window or coiled in
a separate compartment that was connected to that groove through a PEEK frit

(L5mmdm)

For aerobic measurements, the analyte solution was loaded into the
electrochemical cavity by placing a small sample volume (5-10 Ill.) in the center
of the working electrode immediately prior to assembling the cell. Then the
volume around the window was ﬁlled with the supporting electrolyte. For
anaerobic measurements, the cell was pre-ﬁlled with electrolyte solution and
assembled aerobically. The cell was then deoxygenated by injecting 1-2 mL of an
oxygen-free buffer using a syringe via a capillary port, which opened onto the
sides of the back window Close to the working electrode. Excess solution was
drained via another port located at a diametrically opposing sides of the window.

Finally, several volumes of oxygen-free analyte were then injected into the cavity

and the cell was sealed.

2.5.2 Potential Step Method

Spectroelectrochemical measurements were made by stepping the

potential from a value where the redox system is stable (e.g., reduced form) to

42

values at which a redox reaction (e.g., oxidation) occurs. An absorbance
spectrum was collected at each potential after an equilibration period (usually 1

min), as depicted in Figure 2.4.

 

 

Potential (V) +
9°19

 

 

 

 

 

Time (sec)

Figure 2.4 Scheme for measuring the steady-state absorbance of a redox
analyte as a function of the potential in a spectroelectrochemical measurement.
An absorbance spectrum was recorded at each potential, as indicated by the
arrows.

Absorbance difference spectra were then generated by subtracting the
spectrum for the unreacted redox system from the spectrum obtained at each
potential. Small absorbance changes that occur during the transition between
redox states are not always detectable in the individual spectra. However, they
are in difference absorbance spectra. Figure 2.5 shows an idealized difference

spectrum with the negative-going bands reﬂective of the initial state, A, and

positive-going bands reﬂective of product state B.11

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 :> 4
Wavenumberlcm'1 Wavenumberlcm'1
I J
I
B-A

1OOXAbs.

Ai\
Y

Wavenumberlcm'1

 

 

 

 

 

Figure 2.5 Principle of difference absorbance spectroscopy. The difference
spectrum B-A is generated by subtracting spectrum of initial state A from
spectrum of product state B.11

For redox proteins with multiple cofactors, sufﬁcient time (~10-20 min)
should be given at each potential before measuring the spectrum. Due to the
length of such an experiment, it is necessary to discriminate reversible
electrochemical response from irreversible background changes (e.g.,
photoreactions). To accomplish this, potential step method was revised.
Absorbance Spectra at a reference potential were recorded before and after each
potential step after an equilibration period (e.g., 20 min), as depicted in Figure
2.6. The difference absorbance spectrum is generated by subtracting the
average spectrum at the reference potential right before and after each potential

step.

44

Potential (V)

 

 

 

 

 

(2----
L

 

 

 

Time (sec)

Figure 2.6 Potential-time proﬁle for potential step studies of Na"-NQR.
Absorbance spectra at the reference potential (dashed arrow) were recorded
before and after each potential step (solid arrow). The difference absorbance
Spectrum was generated by subtracting the average of the absorbance spectra at

reference potential from absorbance spectrum at each potential (e.g., AA=A2-
(A1+A3)/2).

2.5.3 UV-Vis Spectroelectrochemical Measurements

The electrochemical measurements were performed at room temperature
using a computer-controlled potentiostat (Model 7603, CH Instruments, Inc.,
Austin, TX). All potentials are reported with respect to the Ag/AgCl electrode (3 M
KCI) unless othenrvise speciﬁed. For chronoamperometric measurements, UV-Vis
spectra were recorded continuously at 0.3-S intervals using a Hewlett Packard
8453 diode array Spectrophotometer (Agilent Technologies, Santa Clara, CA).
The start of the optical and amperometric measurement was electronically
synchronized. For spectroelectrochemical studies of Nai-NQR, UV-Vis

measurements were performed using a custom-built UV-Vis spectrophotometer

45

with a CCD detector. This setup, with a low intensity probe light minimized photo-

induced damage in the thin layer.

2.6 Global Regression Analysis

Global nonlinear regression analysis was employed to resolve redox
potentials and spectra of multiple redox centers with Similar potentials and
spectral signatures. Several model spectra are ﬁrst generated with various
intensities and shapes at different wavelength. Second, Nemst functions are

used to describe the relationship of several variables: applied potential (E), redox
potential (Em), number of electrons transferred (n), and temperature (T). Then a

model 3D surface can be created by combining the model spectra and Nemst
functions. The spectroelectrochemical data were simulated with the modeled
surface. Speciﬁcally, in this research, several models of three to ﬁve redox
transitions involving one to three transferred electrons were applied to the high

resolution spectroelectrochemical data.

2.7 Chemicals

All chemicals were reagent grade quality, or better, and were used without
further puriﬁcation. Solutions of potassium ferrocyanide (Aldrich) in 1 M
potassium chloride (CCI) were prepared fresh daily with ultrapure water (~18

MQ-cm) from a commercial water puriﬁcation system (Bamstead E-pure).

46

Stock solutions of cytochrome c (in 0.3 M NaCl, 10 mM Tris-HCI buffer, pH
7) were prepared by chromatographically purifying and concentrating horse heart
cytochrome 0 (Sigma Aldrich Chemical Co.,).12' ‘3 Cytochrome 0 concentrations
were determined spectrophotometrically at 550 nm from the difference in the
absorbance of the ferrous and ferric forms of the protein using A8550=21 mM'icm‘
1.14'15 Ferricytochrome c was reduced by adding a non-quantitative small amount
of sodium dithionite. The Tris-HCI buffer can be exchanged by repeated dilution
and centrifugations using an Amicon® Ultra-15 Centrifugal ﬁlter device (Millipore).
The phosphate buffer solutions were prepared by mixing appropriate amounts of
K2HPO4 (CCI) and KH2PO4 (Spectrum) to obtain the desired concentration and
pH. It is worth mentioning that the glassware and o-rings exposed to the
cytochrome c solutions were rinsed with 0.1 M HCI solution before the ﬁnal
cleaning step. For cytochrome c-cyanide measurements, a small aliquot of stock
KCN solution was added to the cytochrome 0 solution and pre-incubated for 30

min prior to the experiment.

Horse heart myoglobin (meth) (Sigma) was dissolved in 0.1 M sodium
phosphate buffer solution (pH 7) and centrifuged for 10 min to remove
undissolved material. The supernatant was then puriﬁed on a Sephadex G-75
column (100 mm x 15 mm). The concentration of meth was determined
spectroscopically, either by reducing an aliquot of meth with solid sodium

dithionite to the ferrous form deoxyMb (3435:121 mM'icm'1) or by converting

deoxyMb further to the CO-bound form MbCO (3424:207 mM’icm'1,e540=15.4 mM'

47

‘cm'1 and 2579:139 mM'icm'i). The meth sample was degassed 6-8 cycles
between Ar gas and mild vacuum while stirring. Meth was reduced to deoxyMb
with a minimal amount Of sodium dithionite solution freshly prepared with oxygen-
free water with the reduction being monitored spectroscopically. Gas-tight
syringes, ﬂushed with Ar gas/diluted sodium dithionite solution, were used in

subsequent sample handling.

The stock solution of Na*-translocating NADH:quinone oxidoreductase
(Na*-NQR) in 50 mM sodium phosphate buffer (or 50 mM Tris buffer) (pH 8), 100
mM NaCl, 1 mM EDTA, 5% glycerol and 0.05% dodecylmaltoside (DDM) was
received from the Barquera group.16 It was diluted to the desired protein and Na+
concentrations for the electrochemical studies. EDTA helps to sequester metal
ion impurities in solution; glycerol is used to minimize damage by ice crystals in
frozen solution; and DDM is used to extract the protein from the membrane and

helps protein solubilization.
All glassware and storage bottles were cleaned in a three-step manner:

KOH/ethanol bath, alconox/ultrapure water solution, and ultrapure water rinse.

The cleaned glassware was then dried in an oven at ~50 °C.

48

2.8

(1)

(3)

(4)
(5)

(6)
(7)

(8)

(9)
(10)

(11)
(12)

(13)

(14)
(15)

(16)

References

Swain, G. M. In Electroanalytical Chemistry, Bard, A. J., Rubinstein, |.,
Eds.; Marcel Dekker, 2004; Vol. 22, pp 182-277.

Stotter, J.; Zak, J.; Behler, Z.; Show, Y.; Swain, G. M. Anal. Chem. 2002,
74, 5924-5930.

Granger, M. C.; Witek, M.; Xu, J.; Wang, J.; Hupert, M.; Hanks, A.;
Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.;
Swain, G. M. Anal. Chem. 2000, 72, 3793-3804.

Show, Y.; Witek, M. A.; Sonthalia, P.; Swain, G. M. Chem. Mater. 2003, 15,

879-888.

Ranganathan, S.; Kuo, T.-C.; McCreery, R. L. Anal. Chem. 1999, 71,
3574-3580.

Nicholson, R. S. Anal. Chem. 1965, 37, 1351-1355.

Yagi, l.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal.
Chem. 1999, 473, 173-178.

Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21,
407-413.

Mori, Y.; Kawarada, H.; Hiraki, A. Appl. Phys. Lett. 1991, 58, 940-941.

MOSS, D.; Nabedryk, E.; Breton, J.; Mantele, W. Eur. J. Biochem. 1990,
187, 565-572.

Zscherp, C.; Barth, A. Biochemistry 2001, 40, 1875-1883.

Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. J. Biol. Chem. 1978,
253, 130-139.

Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E.; Sidney, F.; Lester, P.
Methods Enzymol. 1978, 53, 128-164.

Massey, V. Biochim. Biophys. Acta 1959, 34, 255-256.

Van Buuren, K. J. H.; Van Gelder, B. F.; Wilting, J.; Braams, R. Biochim.
Biophys. Acta 1974, 333, 421-429.

Barquera, 3.; Zhou, W.; Morgan, J. E.; Gennis, R. B. Proc. Natl. Acad. Sci.
U. S. A. 2002, 99, 10322-10324.

49

CHAPTER 3
DIRECT ELECTROCHEMISTRY OF CYTOCHROME

c AT BORON-DOPED DIAMOND ELECTRODES

3.1 Introduction

3.1.1 Electrochemical Studies of Cytochrome c

Electron-transfer reactions play an important role in many biological
processes. Cytochrome c is a small (12,384 Da), water-soluble protein that
functions as an electron carrier in the ﬁnal step of electron transport during
oxidative phosphorylation. It has been studied extensively as a model system for
biological electron transfer. It contains a c-type heme prosthetic group, which is
covalently linked to the polypeptide chain via thioether bonds through two
cysteine residues. The structure of the heme c is shown in Figure 3.1. The Fe
atom of heme c is also bonded to two axial ligands (histidine and methionine),
above and below the heme plane, in addition to the four nitrogen atoms of the

planar porphyrin ring system.

50

 

Figure 3.1 Heme 0 structure of cytochrome c. (A) Planar porphyrin ring system;
(B) heme Fe is also bonded to histidine (His) and methionine (Met) axial ligands.
The heme group is largely embedded within the hydrophobic interior of the
globular polypeptide, leaving an edge exposed to solvent.“ 2 The Fe readily
accepts and releases an electron and the electron transfer is believed to occur at
the solvent-exposed edge of the heme by an outer-sphere mechanism,2 which is
only 0.6% of the total surface area of the molecule.3 The large dipole moment of
cytochrome c and the electrostatic ﬁeld of its physiological redox partners cause

the heme to be guided into the proper orientation for effective electron transfer.“ 5

The importance of cytochrome c in the respiratory chain of mitochondria
has inspired biologists and biochemists to elucidate the mechanism of
interprotein electron-transfer process.6 The ﬁrst reports on direct electron transfer

between cytochrome c and a solid electrode appeared in 1977. Eddowes and Hill

51

demonstrated, by cyclic voltammetry, quasi-reversible electron transfer at a 4,4'-
bipyridyl-modiﬁed gold electrode.7 At the same time, Yeh and Kuwana showed
by cyclic voltammetry that direct electron transfer was possible at a tin-doped
indium oxide electrode.8 Since their pioneering work, signiﬁcant advances in the

electrochemistry of redox proteins have been made.

Surface modiﬁcation with promoters is the most popular method employed
for achieving quasi-reversible electrode kinetics. Cytochrome c has been studied
using voltammetric methods under diffusion control and in a surface conﬁned
state. Numerous chemically-functionalized electrode materials have been used:
self-assembled monolayer(SAM)-, 9'12 polymer brush-,13 or gold nanoparticles-14'
’5 modiﬁed gold, single-’G/multi-"walled carbon nanotubes-, fullerene ﬁlms-’8 or
DNA-’9 modiﬁed glassy carbon, clay-modiﬁed pyrolytic graphite,20 amine-
terminated nanodiamonds21 and NHS ester-modiﬁed boron-doped
nanocrystalline diamond.22 The modiﬁers provide speciﬁc binding sites for the
redox protein and control the spacing between the electrode and the protein
(reduce tunneling length), thus facilitating the electron transfer.23 Kinetic studies
at SAM-modiﬁed electrodes showed that the presence of carbonyl functionalities
facilitates the orientation conducive for a reversible electron transfer between the

protein and electrode surface.

Alternatively, other researchers have taken the approach of studying

cytochrome c using bare electrodes without promoters. Gold,24 silver,25' 26 HO,”

52

24, 29 23, 30

28 glassy carbon, and boron-doped diamond“ 32 are the most

graphite,
studied bare electrodes. Highly puriﬁed protein is required to avoid any
contamination of the bare electrode with defunct protein.33 Armstrong and
coworkers reported that the electron-transfer kinetics for cytochrome c at the
edge plane of pyrolytic graphite are much higher compared to the basal plane
due to the high oxygen/carbon (O/C) ratio of the edge plane.3°' 3‘ In general, two
factors seem to be necessary for rapid electron transfer for cytochrome c at bare

electrodes: (i) a pure cytochrome 0 solution and (ii) electrode pretreatment to

produce a clean, hydrophilic surface.” 35

Previously, our group reported a different ﬁnding Of quasi-reversible,
diffusion-controlled voltammetry for cytochrome c at an as-deposited boron-
doped ultrananocrystalline diamond thin-ﬁlm electrode possessing a hydrophobic,
hydrogen-terminated surface.32 Rivas et al. also reported immobilization of
Cytochrome c on Ag electrodes that were coated with a hydrophobic self-
assembled monolayer (SAM) n-alkanethiol HS-(CH2)n-CH3.36 They supposed that
h)ldI'Ophobic interactions with the electrode were important for orienting the
Protein in such a way as to facilitate electron transfer. Comparing with
e'eCtrostatically-bound cytochrome c, the hydrophobic interactions lead to the
Same structural changes of the heme and larger rate constants for
Conformational transitions.36 Marken et al., on the other hand, reported direct
Cytcmhrome c electrochemistry at boron-doped diamond electrodes after

polishing with alumina or cathodic cycling; but no voltammetric response was

53

detected after anodic cycling.31 This observation was explained in terms of
favorable interactions between the pretreated, hydrophilic diamond surface and
the reactive region of the cytochrome 0 molecule.31 The pretreatment was also
proposed to impact fouling resistance to the electrode. Zhou et al. reported direct
electrochemistry of covalently immobilized cytochrome c at carboxyl-tenninated
boron-doped diamond electrodes.” The modiﬁed electrode showed the ability to
electrocatalyze the reduction of hydrogen peroxide.22 Similarly, Geng et al.
designed a sandwich structured SiO2/Cytochrome c/SiO2 gel on a boron-doped
diamond ﬁlm electrode for used as an electrochemical nitrite biosensor.37 More
detailed investigation of electrochemistry of cytochrome c at boron-doped
diamond electrodes is needed in order to better understand what factors are
most important for rapid electron transfer for redox proteins. Speciﬁcally, cyclic
voltammetric data as a function of the diamond ﬁlm morphology, surface
chemistry and solution pH were obtained and analyzed in order to better

understand the redox reaction at diamond electrodes.

3.1.2 Oxygen-Terminated Boron-Doped Diamond Electrodes

Boron-doped diamond ﬁlms are normally deposited under H2-rich
conditions, so the as-deposited ﬁlms are typically hydrogen-terminated. The
surface can straightforwardly be converted to an oxygen-termination by any one
of three methods: (1) chemical oxidation (e.g., hot acid), (2) oxygen plasma

treatment, and (3) electrochemical oxidation.38

54

The electrochemical properties of oxygen-terminated diamond electrodes
have been studied by Fujishima and coworkers.‘°’9"‘2 The type of carbon-oxygen
functional groups formed can be quite distinct across the surface according to
both XPS results and electrochemical response changes after certain chemical
treatments. In general, carbonyl (C=O) and ether (C-O-C) groups are formed on

a diamond (100) surface,“ 43

whereas hydroxyl groups (C-OH) are generated on
a diamond (111) surface/'4' 45 Figure 3.2 shows the terminal atomic structure for

hydrogen-terminated and oxygen-terminated diamond (100) and (111) surfaces.

(100) (111)
r3 .1
0909900 9 3 2
OOQQQOQ QQ‘QV

0719-. 8 8 3 ii, 3 <3): Ft
059 Q 9 OQOQOUO OTOQO
2) 4;) Q Q Q L“) 000

carbon: I.) oxygen: 0 hydrogen: Cs

Figure 3.2 Terminal atomic structure for hydrogen- and oxygen-terminated
diamond (100) and (111) surfaces.46

55

Due to the change of surface hydrophobicity, wider working potential
windows and larger voltammetric background currents were observed for
oxygen-terminated diamond electrodes as compared to hydrogen-terminated
electrodes.39 The Fujishima group also reported that redox analytes with a
negative charge exhibit decreased apparent electron-transfer rates at oxygen-
terminated diamond electrodes, while the apparent electron-transfer rates for
redox analytes with positive charge either did not change or were accelerated.
An electrostatic explanation was offered such that anionic complexes are
repelled from the oxidized surface while cationic complexes are attracted. They
proposed that these effects are due to ion-dipole interactions of the charged
redox system with either the carbon-hydrogen surface groups, which have
dipoles with the positive ends pointing away from the electrode surface, or with
the carbon-oxygen groups, which have oppositely oriented dipoles.” 47 In
general, there are two merits of the oxidized diamond surface. First, the
electrochemical properties of diamond electrodes are affected by means of
electrostatic interactions between the electrode surface and redox charged
system. Second, the oxygen-terminated surface can facilitate the introduction of
other functional groups. Oxygen-terminated diamond electrodes have been

widely used in electroanalysis.42' ‘8

In order to understand the electron transfer process between cytochrome

c and the diamond electrode, we studied the electrochemical behavior of this

protein at boron-doped diamond electrodes. The rate of electron transfer was

56

investigated by cyclic voltammetry as a function of (i) ﬁlm morphology
(ultrananocrystalline diamond (UNCD) vs. microcrystalline diamond (MCD) thin
ﬁlms; (ii) surface chemistry (oxygen- vs. hydrogen-termination); and (iii) the

solution pH.

3.2 Results

3.2.1 XPS Measurements

X-ray photoelectron spectroscopy (XPS) was used to characterize the
elemental composition of the diamond surface. Typical XPS spectra for as
deposited, oxygen-terminated (after chemical oxidation) and hydrogen-
terrninated (after chemical oxidation and then rehydrogenation) UNCD surfaces

are shown in Figure 3.3.

57

 

, A C1s

15-

1'104 Counts
3

 

 

 

 

 

 

 

     

 

 

 

 

 

 

 

 

20 I- B
15 - C15
8
c
a
8
v 10 *- 01$
2
5
1000 800 600 400 200 0
Blndlng Energy (eV)
20 - c
C1s
15 -
3
C
5
o
o 10 .
§
2
5 _ CKLL O15
OKLL L
o n . 1 L l L l . L A
1000 800 600 400 200 0
Binding Energy (eV)

Figure 3.3 XPS spectra for (A) as-deposited, (B) oxygen-terminated, and (C)
hydrogen-terminated UNCD electrode surfaces.

58

The spectrum for the as-deposited ﬁlm showed a sharp C 1s peak at 284
eV and a small discernable 0 13 peak at 532 eV. The O/C atomic ratio for this
sample was determined to be 0.05. The plasmon loss feature (34 eV higher in
energy relative to the C 15 peak) results from energy losses of the outgoing
photoelectrons due to excitations of the bulk and surface plasmons of diamond.49
This is a good indication of high purity of the diamond ﬁlm.49 No peak
corresponding to the excitation of the graphite n-n“ plasmon (7 eV higher in
energy relative to the C 15 peak) was observed. After chemical oxidation, the O
13 peak intensity increased considerably, indicating greater oxygen coverage. In
other words, chemical oxidation introduces oxygen functional groups onto the
diamond surface. The O/C value was determined to be 0.17, similar to the
reported 0.18 for a diamond electrode treated in an oxygen plasma.39 The
rehydrogenation step converted the electrode surface back to a hydrogen-
ten'nination surface by removing oxygen functional groups, as indicated by the

reduced O/C ratio of 0.02.

After exposure to the cytochrome c solution, the XPS spectrum for the
oxidized UNCD electrode showed the appearance of a N 1s peak from the
residual cytochrome C on the surface, as shown in Figure 3.4. Similar trends
were seen for oxidized MCD. The XPS data for electrodes with different
terminations before and after cytochrome c exposure are listed in Table 3.1. It is
clear that the nitrogen signal appears after the protein exposure, indicating the

existence of absorption of cytochrome c at speciﬁc Sites.

59

15

310

c

a

O

U

V

<o

75
0
15

.3 10

c

a

O

U

V

(o

75

 

Y

' CKLL

 

O13

C1s

 

    

 

 

 

 

 

 

 

1000' 8004 600 l 400 200 o
Blndlng Energy (eV)

3

C13
013

. CKLL OKLL N1,

1000.300 600-400 200 0
Binding Energy (eV)

Figure 3.4 XPS spectra for UNCD electrode surfaces after potential cycling in
cytochrome c solution. (A) Hydrogen-terminated surface; (B) oxygen-terminated

 

 

 

 

 

 

 

surface.
Table 3.1 Summary of the surface composition on various diamond electrode
surfaces.
XPS UNCD MCD
C1s 013 MS O/C C1S O1S N1s OlC
As deposited 95.04 4.96 0.05 97.96 2.04 0.02
H-terminated 97.56 2.09 0.02 98.07 1.93 0.02
H-terminated+cyt c 88.76 6.41 3.35 86.96 8.81 3.61
O-terminated 80.32 13.58 0.17 87.52 10.79 0.12
O-tenninated+cyt c 82.30 12.52 4.33 88.73 10.88 4.02

 

 

 

 

 

 

 

 

 

6O

3.2.2 Cyclic Voltammetric Studies

In contrast to the previously reported quasi-reversible, diffusion-controlled
voltammetric response at the as-deposited boron-doped diamond electrode,
there was no deﬁned cyclic voltammetric reduction or oxidation peak observed
for cytochrome c (Figure 3.5) at hydrogen-terminated boron-doped diamond. The
increase in the cathodic current at -0.2 V can be attributed to the reduction of
dissolved oxygen catalyzed by a polymerized Fe-porphyrin protein, as will be

discussed later.5°' 5‘

 

 

.1.

-2 I‘

Current! (IA

 

 

-0.2 i -0.1 0.0 ‘ 0.1 + 0.2 L 0.3
Potential (V vs. AglAgCl)

L

 

Figure 3.5 Cyclic voltammetric i-E curve of 0.1 mM cytochrome c in 0.1 M

NaCl, 10 mM Tris-HCI (pH 7) solution at a hydrogen-terminated UNCD electrode.
Scan rate=20 mV/s.

However, a completely different response was observed for the oxygen-
terrninated electrodes. Figure 3.6 shows cyclic voltammetric i—E curves for

cytochrome c at oxygen-terminated UNCD and MCD electrodes as a function of

61

the cycle number. Two well-resolved oxidation and reduction peaks are seen for
both electrodes with no decrease in peak current or change in AEp during

repeated cycling. This indicates that no signiﬁcant protein denaturation occurs

that could foul the electrode.

1.0

 

0.5 -

0.0 -

Current (M)

-0.5 -

 

-1.0 -

 

 

‘—

 

-0.2 A -0.1 0.0 A 0.1 0.2 . 0.3
Potential (V vs. AgIAgCI)

 

0.5 -

0.0 -

Current (ILA)

-0.5 -

 

 

 

_1 .0 J J 1 . l . I . l . L
-0.2 -0.1 0.0 0.1 0.2 0:3
Potentlal (V vs. AgIAgCl)

 

Figure 3.6 Cyclic voltammetric i-E curves for 0.1 mM cytochrome c in 0.1 M
NaCl, 10 mM Tris-HCI buffer (pH 7) at oxygen-terminated (A) UNCD and (B)

MCD electrodes. Curves are shown for different scan numbers. Scan rate=20
mV/s.

62

Stable voltammetric traces are indicative of stable protein structure near
the electrode surface. Cyclic voltammetric i-E background curves for oxygen-
terminated UNCD before and after electrode cycling in the presence of
cytochrome c are shown in Figure 3.7. A small increase in the cathodic current at
-0.2 V is seen for the electrode after potential cycling in the presence of the
protein. Similar curves were also observed for oxygen-terminated MCD
electrodes. We suppose that this “extra” cathodic current is due to the reduction
of dissolved oxygen, catalyzed by a polymerized Fe—porphyrin protein residue on
the electrode.” 51 Indeed the current diminished after a 10 min deoxygenation

step involving N2 purging.

 

 

 

0.0 -
i .......
E -0.2 -
a —before
0 - --after
0.4 - ----- purge with N2

 

 

 

4

’0.6 L L g g L L n n . 1
-0.2 -0.1 0.0 0.1 0.2 0.3

Potential (V vs. AglAgCl)

 

Figure 3.7 Cyclic voltammetric background i-E curves for an oxygen-
terrninated UNCD electrode. Cyclic voltammograms were measured in 0.1 M
NaCl and 10 mM Tris-HCI (pH 7) before (solid line), after (dashed dot line)
cytochrome 0 potential cycling and after purging with N2 (dashed line) following
cytochrome c measurements. Scan rate=20 mV/s.

63

Figure 3.8 Shows cyclic voltammetric i-E curves for cytochrome c at an
oxygen-terminated UNCD electrode at different scan rates. The midpoint
potential (E1/2), measured as the average between the reduction and oxidation
peak potentials, is 0.032 V vs. Ag/AgCl (0.229 V vs. NHE). EH2 is unchanged
over the probed scan rate range. The cathodic peak currents, ipc, were found to

increase proportionally with the square-root of the potential scan rate, V112, and

with the protein concentration, C*, as Shown in Figure 3.9A and B. The linear

relationship between ip and V” is typical of a diffusion-controlled electrochemical
reaction according to the Randles-Sevcik equation,

ID = (2.69x105)n3/2AD1/2C*v1/2,
where n is the number of electrons, A is the electrode area (cmz), C* is the bulk
concentration of analyte (mol/cma), D is the diffusion coefﬁcient (cm2/s), and v is
the scan rate (V/s). The diffusion coefﬁcient, Dox, was determined from the Slope
of the ipc-v“2 curve and was calculated to be 2.63 x10'6 cmzls for UNCD (2.09

x10‘6 cm2/s for MCD). The apparent heterogeneous electron-transfer rate
constant, kgpp, was calculated from the scan rate dependence of AED using
Nicholson’s theoretical treatment, and assuming a Charge-transfer coefﬁcient

(1:0552 The nominal values of kgpp (34811.25 X10“3 cm/s for UNCD and

23810.72 x10'3 cm/s for MCD) are slightly higher than but close to the rate
constant reported for other bare electrodes (10:01 x10'3 cm/s).53 These rate

constants were calculated using diffusion coefﬁcient value 1.1><10'6 cm2/s

64

determined by other methods.“ 55 The fact that the calculated kgpp value is

statistically the same at all scan rates (Table 3.2), suggests that ohmic resistance

within the electrode or the cell does not inﬂuence the voltammetric curve Shape

(i.e., AEp).

 

  
 

— 2 MW:
— 5 mVIs

Current (ILA)
O

— 10 mVls

 

 

 

. — 20 mVls
.3 . -— 50 mVIs
, — 100 mVIs
4 l L L L 1 A 1 LL 1 A l
-0.2 -0.1 0.0 0.1 0.2 0.3

Potential (V vs. AglAgCI)
Figure 3.8 Cyclic voltammetric i-E curves for 0.1 mM cytochrome c in 0.1 M

phosphate buffer (pH 7.5) at UNCD electrodes at different scan rates. Electrode
area=0.2 cmz.

65

Reduction Peak Current (ILA)

Reduction Peak Current (pA)

 

 

    

 

 

 

 

  
 

 

 

2.5 -
2.0 -
1.5 -
‘ y = 8.73 x -0.01
1'0 ' R2 = 0.9999
0.5 -
0.0 ' . ' r '
0.0 0.1 0.2 0.3
Scan Rate1lz Ms)"2
2.5
2.0 -
1.5 -
1.0 _ y=10.93x+0.10
R2 = 0.9991
0.5 -
0.0 . 1 j l A l .
0.00 0.05 0.10 0.15 0.20 0.25
Concentration (mM)

Figure 3.9 Cyclic voltammetric data for cytochrome c at UNCD electrodes.
Electrode area=0.2 cm2. (A) Plot of reduction peak current for 0.1 mM
cytochrome 0 solution versus the square root of the scan rate; (B) Plot of
reduction peak current versus cytochrome C concentration from 0.025 to 0.2 mM
in 0.1 M phosphate buffer (pH 7.5) at 20 mV/s.

66

Table 3.2

calculated from cyclic voltammetric data.

Apparent heterogeneous electron-transfer rate constant, kgpp ,

 

 

 

Scan rate kgpp (“10-3 cm/s)*
V/S UNCD MCD
0.005 3.55 :I: 1.18 1.98 :I: 0.31
0.01 2.90 :I: 0.58 2.26 i 0.34
0.02 3.28 :I: 0.72 2.55 :I: 0.35
0.05 3.29 :1: 0.94 2.33 i 0.34
0.1 3.46 :I: 1.23 2.80 :I: 0.30
Avg. 3.48 :I: 1.25 2.38 i 0.72

 

* Calculations were performed with T=298 K, D,,=DR=1.1x10'6 cmZ/s and o=0.5.

The effect of pH on the cytochrome c voltammetric response was
investigated between pH 6 and 9. A slight shift in E1,2 toward more negative

values by ca. 10 mV over the pH range was found, as shown in Table 3.3. The
peak separation AEp showed a slightly decrease followed by an increase as the

pH was increased from 6 to 9. This trend was more obvious for the MCD
electrodes. This indicates that there is an optimum pH range for the electron
transfer kinetics. Figure 3.10 shows the dependence of the reduction peak
current on the solution pH. The maximum reduction peak current was obtained
between pH 7 and 7.5, which was the same as the biological media pH. This
might be caused by a decrease of the electrostatic interaction between the

cytochrome c and the electrode surface at pH<7 and pH>7.5.

67

Table 3.3 pH dependence Of the midpoint potential E1,2 of cytochrome c at
boron-doped diamond electrodes. Scan rate=20 mV/s. Electrode area=0.2 cmz.

 

 

 

 

 

 

 

 

 

pH E1,2 (mV) AEp (mV) AEp (mV)
vs. Ag_/A;gCI UNCD MCD
6 40 82 98
7 33 72 79
7.5 32 73 77
8 32 71 77
9 30 77 82
-1.3
-1.2 I
i .11 -
_s
-1.0 F
_o.9 1 I l 1
6 7 8 9
pH

Figure 3.10 Effect of the solution pH on the cytochrome 0 reduction peak
current at boron-doped diamond electrodes. The solutions were 0.1 mM
cytochrome c in 0.1 M phosphate buffer at different pH. Scan rate=20 mV/s.
Electrode area=0.2 cm2.

3.3 Discussion

The key ﬁnding from this work is that there is no observed voltammetric
response for cytochrome c at the hydrogen-terminated boron-doped diamond
electrode, but there is for the oxygen-terminated electrode. This is contrary to our

previously reported results.32 Haymond et al. reported quasi-reversible, diffusion-

68

controlled electron transfer for cytochrome c at as-deposited boron-doped
diamond electrodes, which were presumed to be hydrogen-terminated.32 The as-
deposited surface was presumed to be low in oxygen, largely hydrogen-
terrninated, however, no independent measurements was made (e.g., XPS) to
conﬁrm this. The different electrochemical response of the as—deposited and
hydrogen-terminated diamond electrodes reported presently probably means that
the as deposited ﬁlms used in the original work contained some surface oxygen.
The results presented herein clearly Show that the oxygen-terminated diamond
electrode is the most active surface for cytochrome c electron transfer. As the
isoelectric point for cytochrome c is near pH 10, ion-dipole interactions between
the positively charged cytochrome c at pH 7 and carbon-oxygen groups, which
have dipoles with the negative ends pointing away from the electrode surface, or
carbon-hydrogen groups, which have oppositely oriented dipoles, can be used to
explain the different electrochemical responses observed at oxygen- and

hydrogen-terminated diamond electrodes.

The diffusion coefﬁcients determined from the slope of the ipc-v"2 curves

(2.63 x106 cm2/s for UNCD and 2.09 x10'6 cmzls for MCD) are about two times
higher than the reported value of 1.1x10'6 cm2/s determined by other methods.“
55 Uncertainty in the magnitude of certain variables, such as the electrode area
(A), the solution concentration (C), and the number of electrons transferred per
mole (n), that were used to calculate the diffusion coefﬁcient, could lead to errors

in the calculated coefﬁcient. Calculation of the diffusion coefﬁcient from the

69

voltammetric data assumes that the peak current is exclusively controlled by
diffusion of the analyte to and from the electrode. We suppose that the higher
calculated diffusion coefﬁcient in our work is caused by some limited protein
adsorption on the electrode surface. In other words, the electrochemical reaction
of the cytochrome c at oxygen-terminated boron-doped diamond electrodes
appears to involve a combination of adsorption and diffusion. It Should be pointed
out though, that we only have an indirect response for protein adsorption; i.e., the
enhanced oxygen reduction current. Rikhie et al. showed that carbonyl
functionalities on graphite promote cytochrome 0 adsorption leading to a
molecular orientation favorable for electron transfer.” We did not determine the
speciﬁc carbon-oxygen functional groups on the oxidized diamond, but Fujishima
and coworkers did.“ 4345 They reported that the carbon-oxygen functional
groups on diamond can be of multiple types: carbonyl (C=O) and ether (C-O-C)
on diamond (100) surface,“43 and hydroxyl (C-OH) on diamond (111) surface.“
45 The carbonyl groups at the oxygen-terminated diamond surface might promote
the pre-concentration of cytochrome c and facilitate the electron transfer.
Compared to other carbon electrode materials, molecular adsorption on diamond
is generally weak and this is what leads to the high level of fouling resistance.
This is in contrast to the fouling caused by adsorbed cytochrome c at the
exfoliated graphite electrode surface in less than 1 h.23 The diamond surface is
quite heterogenerous structurally and chemically. There are different
crystallographic orientations with different functional groups. There are grain

boundaries and the spZ-bonded carbon that resides there. There are boron

70

clusters aggregated in the grain boundaries and likely boron sites at the surface
of the diamond. Therefore, there are multiple sites where adsorption can occur. It
appears that electron transfer proceeds in a diffusion controlled manner at most
Sites on the electrode. There are sites that are presumably free of adsorbed
proteins. There are other sites where some protein adsorption occurs. Some of
this protein is electrochemically active leading to Slightly larger voltammetric peak
currents than those for the diffusion-controlled reaction. There also appears to be
some of this adsorbed protein that is electrochemically inactive because of

denaturation, but the exposed heme catalyzes the oxygen reduction reaction.

The midpoint potential, E1/2, measured at the oxygen-terminated diamond
electrodes (022910.007 V vs. NHE) is more negative than the 025710.017 V vs.
NHE value reported for cytochrome c in solution, but close to 022110.007 V vs.
NHE for immobilized cytochrome c.59 57 An open heme conformation proposed
by Hildebrandt and coworkers might explain the negative shift of the formal
potential.36' 56' 58 They demonstrated the existence of two conformational states
(B1 and B2) of immobilized cytochrome c on a silver electrode using surface
enhanced resonance Raman spectroscopy.36' 58' 59 In state B1, the native
structure of the heme protein is fully preserved, while in conformational state B2,
the heme iron exists as a mixture of ﬁve-coordinated high-spin and Six-
coordinated low-spin conﬁgurations in thermal equilibrium. The B2lB1
conformational equilibrium is potential dependent. The differences of the two

states, in heme pocket structure and coordination conﬁguration, result in different

71

electron-transfer properties.59 The sthcturaI changes in state B2 are restricted to
the heme crevice, which leads to a more open conformation compared to the
closed structure in state B1. In our case, the pre-concentrated cytochrome c
might exhibit a more open conformational than cytochrome c in the solution, and

results in the negative shift of formal potential.

As mentioned, our evidence for adsorption is indirect and comes from the
voltammetric data presented in Figure 3.7 for the residual oxygen reduction
current. XPS data were recorded for the oxidized UNCD electrode after potential
cycling in a cytochrome 0 solution. The Spectrum showed a N 1s peak at 400 eV,
presumably from the heme of adsorbed cytochrome 0 molecules on the surface.
Similar observations were made for MCD. However, there was no detectable
Signal for Fe (2p3, 710 eV) or S (2p3, 165 eV) atoms from the heme moiety as
one might expect. This can be explained in the following manner: (1) the high
number of N atoms relative to Fe in the heme and (2) the Fe in the heme may be
buried within the tertiary structure of the protein such that the probability of a
photoelectron from the Fe escaping is low. D’Souza et al. also reported very
weak XPS signals for Fe and S atoms of immobilized cytochrome c on fullerene
ﬁlm-modiﬁed electrodes.18 The cyclic voltammetric background curve after
electrode cycling in the presence of cytochrome 0 showed an increased cathodic
current at -0.2 V, which was attributed to the reduction of dissolved oxygen
catalyzed by the porphyrin of adsorbed cytochrome c.50 The absence of

cytochrome c redox peaks in the background voltammograms could be because

72

the protein is adsorbed, but in a conﬁguration less favorable for electron transfer.
The absence of redox peaks might also be due to the amount of adsorbed
cytochrome c or heme unit is too low to be electrochemically detected but is

enough to catalyze the oxygen reduction reaction.

3.4 Conclusions

Room temperature cyclic voltammetric data as a function of the surface
chemistry, ﬁlm morphology and solution pH were presented at boron-doped
diamond electrodes. Quasi-reversible, diffusion-controlled electrochemical
responses were Observed for oxygen-terminated and not for hydrogen-
terminated diamond electrodes. No decrease in peak currents or change in AEp
during repeated cycling indicates that no signiﬁcant protein denaturation occurs
at the electrode surface. The pH studies Show that a maximum peak current and
relatively low peak separation appears between pH 7 and 7.5. No
electrochemical response was observed for hydrogen-terminated diamond
electrodes. The different electrochemical responses of aS-deposited and
hydrogen-terminated diamond electrodes can be explained by different atomic
ratios of O/C. In conclusion, the key ﬁnding was that the most reproducible and
reversible response was observed for the oxygen-terminated, hydrophilic

diamond electrode.

73

3.5

(1)
(2)

(3)
(4)
(5)

(5)

(7)

(8)

(10)

(11)
(12)
(13)
(14)
(15)

(15)

(17)

References

Moore, G. R.; Williams, R. J. P. Eur. J. Biochem. 1980, 103, 523-532.
Moore, G. R.; Huang, Z.-X.; Eley, C. G. S.; Barker, H. A.; Williams, 6.;
Robinson, M. N.; Williams, 0. J. P. Faraday Discuss. Chem. Soc. 1982, 74,
311-329.

Stellwagen, E. Nature 1978, 275, 73-74.

Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1982, 257, 4426-4437.

Matthew, J. B.; Weber, P. C.; Salemme, F. R.; Richards, F. M. Nature
1983, 301, 169-171.

Liu, H.-H.; Lu, J.-L.; Zhang, M.; Pang, D.-W.; Abruna, H. D. J. Electroanal.
Chem. 2003, 544, 93-100.

Eddowes, M. J.; Hill, H. A. O. J. Chem. Soc. Chem. Commun. 1977, 771 -
772.

Yeh, P.; Kuwana, T. Chem. Lett. 1977, 1145-1148.

Yue, H.; Waldeck, D. H.; Petrovic, J.; Clark, R. A. J. Phys. Chem. B 2006,
110, 5062-5072.

Ji, X.; Jin, B.; Jin, J.; Nakamura, T. J. Electroanal. Chem. 2006, 590, 173-
180.

Zhong, T.; Qu, Y.; Huang, 8.; Li, F. Microchimica Acta 2007, 158, 291-297.

Wu, Y.; Hu, S. Bioelectrochemistry 2006, 68, 105-1 12.
Zhou, J.; Lu, X.; Hu, J.; Li, J. Chem. Eur. J. 2007, 13, 2847-2853.
Wang, L.; Wang, E. Electrochem. Commun. 2004, 6, 49-54.

Ding, X.; Yang, M.; Hu, J.; Li, 0.; McDougall, A. Microchimica Acta 2007,
158, 65—71.

Yin, Y.; Wu, P.; Li], Y.; Du, P.; Shi, Y.; Cai, C. J. Solid State Electrochem.
2007, 11 , 390-397.

Zhao, G.-C.; Yin, Z.-Z.; Zhang, L.; Wei, X.-W. Electrochem. Commun.
2005, 7, 256-260.

74

(13)

(19)
(20)
(21)
(22)

(23)
(24)
(25)
(25)

(27)

(28)
(29)
(30)

(31)

(32)

(33)

(34)

(35)
(35)

(37)

D'Souza, F.; Rogers, L. M.; O'Dell, E. S.; Kochman, A.; Kutner, W.
Bioelectrochemistry 2005, 66, 35-40.

Liu, Y.-C.; Cui, S.-Q.; Yang, Z.-S. Anal. Sci. 2006, 22, 1071-1074.
Sallez, Y.; Bianco, P.; LOjou, E. J. Electroanal. Chem. 2000, 493, 37-49.
Huang, L. C.; Chang, H. C. Langmuir 2004, 20, 5879-5884.

Zhou, Y.; Zhi, J.; Zou, Y.; Zhang, W.; Lee, S.-T. Anal. Chem. 2008, 80,
4141-4146.

Rikhie, J.; Sampath, S. Electroanalysis 2005, 17, 762-768.
Szucs, A.; Novak, M. J. Electroanal. Chem. 1995, 383, 75-84.
Reed, D. E.; Hawkridge, F. M. Anal. Chem. 1987, 59, 2334-2339.

Hill, H. A. 0.; Hunt, N. |.; Bond, A. M. J. Electroanal. Chem. 1997, 436, 17-
25.

Kasmi, A. E.; Leopold, M. C.; Galligan, R.; Robertson, R. T.; Saavedra, S.
S.; Kacemi, K. E.; Bowden, E. F. Electrochem. Commun. 2002, 4, 177-181.

Runge, A. F.; Saavedra, S. S. Langmuir 2003, 19, 9418-9424.
Hagen, W. R. Eur. J. Biochem. 1989, 182, 523-530.

Armstrong, F. A.; Hill, H. A. 0.; Oliver, B. N. J. Chem. Soc., Chem.
Commun. 1984, 976-977.

Marken, F.; Paddon, C. A.; Asogan, D. Electrochem. Commun. 2002, 4,
62-66.

Haymond, S.; Babcock, G. T.; Swain, G. M. J. Am. Chem. Soc. 2002, 124,
10634-10635.

Bowden, E. F.; Hawkridge, F. M.; Chlebowski, J. F.; Bancroft, E. E.;
Thorpe, C.; Blount, H. N. J. Am. Chem. Soc. 1982, 104, 7641-7644.

Armstrong, F. A.; Cox, P. A.; Hill, H. A. O.; Lowe, V. J.; Oliver, B. N. J.
Electroanal. Chem. 1987, 21 7, 331 -366.

Daido, T.; Akaike, T. J. Electroanal. Chem. 1993, 344, 91-106.

Rivas, L.; Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2002, 106,
4823-4830.

Geng, R.; Zhao, G.; Liu, M.; Li, M. Biomaterials 2008, 29, 2794-2801.

75

(38)

(39)

(40)

(41)

(42)

(43)
(44)

(45)

(45)

(47)
(48)

(49)

(50)

(51)

(52)
(53)

(54)

Liu, F. B.; Wang, J. D.; Liu, 8.; Li, X. M.; Chen, D. R. Diem. Relat. Mater.
2007, 16, 454-460.

Yagi, l.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal.
Chem. 1999, 473, 173-178.

Rao, T. N.; Yagi, l.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal. Chem. 1999,
71, 2506-2511.

Notsu, H.; Yagi, I.; Tatsuma, T.; Tryk, D. A.; Fujishima, A. J. Electroanal.
Chem. 2000, 492, 31-37.

Tryk, D. A.; Tachibana, H.; lnoue, H.; Fujishima, A. Diam. Relat. Mater.
2007, 16, 881-887.

Pehrsson, P. E.; Mercer, T. W. Surf. Sci. 2000, 460, 49-66.

Loh, K. P.; Xie, X. N.; Yang, S. W.; Zheng, J. C. J. Phys. Chem. B 2002,
106, 5230-5240.

Notsu, H.; Fukazawa, T.; Tatsuma, T.; Tryk, D. A.; Fujishima, A.
Electrochem. Solid-State Lett. 2001, 4, H1-H3.

Fujishima, A.; Einaga, Y.; Rao, T. N.; Tryk, D. A. Diamond
Electrochemistry, 2005.

Rutter, M. J.; Robertson, J. Phys. Rev. B 1998, 57, 9241.

Rao, T. N.; Loo, B. H.; Sarada, B. V.; Terashima, C.; Fujishima, A. Anal.
Chem. 2002, 74, 1578-1583.

Steinmuller-Nethl, D.; Kloss, F. R.; Najam-Ul-Haq, M.; Rainer, M.; Larsson,
K.; Linsmeier, C.; Kohler, G.; Fehrer, C.; Lepperdinger, G.; Liu, X.;
Memmel, N.; Bertel, E.; Huck, C. W.; Gassner, R.; Bonn, G. Biomaterials
2006, 27, 4547-4556.

Su, Y. 0.; Kuwana, T.; Chen, S.-M. J. Electroanal. Chem. 1990, 288, 177-
195.

Okunola, A.; Kowalewska, B.; Bron, M.; Kulesza, P. J.; Schuhmann, W.
Electrochim. Acta 2009, 54, 1954-1960.

Nicholson, R. S. Anal. Chem. 1965, 37, 1351-1355.

Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. Electroanal. Chem. 1984,
161, 355—376.

Theorell, H. Biochem. Z. 1936, 285, 207-218.

76

(55)
(55)

(57)
(58)
(59)

Ehrenberg, A. Acta Chem. Scand. 1957, 11, 1257-1270.

Petrovic, J.; Clark, R. A.; Yue, H.; Waldeck, D. H.; Bowden, E. F.
Langmuir 2005, 21, 6308-6316.

Hawkridge, F.; Kuwana, T. Anal. Chem. 1973, 45, 1021-1027.
Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710-6721 .

Wackerbarth, H.; Murgida, D. H.; Oellerich, S.; Dbpner, S.; Rivas, L.;
Hildebrandt, P. J. Mol. Struct. 2001, 563-564, 51-59.

77

CHAPTER 4
SPECTROELECTROCHEMICAL BEHAVIOR OF
REDOX PROTEINS IN A THIN-LAYER

TRANSMISSION CELL

4.1 Introduction

Spectroelectrochemistry is a hybrid combination of spectroscopy and
electrochemistry, and has become an essential tool in the study of redox reaction
mechanisms, particularly these for proteins and enzymes."7 There are two
essential components for transmission spectroelectrochemistry: an optically
transparent electrode (OTE) and a thin-layer transmission electrochemical cell.
There are two common types of OTEs: thin ﬁlms of metal oxides and carbon. The
development and application of carbon OTES has recently been reviewed.8 The
main advantage of a thin-layer cell design is that exhaustive electrolysis of a
redox analyte can occur within a short time (typically 20-120 s). Thin-layer cells
should have a large electrode area (A) to solution volume (V) ratio, which can be
achieved by conﬁning a very small volume (e.g., a few pL) into a thin layer (< 100
pm) adjacent to an electrode surfacea' 9 As long as the solution thickness, l, is

less than the diffusion layer thickness, which depends on the analyte’s diffusion

78

coefﬁcient and time scale of measurement, l<< JZDt, ﬁnite diffusion will exist

and the solution can be electrolyzed quickly and completely.

4.1.1 Ideal Thin-Layer Behavior

The theoretical thin-layer cyclic voltammetric i-E curve for a reversible,

one-electron redox reaction is shown in Figure 4.1.

 

 

Current (A)

 

 

 

 

 

Potential (V)

Figure 4.1 A theoretical cyclic voltammetric i-E curve for a reversible, one-
electron redox reaction in a thin-layer cell.

For ideal thin-layer behavior, the fonlvard and reverse scans are in
Gaussian shaped and mirror images of each other due to the exhaustive

transformation between the reduced and oxidized forms. The cyclic voltammetric

peak current, ip, is equal to:10

79

I _ n2F2VC.v
p 4RT

=(9.39x105)n2VC*v

in which V is the cell volume (cm3), C* is the bulk concentration of the redox
molecule (mol/cma), and v is the scan rate (V/s). n, F, R and T have their usual
meanings. The peak current is predicted to vary linearly with the scan rate and
the analyte concentration. The integrated area under the cyclic voltammetric
peak is the faradaic charge, Q (Coulomb), which is independent of the scan rate
according to the following expression:10

Q = nFN = nFVC' = nFALC'

in which A is the electrode area (cm2) and L is the thickness of the solution layer
(cm). It can be seen that the charge is invariant with the scan rate, but depends
on the number of moles of analyte electrolyzed and the number of electrons
transferred per mole of analyte. From the charge associated with exhaustive

electrolysis of a reactant, one can calculate the cell volume and compare it with

the theoretical cell volume based on the cell dimensions (area x thickness). For a
reversible system (i.e., kinetically fast), the oxidation, ip°", and reduction, ip'ed,
peak currents, are predicted to be equal in magnitude. Also, the peak separation,

AEp, is expected to be 0. However, if there is signiﬁcant ohmic resistance

between the working and counter electrodes or within the working electrode, or

the electron-transfer kinetics are sluggish, the voltammogram will have a
distorted shape and AEp>0. Slow scan rates and low analyte concentrations can

be used to minimize ohmic distortion of the cyclic voltammogram.

80

4.1.2 Ideal Thin-Layer Transmission Cell

An ideal thin-layer transmission cell should possess several features in
order for good correlation between the electrochemical and Spectroscopic signals:
a) rapid and complete electrolysis of the redox analyte, and b) agreement
between the experimental and theoretical values. Moreover, the cell needs to c)
accept different electrode materials, including thin-ﬁlm electrodes, (I) require a
small operational sample volume, especially for anaerobic conditions, and e)
provide reproducible, quantitative portability between optical and infrared

domains (physically and spectrally) for direct spectral correlations.

4.1.3 Electrochemical Studies of Myoglobin

Myoglobin (Mb) plays a critical role in oxygen transport and storage in
muscle cells. It is a small (MW ~18,800 g/mol), heme-containing protein. It
functions to maintain full muscular working capacity and a high level of oxidative
phosphorylation under long-term oxygen deﬁciency.” '2 It is a globular protein
with a single polypeptide chain that is made up of 153 amino acids. It contains a
single heme b prosthetic group, as Shown in Figure 4.2. Histidine is one of the
iron’S axial ligands, while the opposite axial coordination site is vacant or weakly
ligated by the solvent. The heme group is largely buried inside the nonpolar

pocket of the polypeptide chain.”15

81

 

Figure 4.2 Heme b structure of myoglobin. (A) Planar porphyrin ring system
and (B) heme Fe iS axially bound to a histidine molecule with the sixth
coordination site remaining unoccupied or weakly ligated by a solvent molecule.
Due to the vacant sixth coordination position, the heme iron can bind
oxygen. It can also easily bind other ligands, such as CO and NO. The UV-Vis
absorption spectra of horse myoglobin, with different bound ligands and different
iron oxidation states, are displayed in Figure 4.3. The corresponding extinction
coefﬁcients for the absorption peaks are listed in Table 4.1.16 The concentration

of myoglobin in solution can be determined spectroscopically from the absorption

spectrum for MbCO.

82

Absorbance

 

 

 

meth

 

 

- - deoxyMb
- - MbO2
—-—MbCO
/).:.\"
0.0 - ' "’ '
300 400 500 600

Wavelength (nm)

 

700

Figure 4.3 UV-Vis spectra for horse myoglobin in different oxidation states with
different bound ligands. Metmyoglobin (meth): MbFe(lll)H2O, deoxymyoglobin
(deoxyMb): MbFe(lI), oxymyoglobin (MbO2): MbFe(Il)O2 and carboxymyoglobin
(MbCO): MbFe(ll)CO.

 

 

Table 4.1 . Extinction coefﬁcients for horse myoglobin at pH 7, ~20 °C.16
meth MbO2 MbCO deoxyMb
Soret l nm 408 424 435
E lmM'1 cm" 188 207 121
visible l nm 502 630 542 580 540 579 560
e I mM“ cm" 10.2 3.9 13.9 14.4 15.4 13.9 13.8

 

 

 

 

 

Due to the vacant coordination position, the ferrous heme iron can bind

oxygen reversibly and carry out its physiological function." As shown in the

following scheme, after the oxygen binding, the oxygen-containing form of MbO2

is easily oxidized to ferric meth with generation of superoxide anion. The

meth can not be oxygenated and is physiologically inactive. The meth

83

reductase in muscle tissues can reduce the meth to the ferrous deoxyMb and

maintains the continuity of myoglobin function in vivo.17

 

oxygenation
MbFe(ll) + 02 ———‘ MbFe(||)02
deoxyMb Mb02
autoxldation
reduction

MbFe(lIl)H20 + 02'
meth

Electrochemical studies of Mb without ligands other than water is believed
to involve a one-electron redox reaction between deoxyMb and meth:18
MbFe(ll) + H2O<:’MbFe(lll)H2O + e' E°’= 46 mV vs. NHE18
deoxyMb meth
The sixth coordination Site is vacant in deoxyMb, while in meth, the sixth
coordination position is ligated by water. Compared with the physiological
reaction, the differences are the oxidation state of the heme iron as well as the
transition of the coordination state. This reaction involves heterogeneous electron

transfer at the electrode followed by a ligand dissociation reaction in solution.18

 

Electrode: MbFe(lIl)H2O + e' —" MbFe(lI)H2O

Solution: MbFe(lI)H2O —’ MbFe(ll) + H20

 

It is well known that Fe(ll) complexes are quickly and irreversibly oxidized in the
presence of oxygen at ambient temperature. To inhibit the autoxldation reaction,
the experiment Should be carried out in an anaerobic environment. Both

MbFe(lll)H2O and MbFe(ll) are known to have high-spin states and the iron is

84

below the heme plane positioned toward the proximal histidine due to the longer
Fe-N bond.18 The intermediate MbFe(lI)H2O is proposed to be low-spin with
shorter Fe-N bonds in the porphyrin plane.18 The relatively slow heterogeneous
electron-transfer kinetics are considered to be due to a large activation energy

barrier for conversion Of high-Spin iron to low-spin states.19

There is considerable interest in achieving direct electron transfer between
an electrode and myoglobin.”26 Rusling and coworkers developed a method to
perform direct protein electrochemistry by using insoluble surfactants to
incorporate the protein onto an electrode.” 27' 28 They showed that protein-
surfactant ﬁlms on carbon, Au and Pt electrodes are stable for weeks in buffer.
The surfactant provides a hydrophobic environment, much like that of the cell
membrane, where the protein natively exists. Taniguchi et al. showed quasi-
reversible voltammetry of horse heart myoglobin with an ultra-clean and

hydrophilic indium oxide electrode.” 26

In this Chapter, the electrochemical and Spectroscopic performance of a
new thin-layer cell is described. Three model systems were used for

demonstration: Fe(CN)6'3"4

as a fast and robust inorganic model; cytochrome c
as a simple, stable and well characterized redox protein; and mygolobin as an
oxygen-sensitive redox protein capable of chemical binding at the active site. It is
well established in the literature that cyanide strongly competes with Metao for

binding to axial heme position in ferric cytochrome 0.29 Taking advantage of the

85

excellent electrochemical properties of boron-doped diamonds (BDD), we
present, for the ﬁrst time, spectral characterization of redox-dependent cyanide
binding to cytochorme c. The effect of UV irradiation on functional integrity of

meth was also investigated.

4.2 Results

4.2.1 Electrochemical Performance of the Cell

.4
Background-corrected cyclic voltammetric i-E curves for Fe(CN)6 in 1 M

KCI at a glassy carbon electrode are shown in Figure 4.4 A-C. Symmetric

oxidation and reduction peaks are seen with no evidence of any diffusional tailing.
The ip°x / ipred and Op"x / Qp'ed ratios are both close to unity, as expected for

reversible electrolysis in the thin-layer cell. This shows that all of the reactant
(Fe(CN)64) is converted to the product (Fe(CN)6-3) on the fonrvard sweep and
then is returned completely to the reactant on the reverse sweep. The E1,2 value
for Fe(CN)6-3M was stable across all the experimental conditions tested
(E1,2=28613 mV). Figure 4.4D shows a plot of the peak potential separation

.4
values (AEp), recorded for Fe(CN)6 using cyclic voltammetry, as a function of

the solution layer thickness. AEp remains stable with increasing analyte

concentration at all Spacer thicknesses. However, AEp values for a 7.5 pm spacer

are higher than those for thicker 25 and 75 pm spacers.

86

Figure 4.4 Background-corrected cyclic voltammetric i-E curves for Fe(CN)(:1
in 1 M KCI in the thin-laye; cell with a glassy carbon electrode. (A) Effect of scag
rate for 1 mM Fe(CN)6 using the 75 pm spacer; (B) effect of Fe(CN)6
concentration using the 75 pm spacer at 1 mV/s; (C) effect of layer thickness for
1 mM Fe(CN)6 .at 1 mV/s; and (D) peak separation as a function of layer
thickness.

87

 

Figure 4.4

Current (ILA)

Current (pA)

Current (pA)

 

 

 

 

1 A J

 

0.0

0.1 A 0.2 0.3 0.4 0.5 - 0.5
Potential (V vs. AgIAgCI)

 

 

 

 

l A I

 

I 0.1 0.2 I 0.3 i 0.4 ‘ 0.5 ‘ 0.6

Potential (V vs. AglAgCI)

 

 

 

 

 

0.1 0.2 0.3 0.4 0.5 0.6
Potential (V vs. AglAgCl)

88

Figure 4.4 continued

 

  

 

 

 

 

45
D
- -- 1 mVls, 0.25 mM
., - o- 1 mVls, 0.5 mM
A 30 " ‘ - —A-1 mVls,1 mM
>
E.
9.
lg ‘ . ‘9-
15 - ‘ ~ ,- " ....... i
o . . l . . . . . . . .
10 100
Spacer Thickness (pm)

The oxidation peak current increased linearly with the scan rate for all
concentrations of Fe(CN)64 in 1 M KCI (Figure 4.5A). The thickness of the thin

layer calculated from the slope is 58 pm, which is slightly smaller than the
specified spacer thickness of 75 pm. The speciﬁed value was indicated by the
manufacture of the material and not independently measured in this work.
Similarly, layer thicknesses were calculated to be 19 and 10 pm, respectively, for
the 25 and 7.5 pm spacers (ﬁgure not shown). The layer thicknesses obtained
from optical measurements of meth using 8403:188 mM'icm'i) according to
Beer’s Law were 60, 18 and 6 urn for the 75, 25 and 7.5 pm Spacers,
respectively. These values are in good agreement with values determined from
the electrochemical measurements. Figure 453 shows a trend Of decreasing

faradaic charge with increasing scan rate for all concentrations tested. The

89

 

decrease was most evident for the 1 mM concentration. Ideal thin-layer behavior

would be evidenced by a constant change with increasing scan rate.

 

20
A — 1 mM
-- 0.5mM
15,. ---0.25mM

   
  

y a 1 .05+3.50x
R sossse

I
J
I

I
, .- ’y - 0.57+1.75x
R =o.9995

Oxidation Peak Current (IA)
3

 

 

 

 

 

5 - .a"
a , ,.,.—---—-"" y=0.27+0.85x
:.r-~' R=0.9995
o . J L j 4 1 . a . I .
0 1 2 3 4 5 6
Scan Rate (mVls)
600
B 0 1 mM
. I 0.5 mM
A 0.25 mM
400 -
6‘
3
o .
E M
3 200
O .
é a x
O n . n . 1 4 l L n

 

 

 

1 2 3 4 5
Scan Rate (mVls)

Figure 4.5 Plots of (A) oxidation peak current and (B) faradaic charge vs. scan
rate for different concentrations of Fe(CN)6 . The Spacer thickness was 75 pm
and the supporting electrolyte was 1 M KCI.

90

4.2.2 Spectroelectrochemcial Kinetics
Figure 4.6 shows the amperometric and simultaneously-recorded optical
.4
absorption measurements at 220 nm for Fe(CN)6 at a gold minigrid electrode

using the 25 pm spacer. The current and absorbance were recorded during a

potential step from -0.1 to 0.6 V. At lower electrolyte concentration (100 mM KCI),
.4
1 and 5 mM Fe(CN)6 was electrolyzed completely in 2 and 10 sec, respectively .

The process was much faster at a higher concentration of supporting electrolyte

(1 M KCI) with bulk electrolysis occurring within instrument sampling time.

91

 

 

 

 

 

 

 

 

6 - 0.00
7 A 3 o
‘ — -0.02
5 —+ E e AAAW
A '0‘0‘.".. . '—-0.04
< 4 _ s...” C
'i "H. . — 100 [TIM KCI _ _0 06
O ‘5 .4 '
t; 3 9.“ o ....... A 1mM Fe(CN)6 in”
5 -. '. - - - e 5 mM Pe(CN)6“‘~ -0.08 o
t
5 _ -010
q —- -0.12
i— -0.14
2.0 _1_ I —F T 1 T I I I I I l l l l I II I I ’— 0.00
B
A - ~0.02
A A A A A AAAW
A 1.5 — . — —0.04
< \
'9. ‘, . — 1 M KCI - -0-06
5 1.0 _ t‘ ....... A 1 mM Fe(CN)6“‘ 008 p)”
g ‘, --- e 5mM Fe(CN)6“ "
' ‘9 —-0.12
I... ‘ .
........ ~_ 0 e 9000M
k .................... -~,...... —-0.14
0.0 _ W
2'» 5 tééiéé' 5 3 {TFHU 5 3
1 10

Tune (sec)

Figure 4.6 4 Time dependence of the current and optical absorbance at 220 nm
for Fe(CN)6 at a gold minigrid electrode in the thin-layer cell during a potential

step from -0.1 to 0.6 V. Data are shown for supporting electrolyte concentration
of (A) 100 mM KCI and (B) 1 M KCI. Lines are for the current on the left and
markers are for the simultaneous optical changes on the right. The spacer

thickness was 25 pm.

Figure 4.7 Shows cyclic voltammetric i-E curve for cytochorme c at a gold

minigrid electrode in a thin-layer cell with a 75 pm spacer. Well-deﬁned oxidation

and reduction peaks are present at 0.132 and 0.011 V. The determined midpoint

potential at 0.071 V is in good agreement with the accepted value of 0.06 V.30

92

The cathodic current at —0.2 V is believed to be due to some residual oxygen

reduction, as the thin—layer cell was not deoxygenated.

 

Current (11A)
:1

 

 

-o.2 ‘ -o.1 0.0 A 0.1 ‘ 0.2 ' 0.3
Potential (v vs. AgIAgCI)

J

 

Figure 4.7 Cyclic voltammetric i-E curve for cytochrome c at a gold minigrid
electrode in a thin-layer cell with a 75 um spacer. The analyte solution was 0.1
mM cytochorme c in 10 mM phosphate buffer (pH 7) and 100 mM KCI. Scan
rate=10 mV/s.

Figure 4.8 shows the difference absorbance spectra for cytochrome c
during a potential step from 0.5 to -0.3 V at a gold minigrid electrode. As the
potential was stepped to -0.3 V, the reduction of the cytochrome c occurred. A
serious of positive-going (reduced form of the protein) and negative-going
(oxidized form of the protein) peaks are observed. The spectra are the same as
those reported previously.6' 3‘ Not only Soret (400-450 nm) and Q (500-600 nm)

bands, but also N and L (200-400 nm) bands, are seen in the spectra. They are

all attributed to porphyrin n—nﬂ transitions in the protein.

93

 

0.03

 

Red-Ox 418

0.02

0.01

A Absorbance

 

 

0.00 -

 

-o.o1 - - .
200 300 400 500 600

Wavenumber (cm‘1)

 

Figure 4.8 Difference absorbance spectra of cytochrome c at a gold minigrid
electrode in a thin-layer cell with a 75 um spacer. The Red-Ox difference spectra
were generated during a potential step from 0.5 to -0.3 V. The analyte solution
was 0.1 mM cytochorme c in 10 mM phosphate buffer (pH 7) and 100 mM KCI.

The spacer thickness was 75 pm.

Figure 4.9 shows the absorption-time curves for 0.1 mM cytochrome c in
10 mM sodium phosphate buffer (pH 7)+100 mM KCI during potential steps from
0.5 to -0.3 V at (A) a gold minigrid electrode and (B) a boron-doped
diamond/quartz electrode using a 75 um spacer. The repetitive potential steps
produced relatively slow, but generally reversible optical changes. The potentials
were selected according to the voltammetric i-E curve for cytochrome c in

aqueous solution. Clearly, cytochrome c exhibited slower electrochemical
.4
kinetics compared to Fe(CN)6 , as evidenced by the longer time required to

reach a steady-state response after application of the potential step. While

relatively fast at the beginning, both the rates and amplitudes of the absorbance

94

changes diminished noticeably in successive redox cycles over several minutes
at a gold electrode. The reduction and oxidation of cytochrome c at a boron-
doped diamond was noticeably slower than at the gold minigrid, but signiﬁcantly
more stable as no obvious change in amplitude or rate was observed over

several redox cycles.

100 W A
+0.5v +0.5v

 

 

 

 

 

 

 

 

 

 

 

 

O 100 200 300 400
Time (sec)
8
30 _ -0.3V +0.5V -0.3V +0.5V _0 3V +0.5V
7o — ' ' " '4 """
'o
3?
g 60 —
<
50 —
w ..............
40 l l l l I
0 500 1000 1500 2000

Time (sec)

Figure 4.9 Absorbance change of cytochrome c on (A) a gold minigrid
electrode and (B) a boron-doped diamond thin ﬁlm at 418 nm. The analyte
solution was 0.1 mM cytochorme c in 10 mM phosphate buffer (pH 7) and 100
mM KCI. The spacer thickness was 75 um. Repetitive potential steps between
0.5 and -0.3 V were applied, as indicated in the ﬁgure.

95

4.2.3 Spectroelectrochemistry of Cytochrome c - Cyanide

Complex

Schejter et al. reported cyclic voltammetric studies of cytochrome c in the
presence of KCN at a modiﬁed gold electrode.29 They determined the redox
potential of ferric/ferrous cytochrome c-cyanide redox couple for the ﬁrst time and
provided a detailed thermodynamic characterization of the complete cycle
(Figure 4.10E).29 Here, we use spectroelectrochemical method to demonstrate
thermodynamic cycle of cytochrome o—cyanide complex. The spectral changes of
cytochrome c in response to potential steps in the presence of CN' are shown in
Figure 4.10. The potential was stepped between +0.3, -0.3, and -0.7 V, as
indicated in Figure 4.10A. These potentials were selected based on the reported
potentials for oxidation and reduction of cytochrome c and cytochrome c-cyanide
at 0.06 and -0.44 V vs. Ag/AgCl, respectively.29 Data are shown as differences
between wavelength pairs to minimize the contribution of baseline variations over
time. The 421/404 nm pair corresponds to maximal peak/trough difference and
reﬂects a combination of processes occurring in the sample. The 438/415 nm
and 432/409 nm pairs are based on apparent isobestic points of two major
observed processes. The difference absorbance spectra between key states
(designated by letters a-g in Figure 4.10A) are shown in Figure 4.10 B-D. Upon
the initial step from +0.3 to -0.3 V (a—>b), a relatively rapid absorbance change
was observed mostly at 438 nm. The corresponding difference spectrum is

reﬂective of typical ferric/ferrous cytochrome c transition (Figure 4.108, b-a). A

subsequent step to -0.7 V (b—>c) produced fast and intense changes at 432/409

96

nm, but caused no immediate response at 438/415 nm. Difference spectrum c-b
was similar to that of the b-a transition except for a distinctive red shift in both
Soret (400-450 nm) and a (500-600 nm) spectral regions. Initial response was
followed by slow, but distinctive reversal of optical changes at 421/404 and
432/409 nm (c—>d). This slow phase was also clearly visible at 438/415 nm. The
corresponding difference spectrum d-c (Figure 4.10D) indicated all an optical
shift from 423 to 411 nm. A second shift was also clearly observed from 557 to
550 nm. In general, the 421/404 nm pair responded to both -0.3 and -0.7 V

potential steps, while the 438/415 nm and 432/409 nm pairs only responded to

one of them.

97

Figure 4.10 Spectroelectrochemical measurements of cytochrome c—cyanide
complex on a boron-doped diamond electrode. (A) Absorption changes at
characteristic wavelengths; (B-D) difference absorbance spectra between key
states as designated by letters a to g; and (E) thermodynamic cycle of
cytochrome c—cyanide complex. The analyte solution was 0.1 mM cytochorme c
in 0.1 M KCN, Tris buffer (pH 7) and 100 mM KCI. Potentials were stepped
between +0.3, -0.3 and -0.7 V as indicated in the ﬁgure.

98

 

 

Figure 4.10

 

 

 

 

 

 

 

 

 

—421/404 nm
50- A ----- 432x409 nm
-o.7v ---438/415nm -o.7v
—o.3v c 0.3V
40—
d
"’2
<3 20—
bg 1,
a ....... '
0 ..........
‘~---J.
\‘~
+0.3v “""
l l l 1 l —1
o 100 200 300 400 500
Time (sec)
3 422

 

 

 

 

 

—d-c
---g.f
423
l l | I l l l
300 350 400 450 500 550 600
Wavelength (nm)

99

Figure 4.10 continued

 

E
E°' = 60 mV
Cyt c Fe(l|l) + e- : Q“ : Cyt c Fe(ll)
CN- ox red CN-
K0 K0

 

 

game,” = —440 mV

Cyt C Fe(|l|)<—CN" + e- : Cyt c Fe(|l)+—CN’

 

Reversing the potential from -0.7 to -0.3V (d—>e) mostly reversed the
sample to state b, with one noticeable exception that the 438/415 nm pair did not
respond to the potential step immediately, but showed a much slower change.
Difference spectrum of the d—se transition (Figure 4.10C, e-d) was similar to that
of b—->c transition (Figure 4.10A, c-b) but at ~2/3 amplitude. The spectral changes
for the next step (e—>f) were similar, but much faster and twice the amplitude
compared to initial a-—>b transitions. This was followed by slow spectral changes
similar to the b—>c step. Corresponding difference spectrum g-f showed

peak/troughs at 418/404 nm. No clear changes were observed in the on region at

this step.

4.2.4 Spectroelectrochemistry of Myoglobin

Electrochemical studies of Mb without ligands other than water is believed
to involve a one-electron redox reaction between deoxyMb and meth (E°’=
0.046 v vs. NHE, -0151 v vs. Ag/AgCI).18 Figure 4.11 shows absorbance-time
proﬁles and absorption spectra for 50 uM myoglobin in 10 mM phosphate buffer

solution (pH 7) and 100 mM KCI at a gold minigrid electrode. The spacer

100

thickness was 75 um. The potential was stepped between +0.4 and -0.7 V for
several cycles, as indicated in the ﬁgure. These potentials were selected based
on the reported redox potential of myoglobin at -0.15 V vs. Ag/AgCl. The
absorbance change depended on whether the protein solution was exposed to
far-UV light or not. When all wavelengths were used for excitation (Figure 4.11 C
and D), the peak for deoxyMb at 434 nm shifted to 412 nm as the potential was
stepped to +0.4 V. However, for subsequent potential steps, the peak shifted to
and stayed at 421 nm irrespective of the number of positive or negative potential
steps. On the other hand, the redox reaction of Mb became fully reversible when
mid-lfar-UV sampling light (M300 nm) was blocked using a glass ﬁlter (Figure
4.11 A and B). A positive potential step led to a shift of the Soret absorption
maximum initially at 434 (reduced form) to 409 nm (oxidized form), characteristic
of a transition from deoxyMb to meth and in agreement with the literature.7
Such changes were reversed upon a potential step from +0.7 to -0.4 V. Spectra
of deoxyMb and meth obtained at the third redox cycle were completely

superimposable with the corresponding initial spectra (Figure 4.1 1 B).

101

Figure 4.11 Absorbance-time proﬁles and absorption spectra of myoglobin
without (A, B) and with (C, D) far-UV light exposure. Spectra were recorded in
the thin-layer transmission cell with a gold minigrid electrode. The spacer
thickness was 75 um. The analyte solution was 50 uM myoglobin in 10 mM
sodium phosphate buffer (pH 7) and 100 mM KCI.

102

 

Figure 4.11

 

 

 

 

 

 

 

 

100 — A
-0.7V -O.7V -0.7V -0.7V
80 ‘ +0.4v +0.4v +0.“,
b d
m 50 -'
'2 a 434 nm
x ,, . "’.’ ~ ‘
< 40 - ‘ ' I .
l . . ‘ ' . , 409 nm
20 — , . ,
c I
0 F l l l l
0 2 4 6 8
Time (x103 sec)
80 — B
409
434 — a
60 - I. --- b
. . . A c
m 40“ .' . + d
'2 ' .. A
2 20 _, . J
"'7'.“ .. \
gss;v ‘ o
0 _ L
-20 -l
l l l l 1 | l l
360 380 400 420 440 460 480 500
Wavelength (nm)
100 —

-0.8V

 

 

 

 

 

Time (x103 sec)

103

Figure 4.11 continued

80 a 421

o 412l % 434

 

 

 

360 380 400 420 440 460 480 500
Wavelength (nm)

4.3 Discussion

Electrochemical characterization of the new thin-layer cell showed that

there were two notable deviations from the expected thin-layer behavior. Some
distortion of the i-E curves lead to non-zero AEp values being observed. AEp

values larger than 0 can be caused by ohmic resistance within the cell and

electrode and or slow electron-transfer kinetics at the electrode surface. The
stability of AEp with increasing analyte concentration, demonstrates that sluggish
electron-transfer kinetics at the electrode surface are the main cause of the
nonzero AEp. The second deviation was that the [EF) was largest under at all

conditions at minimal layer thickness (7.5 pm). This behavior can be rationalized

by considering the diffusion of analyte in the electrolyte volume above the

electrode that connected the thin layer with reference and counter electrodes

(Figure 2.3). For thick layers (e.g., 75 pm), this volume is negligible compared to

104

is
F.
i.
E
g,

 

the volume of the thin layer. As the layer thickness decreases to 25 and 7.5 urn,

the relative contribution of this volume increases to a signiﬁcant level.

Spectroelectrochemical measurements can provide the information about
current and absorbance change at the same time. The current can be recorded
as soon as the electron transfer occurs at the electrode surface. However, it
needs some time for the product to build up in the cell to be detected by
spectrophotometer. This explains the delayed absorbance response is observed
in Figure 4.6. Diamond electrode offers several major beneﬁts in biological
applications, such as outstanding chemical inertness, stability, as illustrated in
Figure 4.9, and a wide potential window.”37 Due to the continuous nature of
diamond ﬁlm electrodes, their ohmic resistance has to be balanced against their
optical transmission. Light transmitted through the ﬁlm electrode can also probe
analytes on the electrode surface, allowing spectroscopic studies of analytes
immobilized on the electrode via chemical or lipid assemblies, thus eliminating

diffusion altogether, which can counterbalance resistance of the ﬁlm electrode.

Cytochorme c—Cyanide Complex. The difference absorption spectra for
the cytochrome c-cyanide complex can be interpreted by a redox-driven ligand
exchange of cytochrome c in the presence of cyanide ion.29 The ferric
cytochrome 0 solution was initially equilibrated with 0.1 M KCN at +0.3 V. When

the potential was stepped to -0.3 V (a—>b), the native ferric cytochrome c was

reduced (E°’Cyt C: 60 mV), the cytochrome c-cyanide complex remained oxidized

105

due to the more negative formal potential (E°'C C.CN= -440 mV). As the potential
in

was decreased to -0.7 V (bac), a 5x larger absorbance change was observed
due to the reduction of the ferric cytochrome c-cyanide complex. The relative
amplitudes of two reduction phases indicate that the equilibrium was shifted
toward the CN'-bound ferric cytochrome c. Afﬁnity of ferrous cytochrome c for
CN' is signiﬁcantly lower (binding constant: 4.7x10'3 M'1 29) and dissociation of
the ferrous cytochrome c-cyanide complex occurred (c—>d) increasing the
population of native ferrous cytochrome c. When the potential was stepped back
to -0.3 V, the oxidation of the remaining ferrous cytochrome c-cyanide complex
occurred, with a reduced amplitude compared to that for (b—>c). The amplitude of
absorbance change for the e—+f step was increased for the same reason. Upon
oxidation of ferric cytochrome 0, binding of CN' to the increased population of
ferric cytochrome c began to re-establish equilibrium in the sample (f—>g) similar

to point a.

Myoglobin. The equilibration time for myoglobin is longer compared to
cytochrome c. The relatively slow heterogeneous electron-transfer kinetics are
considered to be due to a large activation energy barrier for conversion of high-
spin iron to low-spin states.19 Fast electrochemical response for myoglobin has
been reported. Rusling and coworkers performed direct protein electrochemistry
by using insoluble surfactants to incorporate the myoglobin onto an electrode.“
27' 28 Taniguchi et al. showed quasi-reversible voltammetry of horse heart

myoglobin with an ultra-clean and hydrophilic indium oxide electrode.25' 26 In

106

Figure 4.11, the far-UV light’s effect, to disable the reversible reaction of
myoglobin, is obvious with the uncommon peak positions. Far-UV light is likely to
cause photoreduction or radical damage of Mb (Figure 4.12) and inhibit the
reversible reaction between deoxyMb and meth. Therefore, it is necessary to
check for far-UV damage to the proteins prior to UV—Vis spectroelectrochemical
measurements. The conversion between deoxyMb and meth with far-UV light

blocked demonstrated the ability of performing anaerobic experiments in the cell.

: E°’=-150mV .

deoxyMbX h" meth
w /

radical damage

 

Figure 4.12 Proposed mechanism for photoreduction of myoglobin.

4.4 Conclusions

The electrochemical characterization of the new thin-layer cell showed
Gaussian-shaped thin-layer behavior and a linear relationship of peak current vs.
scan rate. The spectroelectrochemical measurements of ferrocyanide showed
that showed the heterogeneous response along the radial coordinate. The fouling
resistance to proteins of boron-doped diamond electrode compared to the gold
minigrid electrode was clearly demonstrated by the non-decreasing signal of

cytochrome c over several cycles. The spectroelectrochemical measurements of

107

the cytochrome c-cyanide complex showed the midpoint potentials for the CN-
bound cytochrome c at more negative values. The conversion between deoxyMb
and meth with far-UV light blocked, demonstrated the ability of anaerobic
experiment in the cell. In conclusion, the thin-layer transmission cell design met

the criteria for the ideal thin-layer transmission cell.

108

4.5

(1)

(2)

(3)

(4)
(5)
(6)

(7)
(8)

(10)

(11)

(12)

(13)

(14)
(15)
(15)

(17)

References

Kuwana, T.; Darlington, K. R.; Leedy, D. W. Anal. Chem. 1964, 36, 2023-
2025.

Anderson, J. L.; Kuwana, T.; Hartzell, C. R. Biochemistry 1976, 15, 3847-
3855.

Bowden, E. F.; Hawkridge, F. M. J. Electroanal. Chem. 1981, 125, 367-
386.

Zak, J.; Porter, M. D.; Kuwana, T. Anal. Chem. 1983, 55, 2219-2222.
Gui, Y.; Kuwana, T. J. Electroanal. Chem. 1987, 226, 199-209.

Moss, D.; Nabedryk, E.; Breton, J.; Mantele, W. Eur. J. Biochem. 1990,
187, 565-572.

Schlereth, D. D.; Mantele, W. Biochemistry 1992, 31 , 7494-7502.
Dai, Y.; Swain, 6.; Porter, M. D.; Zak, J. Anal. Chem. 2008, 80, 14-22.

Haymond, S.; Zak, J. K.; Show, Y.; Butler, J. E.; Babcock, G. T.; Swain, G.
M. Anal. Chim. Acta 2003, 500, 137-144.

Bard, A. J.; Faulkner, L. R. In Electrochemical Methods: Fundamentals
and Applications, 2 ed.; John Wiley & Sons: New York, 2001.

Wittenberg, B. A.; Wittenberg, J. B.; Caldwell, P. R. J. Biol. Chem. 1975,
250, 9038-9043.

Postnikova, G. B.; Tselikova, S. V.; Kolaeva, S. G.; Solomonov, N. G.
Comp. Biochem. Physiol., PartA 1999, 124, 35-37.

Kendrew, J. C.; Watson, H. C.; Strandberg, B. E.; Dickerson, R. E.;
Phillips, D. C.; Shore, V. C. Nature 1961, 190, 666-670.

Takano, T. J. MOI. Biol. 1977, 110, 537-568.
Takano, T. J. Mol. Biol. 1977, 110, 569-584.

Antonini, E.; Brunori, M. Hemoglobin and myoglobin in their reactions with
ligands; North-Holland Pub. 00.: Amsterdam, 1971.

Shikama, K. Chem. Rev. 1998, 98, 1357-1374.

109

 

 

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(25)

(27)
(28)
(29)

(30)
(31)

(32)

(33)
(34)

(35)

King, B. C.; Hawkridge, F. M.; Hoffman, B. M. J. Am. Chem. Soc. 1992,
114, 10603-10608.

Cohen, D. J.; King, B. C.; Hawkridge, F. M. J. Electroanal. Chem. 1998,
447, 53-62.

Armstrong, F. A.; Hill, H. A. 0.; Oliver, B. N. J. Chem. Soc., Chem.
Commun. 1984, 976-977.

Armstrong, F. A.; Cox, P. A.; Hill, H. A. O.; Lowe, V. J.; Oliver, B. N. J.
Electroanal. Chem. 1987, 217, 331-366.

Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21,
407-413.

Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. Soc. 1993, 115, 11891-
11897.

Nassar, A. E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386-
2392.

Taniguchi, |.; Watanabe, K.; Tominaga, M.; Hawkridge, F. M. J.
Electroanal. Chem. 1992, 333, 331-338.

Tominaga, M.; Kumagai, T.; Takita, 8.; Taniguchi, l. Chem. Lett. 1993,
1771-1774.

Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369.
He, P.; Hu, N.; Rusling, J. F. Langmuir 2004, 20, 722-729.

Schejter, A.; Ryan, M. D.; Blizzard, E. R.; Zhang, C.; Margoliash, E.;
Feinberg, B. A. Protein Science 2006, 15, 234-241.

Hawkridge, F.; Kuwana, T. Anal. Chem. 1973, 45, 1021-1027.

Stotter, J.; Haymond, S.; Zak, J. K.; Show, Y.; Cvackova, Z.; Swain, G. M.
Interface 2003, 12, 33-38.

Xu, J.; Granger, M. C.; Chen, 0.; Strojek, J. W.; Lister, T. E.; Swain, G. M.
Anal. Chem. 1997, 69, 591A-597A.

Swain, G. M.; Anderson, A. B.; Angus, J. C. MRS Bulletin 1998, 23, 56-59.

Rao, T. N.; Loo, B. H.; Sarada, B. V.; Terashima, C.; Fujishima, A. Anal.
Chem. 2002, 74, 1578—1583.

Fujishima, A.; Rao, T. N.; Try, D. A. Proc.-Electrochem. Soc. 2000, 99—32,
383-388.

110

 

(36) McGaw, E. A.; Swain, G. M. Anal. Chim. Acta 2006, 575, 180-189.

(37) Dai, Y.; Proshlyakov, D. A.; Zak, J. K.; Swain, G. M. Anal. Chem. 2007, 79,
7526-7533.

111

 

CHAPTER 5
SPECTROELECTROCHEMICAL STUDIES OF Na+-
TRANSLOCATING NADHzQUINONE

OXIDOREDUCTASE FROM VIBRIO CHOLERAE

5.1 Introduction

Na*-translocating NADH:quinone oxidoreductase (Na“-NQR) acts as a
redox-driven sodium pump in prokaryotes, such as marine bacteria (e.g., Vibn'o
a/ginolyticus) and human pathogenic bacteria (e.g., Vibrio cholerae).1 Na*-NQR
is a membrane-bound enzyme that couples electron transfer from NADH to
ubiquinone with Na+ translocation across the membrane. The efﬁciency of Na“
translocation by the Na*-NQR has been shown to be 1 Na‘/ 1 e'.2 The
electrochemical sodium gradient generated by Na"-NQR provides the energy for
cellular metabolic functions, such as driving bacterial ﬂagellum and transporting

nutrients.3' 4

Na"-NQR has a molecular mass of ~210 kDa and is an assembly of six
polypeptide subunits (quA-F): hydrophobic subunits quB, quD, quE and
relatively hydrophilic subunits quA, quC, quF.2'4 The known prosthetic groups

of Na+-NQR include a noncovalently bound ﬂavin adenine dinucleotide (FAD)

112

and a [2Fe—28] cluster in quF where the binding site for NADH is also located;
two covalently-bound ﬂavin mononucleotides (FMNs) that are attached by
phosphoester bonds to threonine residues in subunits quB and quC
respectively; a noncovalently bound riboﬂavin that has not been deﬁnitively
localized in the structure of the enzyme." 59 The chemical structures of riboﬂavin,

FMN, FAD and ubiquinone-8 are showed in Figure 5.1.

 

 

 

""""""""""" ‘1 2
u.
z H3CO CH3
— z
> E
a u. H3CO \ H
8 0 CH3 8
E ubiquinone-8

 

Figure 5.1 Chemical structures of riboﬂavin, FMN, FAD and ubiquinone-8.

The quF catalyzes the initial oxidation of NADH: a two-electron reduction
of the noncovalently bound FAD on the C-terminal domain of the quF, followed

by one-electron transfer to the [2Fe—28] cluster in the N-terminal Fe-S domain of

quF.1'5' 10,11

113

 

NADH/Mm 2.6.. FAD/FADH2 49.» [2Fe-2S]ox/[2Fe-ZS],ed

The electron transfer pathway from the [2Fe-2S] cluster to other cofactors in the
enzyme and the substrate quinone is unclear and this has been discussed
controversially in the literature. Juarez et al. recently proposed that the electrons
ﬂow in the following order using a stopped ﬂow measurement:7
FAD—> [2Fe-23] —> FMNC —-> FMNB —> riboﬂavin
However, they found that the cofactors were reduced in the following order:
FAD/FADH2—>[2Fe-ZS]ox/[2Fe-28],ed—>riboﬂavin
—>2(FMN/FMN")—>FMNC"/ FMNcHz
Two FMNs were ﬁrst transformed to two anionic ﬂavosemiquinone state FMN"
but only FMNC” was ﬁnally converted to FMNH2. They also concluded that the
reduction of the FMNc to its anionic ﬂavosemiquinone state FMNC” is the ﬁrst

Na*-dependent step and could be linked to Na” uptake.

Na"-NQR, which contains four ﬂavins, one [2Fe-28] center, and
ubiquinone-8 as cofactors, is the entry point for the electrons into the aerobic
respiratory chain of marine bacteria. Further studies are needed in order to better
understand how the energy conversion is coupled with the transmembrane Na+
translocation. To understand the mechanism of Na+ pumping, it is important to
characterize the redox potentials of all cofactors in Na“-NQR and to determine
their dependence on the Na+ concentration. In this chapter,
spectroelectrochemical studies of Na+-NQR are presented together with the

global regression analysis results. The electrochemistry of Na+-NQR was ﬁrst

114

studied using a glassy carbon electrode. The spectroelectrochemical
measurements were then performed with a diamond/quartz OTE in the thin-layer
cell using a potential step method. Global nonlinear regression analysis was
applied to the high resolution (10 mV/step) spectroelectrochemical data to

resolve redox centers in the Na*-NQR and determine their sodium sensitivity.

5.2 Results

The UV-Vis absorption spectra of the puriﬁed Na+-NQR after air
oxidization and reduction with sodium dithionite are shown in Figure 5.2. The
broad peaks centered around 370 and 460 nm are characteristic of
ﬂavoproteins.4 The absorption peak around 460 nm is due to the ﬁrst singlet state
transition So—>S1, and the 370 nm band is associated with the $0982 transition.12
The absorption by the [2Fe-ZS] cluster is also likely to be included in the broad
peaks, as the visible spectra of [2Fe-28] cluster is usually featureless due to

multiple overlapping S—>Fe charge transfer transitions.13

115

0.4

 

  

 

 

 

 

 

 

 

A“ — Oxldlzed Na‘*-NQR
0.3 - - Reduced Na+-NQR
G
0
E
o 0.2
a
.D
<
0.1
0.0 . l . l . T ~ .
300 400 500 600 700
Wavelength (nm)
0.00
. B
, '0'05 ‘ —-Red-Ox
0
c b
N
”E -o.1o -
B
5
-o.15 -
-o.2o --L--'-'
300 400 500 600 700
Wavelength (nm)

Figure 5.2 (A) UV-Vis absorption spectra for air-oxidized (solid line) and
sodium dithionite reduced (dashed line) Na*-NQR puriﬁed from Vibrio cholerae.
The solution was 0.01 mM Na*-NQR in 50 mM sodium phosphate buffer (pH 8),
100 mM NaCl, 1 mM EDTA, 5% glycerol and 0.05% dodecylmaltoside; (B)
difference absorbance spectrum (Red-Ox) for Na*-NQR.

Figure 5.3 presents background-corrected cyclic voltammetric i-E curves
for 0.1 mM Na+-NQR in sodium phosphate buffer using glassy carbon and
diamond/quartz OTE in the thin-layer cell with a 75 pm spacer. A low potential

scan rate of 0.5 mV/s was employed. Both i-E curves show two pairs of

116

 

 

oxidation/reduction peaks with similar peak potentials. Cyclic voltammetry was
also performed in small potential ranges (0.2 to -0.2 V and -0.2 to -0.7 V) to
investigate the relationship of these two redox couples and the results indicated
that they reacted separately. However, Na"-NQR contains four ﬂavins and one
[2Fe-ZS] as cofactors, it is proposed that the redox potentials for some redox
centers are similar and can not be resolved by cyclic voltammetry. In general, the
voltammetric results show that the potential range desired for the measuring Na*-

NQR redox activity is from 0.2 to -0.7 V.

117

 

 

 

 

 

 

 

 

 

 

i=1.
‘5
E
3
U
.1.2 . 1 . n . n .
-0.6 -0.4 -0.2 0.0 0.2
Potential (V vs. Ag/AgCI)
3
E
E
3
O
0.2 . I . l . 1 .
-O.6 -0.4 -0.2 0.0 0.2

Potential (V vs. AgIAgCl)
Figure 5.3 Background-corrected cyclic voltammetric i-E curves for Na+-NQR
at (A) a freshly polished glassy carbon electrode and (B) a diamond/quartz
electrode in a thin-layer cell with a 75 um spacer. The analyte solution was 0.1

mM Na*-NQR in 50 mM sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM
EDTA, 5% glycerol and 0.05% dodecylmaltoside. The scan rate was 0.5 mV/s.

Figure 5.4 shows the spectroelectrochemical response of Na+-NQR at 460
nm with a diamond/quartz OTE in a transmission cell using a 75 um spacer. 460
nm was selected due to the maximum spectral response changes. Potential

steps between 0.2 and -0.7 V (as indicated in the figure) produced relatively slow,

118

 

 

but generally reversible optical changes. It is obvious that the oxidation is faster
than the reduction reaction, based on the observation that the steady-state
optical response for the reduced form is reached faster after application of the
potential than for the response for the oxidized form. Figure 5.5 shows the Ox-
Red and Red-Ox UV-Vis difference absorption spectra for 0.1 mM Na‘-NQR in
50 mM sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside. The difference spectra were generated from the difference
between the spectra for the oxidized (0.2 V) and reduced (-0.7 V) forms of the
enzyme. The spectra are mirror images of each other, which is consistent with a

reversible redox reaction and complete electrolysis of the analyte in the thin-layer

 

 

 

 

cell.
5 .—
0.2 v 0.2 v
0 —l
c.I’C‘D
:0
s -5 -
E
-10 _
-o.7 v
'15 I I l I l l
0 2 4 6 8 10

Time (103 sec)

Figure 5.4 Absorbance change of Na“-NQR (460nm) at a diamond/quartz OTE
in a transmission cell with a 75 pm spacer. Potential steps to 0.2 and -0.7 V were
applied as indicated in the ﬁgure. The analyte solution was 0.1 mM Na+-NQR in
50 mM sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside.

119

 

 

L‘ 0.005

AAbs

 

 

 

L l A l A l

300 400 500 600 ‘ 700
Wavenumber (cm'1)

 

Figure 5.5 UV-Vis difference absorbance spectra measured for Na*-NQR at a
diamond/quartz OTE in a transmission cell with a 75 um spacer. Ox-Red and
Red-Ox difference spectra were generated from the oxidized and reduced
spectra collected at 0.2 and -0.7 V. The analyte solution was 0.1 mM Na‘-NQR in
50 mM sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside.

The difference absorbance-potential (voltabsorptometry) curve was also
recorded during one cyclic voltammetric cycle and is shown in Figure 5.6. The
scan was initiated at 0.2 V and scanned in the negative direction to -0.8 V at a
scan rate of 2 mV/s. During the fonNard scan, there was no change in
absorbance until -0.5 V at which point there was a decrease in absorbance with
increasing negative potential. On the reverse scan, there is considerable
hysteresis. The absorbance is constant on the reverse scan until 04 V at which
point the absorbance increases progressively to 0.2 V limit. The absorbance at

this potential is less than that of the solution initially at the same potential. This

could be due to incomplete electrolysis on the time scale of the measurement.

120

 

 

0.000

A Absm
.6
8
0|
\

-0.01 0

 

 

-o.a A -0.6 -o.4 ‘ -o.2 0.0 ‘ 0.2
Potential (V vs.Ag/AgCl)

 

Figure 5.6 Difference absorbance-potential curve (460 nm) for Na"-NQR at a
diamond/quartz OTE in a transmission cell with a 75 um spacer. The analyte
solution was 0.1 mM Na*-NQR in 50 mM sodium phosphate buffer (pH 8), 500
mM NaCl, 1 mM EDTA and 0.05% dodecylmaltoside. The data were collected at
a scan rate of 2 mV/s.

Figure 5.7 shows the absorbance changes during a series of potential
steps from 0.2 to -0.7 V and then back to 0.2 V in 100 mV increments. Both the
reduction and oxidation changes are plotted. Sufﬁcient time (20 min) was allowed
at each potential to ensure that equilibration conditions were reached. The
oxidation process reversed the optical changes of the reduction process and their
optical changes were almost identical at all potentials. Background-corrected
cyclic voltammetry of Na+-NQR in Figure 5.3 showed an oxidation peak around 0

V, however, no optical changes were detected around this potential. This might

be due to the tightly bound ubiquinone-8 with expected potential of -150 mV.

121

0.01

Cathodic steps

AAbs (Red-OX)

AAbs (Red-OX)

 

Figure 5.7 UV-Vis difference absorbance spectra of Na"-NQR during cathodic
and anodic potential steps at a diamond/quartz OTE in a transmission cell with a
75 um spacer. The difference spectra were generated from the oxidized spectra
collected at reference potential 0.2 V for reduction and oxidation potential steps
every 100 mV and 20 min. The analyte solution was 0.1 mM Na+-NQR in 50 mM

sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM EDTA and 0.05%
dodecylmaltoside.

122

In order to resolve redox potentials and spectra of multiple redox centers
with similar potentials and spectral signature in Na*-NQR with global regression
analysis, spectroelectrochemical measurements were performed during much
smaller potential steps. Reduction potential steps were applied from -0.15 to -
0.55 V at 10 mV, 20 min/step using reference potential at -0.1 V. Similarly,
oxidation potential steps were applied from -0.55 to -0.15 V using reference
potential at -0.6 V. Speciﬁcally, several models of three to ﬁve redox transitions
involving one to three transferred electrons were applied to the high resolution
spectroelectrochemical data. The optical changes at selected wavelengths during
the reduction and oxidation of Na*-NQR, and the corresponding global ﬁtting
results are shown in Figure 5.8. The solid lines represent the result of the global
ﬁtting for the experimental data shown in markers. The reduction and oxidation of
the protein were performed at the same potential range, and show similar
absorbance changes. However, the asymmetry in the course of the two
processes was clearly seen. The resolved spectra of Na+-NQR at the determined

redox potentials are shown in Figure 5.9.

123

 

    

—- global ﬁt
0 C] A v reduction
0 l A v oxidation

 

 

I l l l
-0.5 -0.4 -0.3 -0.2

Potential (V vs. Ag/AgCl)

 

Figure 5.8 Absorbance changes of Na+-NQR upon reduction (vs. -0.1 V) and
oxidation (vs. -0.6 V) potential steps every 10 mV and 20 min. Markers:
experimental data, solid line: global ﬁtting results. The analyte solution was 0.1
mM Na"-NQR in 50 mM sodium phosphate buffer (pH 8), 500 mM NaCl, 1 mM
EDTA and 0.05% dodecylmaltoside.

Oxidation Reduction
-290 mV, n=1

      
  

  
  

 

-286 mV, n=1

‘350 "‘V- "=2 4139 rnV n=2

-362 mV, n82

-387 mV, n=2 or 3

     

 

 

0.002
-430 mV, n=1
l l I F T l T F 1 l l l l l
250300350400450500550 250300350400450500550
Wavelength (nm) Wavelength (nm)

Figure 5.9 Resolved spectra of redox transitions in Na*-NQR.

124

Na+ sensitivity was also examined at 50 and 0.5 mM Na“. However, the
global regression analysis of spectroelectrochemical data did not show Na+
dependence of the redox potentials (60 mV per 10-fold change of Na+

concentration).

5.3 Discussion

The spectra at -290 or -286 mV (n=1) show small visible features and UV

 

changes, and this could be attributed to riboﬂavin transitions. The -350 or -339

 

mV (n=2) spectra show broad peaks around 450 and 285 nm, which may arise
from FAD transition. The reduction spectra at -362 (n=2) and -430 (n=1) mV are
similar in shape with broad peaks at 390 and 470 nm, which is likely due to the
two FMNs transition and [2Fe-28]; however, the oxidation spectra of FMN is a
single phase at -387 mV (n=2 or 3). The determined redox potentials for the

reduction and oxidation of the Na"-NQR are summarized in Table 5.1.

Table 5.1 Determined redox potentials for Na"-NQR.

 

 

Cofactor Redox Transition l-Em (mV)* Em (mV)*
Oxidation Reduction
FADqup FAD/FADHZ (n=2) _390 -340
[ZFG-ZS] quF [2Fe-2S]ox/[2Fe-28]red(n=1 ) 430
FM Nqugmrc 2(FMN/FMN")2(n=1) -350 -360
Rqun RfH'lRfH“(n=1) -290 -290

 

*E was reported vs. Ag/AgCI, saturated KCI.

125

Bogachev et al. recently reported spectroelectrochemical redox titration of
Na*-NQR and made a global ﬁtting based on one two-electron and ﬁve one-
electron transitions.” 15 They determined the redox potentials with corresponding
redox transitions in Na+-NQR, as listed in Table 5.2, and possible electron
transfer pathways in Na+-NQR: FAD/FADH"—+[2Fe-ZS]0x/[2Fe-ZS],ed—>
(FMN/FMN")c—> (FMN/FMN")3-> Rﬂ-l'lRfH‘. They also reported that the redox

potentials of all the redox transitions were independent of Na+ concentration.

Table 5.2 Redox transitions for known cofactors in Na*-NQR determined by
Bogachev.‘5

 

 

Cofactor Redox Transition Em* (mV)
FADquF FAD/FADH' (n=2) -400
[2F e-2S] qup [2F e-2S]ox/[2Fe-2S],ed(n=1 ) ~470
FMNquc FMN"/ FMNH'(n=1) -450
F MNquc FMN/FMN"(n=1) -400
FMNqua FMN/FMN"(n=1) -330
Rngr? Rﬂ-l '/Rﬂ-l‘(n=1) -210

 

*E was reported vs. Ag/AgCl, saturated KCI.

The global nonlinear regression analysis employed in this chapter is
similar to the procedure used by Bogachev. However, a low-order polynomial
baseline correction was made for the low intensity of optical changes due to
small protein volume ~5 pL and short pathlength ~75 pm (compared to 40 pL and
600 pm in Bogachev’s work). Our spectroelectrochemical data and global ﬁtting
results are somewhat different from Bogachev’s ﬁndings. The data reported by
Bogachev were generated using Na+-NQR from Escherichia coli, while in this

work all experiment data were performed with Na*-NQR from Vibrio cholerae.

126

 

 

 

Our results indicate that the spectral changes recorded during the reduction and
oxidation potential steps are not mirror images of each other, even though they
showed similar total absorbance changes. However, practically identical
reduction and oxidation titrations, with hysteresis less than 10 mV, was reported
by Bogachev.6 In general, the combination of the spectroelectrochemical method
and global nonlinear regression analysis provides a sensitive method for the

analysis of a multicomponent redox system.

Juarez et al. recently reported that the reduction of the FMNc to its anionic
ﬂavosemiquinone state was the ﬁrst Na*-dependent step and could be linked to
Na+ uptake using kinetic measurements.7 The reason for the absence of Na*-
dependent in both Bogachev and our work is that Na“-NQR might be a kinetic
pump. Na” dependence is carried out only at a speciﬁc stage of the catalytic
cycle and is not accessible under thermodynamic conditions. Future
spectroscopic measurements of vibrational modes could help us to better
understand any Na+ concentration dependence. It is expected that the spectral
analysis will show the contributions of all cofactors to the individual electron-

transfer steps.

5.4 Conclusions

An analytical method was developed and used for the study of the
multicomponent redox protein: Na*-NQR. The UV-Vis difference spectra,

recorded with a diamond/quartz OTE, indicated that reversible and complete

127

 

 

electrolysis occurs in the thin-layer cell. This is the ﬁrst spectroelectrochemical
study of this protein without the use of mediators. Global regression analysis
results resolved redox transitions and corresponding spectral changes in Na“-
NQR. The determined redox potentials of the cofactors are important for the
vibrational measurements to fully characterize the protein. The thermodynamic
measurements did not reveal any Na+ dependence of the formal potential for the
redox transitions. In conclusion, the combination of spectroelectrochemical data
and global regression analysis showed the ability of resolving redox centers in

Na”-NQR.

128

 

5.5

(1)

(2)

(3)

(4)

(5)

(5)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

References

Turk, K.; Puhar, A.; Neese, F.; Bill, E.; Fritz, G.; Steuber, J. J. Biol. Chem.
2004, 279, 21349-21355.

Bogachev, A. V.; Bertsova, Y. V.; Aitio, O.; Permi, P.; Verkhovsky, M. l.
Biochemistry 2007, 46, 10186-10191 .

Barquera, 8.; Zhou, W.; Morgan, J. E.; Gennis, R. B. Proc. Natl. Acad. Sci.
U. S. A. 2002, 99, 10322-10324.

Barquera, B.; Hellwig, P.; Zhou, W.; Morgan, J. E.; Hase, C. C.; Gosink, K.
K.; Nilges, M.; Bruesehoff, P. J.; Roth, A.; Lancaster, C. R. D.; Gennis, R.
8. Biochemistry 2002, 41, 3781 -3789.

Tao, M.; Fritz, G.; Steuber, J. J. Inorg. Biochem. 2008, 102, 1366-1372.

Bogachev, A. V.; Bertsova, Y. V.; Bloch, D. A.; Verkhovsky, M. l.
Biochemistry 2006, 45, 3421 -3428.

Juarez, 0.; Morgan, J. E.; Barquera, B. J. Biol. Chem. 2009, 284, 8963-
8972.

Bogachev, A. V.; Belevich, N. P.; Bertsova, Y. V.; Verkhovsky, M. I. J. Biol.

Chem. 2009, 284, 5533-5538.

Juarez, O.; Nilges, M. J.; Gillespie, P.; Cotton, J.; Barquera, B. J. Biol.
Chem. 2008, 283, 33162-33167.

Lin, P.-C.; Puhar, A.; Turk, K.; Piligkos, 8.; Bill, E.; Neese, F.; Steuber, J. J.

Biol. Chem. 2005, 280, 22560-22563.

Lin, P.-C.; Turk, K.; Hase, C. 0.; Fritz, G.; Steuber, J. J. Bacteriol. 2007,
189, 3902-3908.

Briggs, W. R.; Spudlch, J. L. Handbook of Photosensory Receptors;
Wiley-VCH, 2005.

Hatzfeld, O. M.; Unalkat, P.; Shergill, J. K.; Cammack, R.; Mason, J. R.
Biochemistry 1996, 35, 7546-7552.

Bogachev, A. V.; Kulik, L. V.; Bloch, D. A.; Bertsova, Y. V.; Fadeeva, M. S.;

Verkhovsky, M. I. Biochemistry 2009, 48, 6291-6298.

Bogachev, A. V.; Bloch, D. A.; Bertsova, Y. V.; Verkhovsky, M. I.
Biochemistry 2009, 48, 6299-6304.

129

CHAPTER 6

CONCLUSIONS AND OUTLOOK

The long-term goal of the project is to develop a transmission
spectroelectrochemical method with optically transparent boron-doped diamond
thin-film electrodes which are useful for the study of redox proteins. Research
conducted in this dissertation shows the progress that has been made towards

this goal.

In Chapter 3, the electrochemical behavior of cytochrome c was
investigated at both UNCD and MCD electrodes. Room temperature cyclic
voltammetry of horse heart cytochrome c was performed as a function of the
surface chemistry, ﬁlm morphology and solution pH. Quasi-reversible, diffusion-
controlled electron transfer for cytochrome c was observed at oxygen-terminated
boron-doped diamond electrodes. A stable electrochemical response during
repeated cycling indicated that no signiﬁcant protein denaturation occurred at the
electrode surface. UNCD electrodes exhibited a similar electrochemical response
as the MCD electrodes, but showed higher peak current due to a relatively large
surface area. The pH studies showed that the maximum peak current and
relative low peak separation was observed between pH 7 and 7.5. On the other

hand, no electrochemical response was observed for hydrogen-terminated

130

 

diamond electrodes. The requirement of surface carbon-oxygen functionalities at
the electrode surface for direct electrochemistry of cytochrome c is in agreement
with Armstrong’s ﬁndings. The characterization of electrochemistry of cytochrome
c at boron-doped diamond electrodes serves as a guide for future studies of

more complicated redox systems.

In Chapter 4, a custom-designed thin-layer transmission cell was
characterized and employed in the spectroelectrochemical measurements. The

electrochemical characterization with a reversible, one-electron redox system
-3/-4
Fe(CN)6 at a glassy carbon electrode in the thin-layer cell showed Gaussian-

shaped thin-layer behavior and a linear relationship of peak current vs. scan rate.
Kinetic measurements indicated rapid electrolysis in the cell and good agreement
between electrochemical and spectral data. The cell was then used to investigate
the spectroelectrochemical behavior of several model redox proteins. The
cytochrome 0 studies showed the advantage of diamond/quartz OTE compared
with the gold minigrid as the non-decreasing signal of cytochrome c over several
cycles was detected on the diamond electrode. The spectroelectrochemical
studies of cytochrome c-cyanide complex demonstrated that the electron transfer
of cytochrome c-cyanide complex and native cytochrome 0 occurred at two
different potentials. The investigation of Mb clearly demonstrated the applicability
of the cell under anaerobic condition. An interesting ﬁnding was that an
irreversible spectroelectrochemical response was observed under UV light. This

might be caused by radical damage or photoreduction of the protein due to UV

131

irradiation. The reproducible and quantitative spectroelectrochemical responses
in the thin-layer cell provide the foundation for the application of the

spectroelectrochemical technique to investigate more complicated redox proteins.

Chapter 5 presented a sensitive analysis method for the study of the
multicomponent redox protein: Na”-NQR. The spectroelectrochemical data with
diamond/quartz OTE without mediators, indicate that the reversible reaction and
complete electrolysis occurs in the thin-layer cell. The potential step method was
used to minimize the baseline change. The spectra were then modeled with a
global regression analysis to resolve the redox centers in Na*-NQR and to
determine the corresponding formal potentials. The combination of
spectroelectrochemical data and global regression analysis can also be used to

resolve redox centers with similar thermodynamic parameters.

The next aim is to characterize the coupling between redox transitions and
Na” uptake and release. The IR spectroelectrochemical measurements may
provide structural changes related to redox reactions of the cofactors. The
vibrational spectra might provide information for the pathways of sodium
translocation. There are a number of obstacles to performing vibrational
spectroelectrochemical measurements: strong water absorbance, limitations in
the optical windows, detector sensitivity and temperature control problems. The

broad water lattice vibrations below 1000 cm'1 are very sensitive, even for a small

pathlength (10 pm) or sampling conditions. The low signal-to-noise ratio was

132

because of the decreased light intensity in the far-IR region, and the smaller
vibrational mode extinction coefﬁcients makes it hard to select the window
material and a detector with the right sensitivity. The baseline distortions due to
the imprecise temperature control can result in thermal bands. Each of these
issues has to be considered in future work. A possible approach is to immobilize
the protein in a vesicle or dehydrate protein ﬁlm on an electrode surface in order
to reduce the solvent background absorption in protein spectroelectrochemistry

in the IR region.

133

ST

”lliljlllljljljllljjljlljjjljlll“