Direct wiring of cytochrome c's heme unit to an electrode: Electrochemical studies

Article abstract of DOI:10.1021/ja025518c

A novel strategy for the immobilization of cytochrome c on the surface of chemically modified electrodes is demonstrated and used to investigate the protein's electron-transfer kinetics. Mixed monolayer films of alkanethiols and ω-terminated alkanethiols (terminated with pyridine, imidazole, or nitrile groups that are able to ligate with the heme) are used to adsorb cytochrome c to the surface of gold electrodes. The use of mixed films, as opposed to pure films, allows the concentration of adsorbed cytochrome to remain dilute and ensures a higher degree of homogeneity in their environment. The adsorbed protein is studied using electrochemical methods and scanning tunneling microscopy.

Full text of DOI:10.1021/ja025518c

Published on Web 07/17/2002
Direct Wiring of Cytochrome c’s Heme Unit to an Electrode:
Electrochemical Studies
Jianjun Wei, Haiying Liu, Allison R. Dick, Hiromichi Yamamoto, Yufan He, and
David H. Waldeck*
Contribution from the Chemistry Department, UniVersity of Pittsburgh,
Pittsburgh, PennsylVania 15260
Received January 3, 2002. Revised Manuscript Received February 16, 2002
Abstract: A novel strategy for the immobilization of cytochrome c on the surface of chemically modified
electrodes is demonstrated and used to investigate the protein’s electron-transfer kinetics. Mixed monolayer
films of alkanethiols and ω-terminated alkanethiols (terminated with pyridine, imidazole, or nitrile groups
that are able to ligate with the heme) are used to adsorb cytochrome c to the surface of gold electrodes.
The use of mixed films, as opposed to pure films, allows the concentration of adsorbed cytochrome to
remain dilute and ensures a higher degree of homogeneity in their environment. The adsorbed protein is
studied using electrochemical methods and scanning tunneling microscopy.
Introduction
surface of Au electrodes and found that the electron-transfer
rate constant could be controlled by changing the thickness of
Electron transfer reactions play a central role in biological
processes, for example, photosynthesis and respiration. In
addition, electron transfer processes are central to the develop-
ment and operation of many biosensors and biocatalytic devices.
Our understanding of electron transfer in proteins has seen great
strides in recent years for both unimolecular and bimolecular
processes. With the recent growth of methods to control and
manipulate the surface chemistry of electrodes, heterogeneous
electron transfer with biomolecules (proteins, nucleotides, etc.)
should see a similar development. This work describes a strategy
for immobilizing biomolecules on a chemically modified
electrode through a specific interaction that provides some
selectivity for the biomolecule’s orientation on the surface. This
strategy is realized for the binding of cytochrome c to the surface
of chemically modified Au electrodes and should enable a range
of fundamental studies on the electron-transfer kinetics.
the monolayer film; however, he did not immobilize the protein
on the surface. Workers 2,3 have immobilized cytochrome c by
electrostatic association of carboxylic acid-terminated alkane-
thiol monolayer films with the positively charged outer surface
of the protein. These systems allow for the implementation of
well-defined electrochemistry and electron transfer rate constant
measurements as a function of the film thickness. However,
the voltammograms obtained from such studies can display a
significant degree of inhomogeneity, presumably a result of
protein aggregation or a distribution of surface sites and
geometries.
Most recently, cytochrome c was immobilized on the surface
of pure monolayers of pyridine-terminated alkanethiols that had
alkane chain lengths of more than six methylenes.5 For chain
lengths below six methylenes, no immobilization was observed.
Presumably, the length requirement results from the need for
the cytochrome to partially penetrate the film so that the pyridine
moiety can interact with the heme. A large negative shift in the
apparent redox potential, as compared to that observed on the
carboxylic acid terminated films, was identified. Although the
immobilization was robust, the electrochemical response was
not very reversible, making these systems unsuitable for detailed
studies of the electron-transfer mechanism.
A number of workers1-3 have investigated the electron
transfer mechanism of cytochrome c on electrodes. The earliest
studies were reported for electrodes modified with a redox
mediator through which the electrode reduces or oxidizes the
cytochrome c. Much of this early work focused on finding
systems in which the electron transfer is facile and preventing
decomposition of the protein on the electrode. More recently,
4
Miller used hydroxyl-terminated alkanethiols to coat the
This work demonstrates the ability to create mixed monolayer
films that associate with a specific part of the protein and allow
electron transfer to its redox center. Figure 1 illustrates the
design of the monolayer system (panel A) and its realization
for the pyridine/cytochrome c system (panel B). By creating
mixed films of pyridine alkanethiols in a diluent of shorter chain
alkanethiols, cytochrome c can be immobilized on the surface
through association of the functionalized longer chain thiols with
* E-mail: dave@pitt.edu.
(1) (a) Lewis, N. S.; Wrighton, M. S. Science 1981, 211, 944. (b) Armstrong,
F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407.
(2) a) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc.,
Faraday Trans. 1997, 93, 1 367. (b) Avilla, A.; Gregory, B. W.; Niki, K.;
Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759.
(3) (a) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847. (b)
Collinson, M.; Bosden, E. F.; Tarlov, M. J. Langmuir, 1992, 8, 1247. (c)
Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993,
97, 6564.
(4) Terrettaz, S.; Cheng, J.; Miller, C. J.; Guiles, R. D. J. Am. Chem. Soc.
1996, 118, 7857.
(5) Yamamoto, H.; Liu, Haiying; Waldeck, D. H. Chem. Commun. 2001, 1032.
9
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J. AM. CHEM. SOC. 2002, 124, 9591-9599
9591

A R T I C L E S
Wei et al.
Cytochrome c (Sigma C 7752, from horse heart, minimum 95%
based on molecular weight 12 384) was purified using a cation exchange
column (CM-52, carboxymethyl-cellulose from Whatman). The puri-
fication was carried out in a cold room at 5 °C, by the reported method.6
A 30-mg portion of cytochrome c was dissolved in 2 mL of 50 mM
phosphate buffer solution at pH 7 (25 mM Na2HPO4 and 25 mM NaH2-
PO4). A small amount of K3Fe(CN)6 was added to the solution to
oxidize the protein. This solution was placed onto a 1.5-cm-diameter
× 30-cm-long column containing carboxymethyl cellulose (Whatman,
CM-52) that was pretreated with 25 mM of the phosphate buffer. The
protein was eluted with 50, 60, 70, and 80 mM phosphate buffer in a
stepwise manner. The center of the last separated portion was collected.
The phosphate buffer was removed from the protein using an ultrafil-
tration membrane (Millipore, YM10) under positive pressure. The
cytochrome c aqueous solution was quickly frozen at -80 °C and dried
in a vacuum. The purified cytochrome c was stored in a freezer with
dry ice under an argon atmosphere until use.
Figure 1. The schematic diagram in panel A illustrates the strategy for
immobilizing a molecule on the monolayer surface through a specific
binding event. The drawing in panel B illustrates the realization of this
approach for immobilizing cytochrome c on the surface.
Chart 1
Electrode Preparation. A gold wire (0.5 mm diameter,99.99%) was
cleaned by reflux in nitric acid (68-70%) at 130 °C overnight and
then was washed with deionized water. The tip of the gold wire was
heated to form a ball of ∼0.06-0.15 cm2 surface area. The gold ball
was reheated in the flame until glowing and then quenched in deionized
water. This annealing process was performed more than 15 times to
make a smooth gold ball. The exposed Au wire was sealed in a glass
capillary tube, and the Au ball tip was annealed and cooled in a high-
purity stream of Ar gas.
Chemically modified electrodes were prepared by immersion in an
ethanol solution that contained 1 mM of 1-(11-mercaptoundecyl)
imidazole and 1-octanethiol (the mole ratio of 1-(11-mercaptoundecyl)
imidazole to 1-octanethiol was 1:9). The electrode remained in this
solution for 2-3 days to form the mixed SAM. The electrode was taken
out from the solution, first rinsed with absolute ethanol, then rinsed
with the supporting buffer solution (20 mM phosphate buffer pH 7),
and finally dried by a stream of dry argon gas. At this stage, the
electrode was used to perform characterization studies of its capacitance
and voltammetric response in the buffer solution. After this character-
ization, the electrode was immersed in a 100 µM cytochrome c solution
(purged with argon gas) for 30 to 60 min in order to immobilize the
cytochrome on the SAM-coated electrode. These electrodes were
immediately used in voltammetry studies.
the heme of the cytochrome. The immobilization is achieved
by an alkanethiol that is terminated with a functionality that
can bind to the heme of the cytochrome. In particular, pyridine
and imidazole were found to interact strongly, and nitrile-
terminated chains, more weakly. Chart 1 shows the materials
used to create these three mixed-film systems. The immobiliza-
tion is demonstrated by electrochemical voltammetry measure-
ments and STM imaging of the surfaces. We further show that
the voltammograms are close to ideal, which indicates well-
defined sites on the surface of the electrode, allowing the elec-
tron-transfer kinetics to be characterized electrochemically. This
strategy for immobilization should be applicable to many sys-
tems and should allow the use of electrochemical methods to
address important issues in protein electron transfer; for exam-
ple, developing structure-function relationships for the reor-
ganization energy, quantifying the relationship between the elec-
tronic coupling and the electron-transfer mechanism, and others.
After the measurements, the monolayer film was removed from the
electrode by immersing it in a “piranha” solution (a mixture of 30%
H2O2 and 98% H2SO4 in a 1:3 volume ratio) for 20 s and rinsed with
dionized water. The surface area of the electrode was then determined
by performing voltammetry in a 0.5 M KCl solution that contained 1
mM K3[Fe(CN)6] and 1 mM K4[Fe(CN)6]. The peak current in this
measurement displayed a linear relation with the square root of the
scan rate.7
The same procedure was used to prepare the pyridine- and nitrile-
terminated thiol monolayers. For the pyridine-terminated films, the 1
mM thiol solution was composed of a 1:9 mixture of 1-(12-mer-
captododecyl)pyridine and 1-undecanethiol, and for the nitrile-
terminated films, the 1 mM thiol solution was composed of 1-(11-
mercaptoundecyl)nitrile and 1-octanethiol. For the control study in
which the diluent film blocked adsorption, the SAM was composed of
1-(12-mercaptodecyl)pyridine and 1-hexadecanethiol.
Experimental Section
Reagents and Materials. Water for experiments was purified by
using a Barnstead-Nanopure system and had a resistivity of 18 MΩ‚
cm. 1,3-Dicyclohexylcarbodiimide, or DCC, (99%) was purchased from
Alfa Aesar. 1-Octanethiol (98.5+%), 1-hexadecanethiol, and 1-unde-
canethiol (98+%) were purchased from Aldrich and used without
further purification. Imidazole (99%), 11-bromo-1-undecanol (98%),
12-mercapto-1-dodecanol (98+%), methanolic iodine (99%), sodium
bisulfite (99%), thiourea (99+%, A.C.S. reagent), K2CO3 (99+%,
A.C.S. reagent), NaOH (97%), and MgSO4 (99%) were purchased from
Aldrich. Absolute ethanol was purchased from Pharmcoproducts, Inc.
Electrochemical Measurements. Cyclic voltammetry on the im-
mobilized cytochrome c was carried out using an EG&G PAR-283
potentiostat, which was controlled by a Pentium computer running
version 4.3 of PARC model 270 software and a GPIB board. The three-
(6) Brutigan, D. L.; Ferguson, S.; Margoliash, E. Methods in Enzymology;
Fleischer, S., Packer, L., Eds.; Academic Press: New York, 1978, Vol.
53, pp 131-132.
(7) Sawyer, D. T.; Sobkowiak, A.; Roberts, J. L., Jr. Experimental Electro-
chemistry for Chemists; Wiley: New York, 1995; pp 74-75. The diffusion
3-/4-
constant of Fe(CN)6
is assumed to be 7.63 × 10-6 cm2/sec.
9
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Direct Wiring of Cytochrome c Heme Unit, Electrode
A R T I C L E S
electrode cell was composed of a platinum spiral counter electrode, a
Ag/AgCl (3 M NaCl) reference electrode, and the SAM-coated Au as
a working electrode. The voltammetry measurements were performed
in 20 mM phosphate buffer solution (pH of 7.0) under an argon
atmosphere.
a white solid. 1H NMR (300 MHz) CDCl3: δ 3.651 (q, J ) 6.36, 4H),
2.689 (t, J ) 7.34, 4H), 1.654 (m, 4H), 1.570 (m, 4H), 1.379-1.255
(m, 32H).
b. Bis[12-((pyridinylcarbonyl)oxy)dodecyl] disulfide. 1,3-Dicy-
clohexylcarbodiimide (DCC) (0.603 g, 2.92 mmol) was added to 20
mL of dichloromethane solution of bis(12-hydroxyodecyl) disulfide
(0.55 g, 1.33 mmol), isonicotic acid (0.327 g, 2.66 mmol) and
4-(dimethylamino)pyridine (32 mg, 0.266 mmol) at 0 °C. After 1 h,
the solution was allowed to warm to room temperature, and stirring
was continued for 4 days. After removal of the precipitated dicyclo-
hexylurea (DCU) by filtration, the solvent was removed under reduced
pressure to yield a crude solid. The solid was recrystallized with ethanol
to yield a white powder product. 1H NMR (300 MHz) CDCl3: δ 8.799
(s, 4H), 7.910 (d, J ) 4.83, 4H), 4.369 (t, J ) 6.60, 4H), 2.685 (t, J )
7.29, 4H), 1.766 (m, 4H), 1.676 (m, 4H), 1.378-1.287 (m, 32H). EI-
HRMS: Calcd, 644.3702 (C36H56N2O4S2). Found, 644.3682.
3. Synthesis of 12-Mercaptododecanenitrile. a. 12-Hydroxydode-
canenitrile. 11-Bromoundecan-1-ol (4.00 g, 15.923 mmol) and sodium
cyanide (1.528 g, 31.84 mmol) were added to 30 mL of DMSO solution
and stirred at 80 °C for 2 days. The resulting solution was extracted
with methylene chloride and washed with a large amount of water to
remove DMSO. The combined organic layers were washed, dried, and
concentrated at reduced pressure. The crude product that resulted from
evaporation of solvent was purified by column chromatography
(methylene chloride) to obtain 12-hydroxydodecanenitrile. H NMR δ
(CDCl3): 3.626 (t, J ) 6.60 Hz, 2H), 2.332 (t, J ) 7.02 Hz, 2H),
1.650 (m, 2H), 1.558 (m, 2H), 1.435 (m, 2H), 1.281-1.208 (broad,
12H).
b. 12-Bromdodecanenitrile. 12-Hydroxydodecanenitrile (1.20 g,
6.09 mmol) was dissolved in 30 mL of dry ethyl ether and cooled to
-10 °C. Subsequently, 0.6 mL of PBr3 was added to the solution and
stirred at room temperature for 3 days. The resulting solution was
washed with 0.1 M Na2CO3 solution and pure water and extracted with
ethyl ether. The combined organic layers were washed, dried, and
concentrated at reduced pressure. The crude product that resulted from
evaporation of solvent was purified by column chromatography
(methylene chloride) to obtain 12-bromodecanenitrile. H NMR δ
(CDCl3): 3.409 (t, J ) 6.81 Hz, 2H), 2.337 (t, J ) 7.10 Hz, 2H),
1.630 (m, 2H), 1.417 (m, 2H), 1.342 (m, 2H), 1.225-1.207 (b, 12H).
c. 12-Mercaptododecanenitrile. 12-Mercaptododecanenitrile was
prepared according to a literature procedure.9 11-Bromoundecanenitrile
(1.079 g, 3.978 mmol) and thiourea (0.899 g, 11.812 mmol) were added
to 50 mL of dry ethanol and refluxed overnight under N2 atmosphere.
The solvent was removed at reduced pressure. A 50-mL portion of
water containing KOH (0.662 g, 11.81 mmol) was added and refluxed
for 6 h. The resulting solution was cooled to room temperature, extracted
with methylene chloride, and washed with water. The combined organic
layers were washed, dried, and concentrated at reduced pressure. The
crude product that resulted from evaporation of solvent was purified
by column chromatography (methylene chloride) to obtain 12-mer-
captododecannitrile. H NMR δ (CDCl3): 2.516 (q, J ) 7.41 Hz, 2H),
2.332 (t, J ) 7.08 Hz, 2H), 1.674 (m, 4H), 1.459-1.276 (b, 17H).
EI-HRMS: Calcd., 213.1551 (C12H23NS). Found, 213.1542.
Impedance measurements (EIS) were performed using a VoltaLab
PGZ407 universal potentiostat to determine the capacitance of the mixed
SAMs before immobilization of the cytochrome c. The experiments
were performed using a three electrode cell and a 20 mM phosphate
buffer solution (pH 7.0).
STM Measurements. For the STM studies, a Au(111) facet of a
single crystalline bead (prepared by Clavilier’s method 8) was used as
the substrate. It was cleaned by immersion in hot piranha solution (1:3
H2O2 and H2SO4) for 1 h, followed by immersion in hot HNO3 for 30
min. After each step, the sample was rinsed by ultrasonication in
ultrapure water (>18.2 MΩ‚cm) from a Barnstead-Nanopure Infinity
system. The crystal was hydrogen flame-annealed, and allowed to cool
to room temperature in air. The preparation of mixed SAMs of 1-(12-
mercaptodecyl) pyridine and 1-undecanethiol (1:9 mole ratio) on the
Au (111) bead for STM was the same as the SAMs prepared for
electrochemical experiments. Two beads were put into the solution
mixture for 2-3 days. One bead was rinsed with ethanol and then
directly used for STM experiments, and the other bead was placed in
a solution of cytochrome c (100 µM) for 30-60 min to immobilize
the protein. This bead was rinsed with supporting buffer solution before
being analyzed by STM. The STM images were obtained using a
PicoScan STM system (Molecular Imaging). STM tips were cut by
using 0.25-mm-diameter Pt-Ir wires (Goodfellow). All of the STM
images were obtained under constant current mode at 50-100 pA and
a tip-sample bias of 0.8-1.0 V.
1. Synthesis of 1-(11-Mercaptoundecyl)imidazole. The 1-(11-
mercaptoundecyl) imidazole was prepared in the following manner:
Imidazole (1.453 g, 21.316 mmol) and 11-bromo-1-undecanol (5.355
g, 21.316 mmol) were added together in 50 mL of dry DMF under
argon atmosphere. K2CO3 (5.898 g, 42.676 mmol) was added to the
mixed solution and stirred for 24 h at room temperature. The resulting
mixture was poured into ice water and extracted with methylene chloride
(350 mL) to remove DMF. The solution was dried with MgSO4, filtered,
concentrated, and purified by column chromatography (silica gel,
chloroform) to obtain 1-(11-hydroxundecyl) imidazole. 1H NMR (300
MHz) CDCl3: 7.503 (s, 1H), 7.064 (s, 1H), 6.911 (s, 1H), 3.933 (t, J
) 7.08, 2H), 3.64 (t, J ) 6.89, 2H), 1.772 (m, 2H), 1.561 (m, 2H),
1.267 (broad, 14 H). The 1-(11-hydroxyundecyl)imidazole (3.259, 12.83
mmol) and thiourea (2.930 g, 38.492 mmol) were added to 35 mL of
hydrobromic acid (48%) and refluxed for a day. The mixture was
neutralized with K2CO3, then NaOH was added (1.539 g, 38.492 mmol),
and the solution was refluxed in an argon atmosphere for 8 h. The
resulting solution was cooled to room temperature, poured into ice
water, and extracted with methylene chloride. The solution was dried
with MgSO4, filtered, concentrated, and purified by column chroma-
tography (silica gel, chloroform) to obtain 1-(11-mercaptoundecyl)
imidazole. 1H NMR (300 MHz) CDCl3: 7.490 (s, 1H), 7.066 (s, 1H),
6.905 (s, 1H), 3.924 (t, J ) 7.13, 2H), 2.522 (q, J ) 7.47, 2H), 1.769
(m, 2H), 1.605 (m, 2H), 1.392-1.264 (broad, 15 H). EI-HRMS: Calcd.,
254.18167 (C14H26N2S). Found, 254.18215.
Results
Structural Characterization. The thickness of the monolayer
films was assessed through capacitance studies. AC impedance
measurements were used to characterize the capacitance of the
monolayer films, and the area of the electrode was determined
in the manner described in the Experimental Section. For the
pyridine system (dodecylpyridine and undecane), an average
capacitance of 1.34 ( 0.17 µF/cm2 was found, and for the
imidazole system (undecylpyridine and octane), an average
2. Synthesis of Bis[12-((pyridinylcarbonyl)oxy)dodecyl] disulfide.
a. Bis(12-hydroxyododecyl) disulfide. 12-Mercapto-1-dodecanol (10
mmol) was dissolved in 50 mL methanol and titrated with 0.5 M
methanolic iodine until the reaction solution turned from colorless to
a persistent yellow. The reaction was quenched with 10% sodium
bisulfite to a colorless solution. The resulting mixture was dissolved
in distilled water and extracted with CH2Cl2. The solvent was removed
under vacuum. Purification of the resulting crude disulfide was
performed by flash chromatography (CH3Cl) to obtain the disulfide as
(9) Napper, A. M.; Liu, H.; Waldeck, D. H.; J. Phys. Chem. B 2001, 105,
(8) Clavilier, J. J..Electroanal. Chem. 1980, 107, 205.
7699.
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A R T I C L E S
Wei et al.
one expects no significant influence of defect sites on the
observed faradaic current.
Scanning tunneling microscopy studies were used to char-
acterize the films. Figure 3A shows STM images of a pyridine
terminated alkanethiol film to which cytochrome c had been
immobilized. The bright spots show positions on the surface
where the protein is adsorbed. The feature that is analyzed here
occupies an area of about 15 nm2 and a height of 0.7-0.9 nm.
Although a bit larger than the cross-sectional area expected for
individual cyctochrome c molecules (7-8 nm2), this size is
consistent with that expected for a protein.13 A range of feature
sizes, somewhat smaller than that shown and significantly larger
ones, can be identified on the surface, however. An analysis of
the image in Figure 3A indicates a surface coverage of ∼2.5%.
It should be emphasized that the distribution of protein on the
surface is not uniform; both regions with higher density of
protein and with lower density of protein were readily identifi-
able. Figure 3B shows images of a monolayer film with no
adsorbed protein (the scale is expanded over that shown in
Figure 3A). Different regions are also evident in this image.
Areas of depression (dark regions) are typically of dimension
20-30 Å across. Such structures represent depressions in the
film that are associated with defects in the underlying Au surface
and have been commonly observed for alkanethiol films on gold
electrodes.14 Although these features are interpreted as defects
in the underlying gold, they are still coated with alkanethiol. In
addition to this structure, elevated regions are also visible. These
elevated regions, which are not present in pure alkanethiol
monolayer films, correspond to 3-4% of the total area and are
assigned to the pyridine-terminated thiols. The vertical/height
length scale shown here for the images is compressed over the
actual physical height. The reasons for this artificial compression
when observing alkanethiols is discussed elsewhere.15 It is
evident from the image that the pyridine is not uniformly
distributed throughout the film. The degree of “phase segrega-
tion” and its dependence on preparation and solvent conditions
has not yet been investigated.
Figure 2. Voltammograms are shown for three different electrodes in
3-/4-
contact with an equimolar Fe(CN)6
solution (a, solid line is bare
electrode; b, dashed line is imidazole mixed-film electrode; c, dotted line
is pyridine mixed-film electrode).
capacitance of 1.96 (0.16 µF/cm2 was found. Using a parallel
plate model for the monolayer film, one obtains a thickness of
15.0 ( 1.9 Å for the pyridine-terminated system and 10.5 (
0.9 Å for the imidazole-terminated system.10 These distances
are in reasonable agreement with expectation. Because the
pyridine-terminated film consists mostly of undecanethiol and
a small fraction of pyridine terminated material, the capacitance
measurement should yield a film thickness that is similar to
that expected for undecanethiol, perhaps slightly thicker. If one
assumes that the alkanethiol chains are tilted at 30° from the
surface normal,11 one obtains a thickness of 12.3 Å for an
undecanethiol film. A corresponding analysis for the imidazole
terminated films, mostly composed of octanethiol, yields a film
thickness of 9.0 Å.
Electrochemical Characterization. Cyclic voltammetry was
performed on mixed monolayer films consisting of ∼97%
alkanethiol and 3% of an alkanethiol chain that was function-
alized with either pyridine, imidazole, or nitrile.16 These SAM-
coated electrodes were incubated in a solution of cytochrome c
for 30-60 min before being placed in a phosphate buffer
solution at pH ) 7.0. When the functionalized alkanethiol chain
was longer than the alkanethiol diluent, cytochrome c was
immobilized on the electrode surface. When the alkanethiol
diluent was longer than the functionalized chain, the cytochrome
c did not adsorb to the film. This conclusion was deduced from
the inability to observe a faradaic current in the mixed films
when the diluent alkanethiol had a chain length comparable to
that of the ω-terminated thiol.
Figure 2 illustrates the good blocking behavior observed for
the mixed monolayer films. The three voltammograms in this
figure were taken with the same redox solution (1 mM [Fe-
(CN)6]3-/[Fe(CN)6]4- in 0.5 M KCl) and the same parameters
(cell geometry and 100 mV/s scan rate). The bare Au electrode
shows a well-defined faradaic response (solid curve, a). In
contrast, the voltammograms for the octanethiol and imidazole
alkanethiol films (dashed curve, b) and the undecanethiol and
pyridine-alkanethiol films (dotted curve, c) show the blocking
behavior that is commonly found for insulating alkanethiol
coated electrodes.12 The blocking behavior indicates that the
films are compact and inhibit penetration of the ferricyanide
and ferrocyanide redox species. Because of the much larger size
of a cytochrome c, as compared to ferricyanide and ferrocyanide,
The electrochemical response was used to demonstrate that
the cytochrome was immobilized on the surface of the mono-
(10) This calculation assumes a parallel plate model for the capacitance of the
film (C ) ꢀ (area)/(thickness)) and takes the alkanethiol dielectric constant
ꢀ to be 2.6.
(13) Cytochrome C: A Multidisciplinary Approach; Scott, R. A., Mauk, A. G.,
Eds.; University Science Books: Sausalito, 1996.
(14) Poirier, G. E. Chem. ReV. 1997, 97, 1117.
(11) (a) Ulman, A. Chem ReV. 1996, 96, 1533. (b) Schreiber, F. Prog. Surf.
Sci. 2000, 65, 151.
(12) (a) Miller, C. J. Physical Electrochemistry: Principles, Methods, and
Applications; Rubinstein, I, Ed.; Marcel Dekker: New York, 1995; p 27.
(b) Finklea, H. O. Electroanalytical Chemistry; Bard, A. J., Rubinstein, I.,
Eds. Marcel Dekker: New York, 1996; p 109. (c) Khoshtariya, D. E.;
Dolidze, T. D.; Zusman, L. D.; Waldeck, D. H. J. Phys. Chem. 2001, 105,
1818.
(15) (a) Weiss, P. S.; Bumm, L. A.; Dunbar, T. D.; Burgin, T. P.; Tour, J. M.;
Allara, D. L. Ann. N.Y. Acad. Sci. 1998, 852, 145. (b) Gorman, C. B.;
Carrol, R. L.; He, Y.; Tian, F.; Fuierer, R. Langmuir 2000, 16, 6312.
(16) Although the deposition solution contained a 9:1 ratio of thiol species, the
STM studies indicate that the composition of the film deviates from this
ratio significantly.
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Direct Wiring of Cytochrome c Heme Unit, Electrode
A R T I C L E S
Figure 3. Panel A shows a topographic image for an electrode that has cytochrome c immobilized on the surface. A cross section through one of the
features is shown for two different directions. The image size is 188 × 188 nm, the bias voltage is 0.5 V, and the current set point is 25 pA. Panel B shows
an image for a pyridine-coated electrode with no cytochrome c adsorbed on the surface. The image size is 36.5 × 36.5 nm, the bias is 0.8 V, and the current
set point is 0.1 nA.
layer film. Figure 4a shows voltammograms obtained for the
imidazole systems both with and without incubating the
electrode in the cytochrome solution. In every case, when a
monolayer-coated electrode was placed directly in the electro-
chemical cell containing only the buffer solution (not exposed
to cytochrome c), the voltammogram displayed no faradaic
response. Subsequently, this same electrode was treated with
cytochrome c, rinsed, and placed in the buffer solution (see
Experimental Section for details). In each case, a well-defined
faradaic response was observed for the electrodes that were
incubated in the cytochrome c solution. The same behavior was
observed for the mixed films that were functionalized with
pyridine, and voltammograms of this sort were shown for
pyridine-terminated films earlier.5
coated with cytochrome and was found to exhibit a linear
dependence, which is consistent with immobilization of the
cytochrome on the surface. These data are presented for both
pyridine and imidazole in panel B of Figure 4. For a redox
couple that is immobilized on the electrode surface, the peak
current is given by
n2F2
4RT
ip )
VN
where n is the number of electrons transferred, F is Faraday’s
constant, V is the voltage scan rate, and N is the number of
redox active sites on the surface. For the imidazole system, the
slope of the peak current versus scan rate plot, with n ) 1,
gives a surface coverage of 2.0 ( 0.1 × 1012 cm-2 (or 3.3 (
0.2 × 10-12 mol/cm2), and for the pyridine system, it gives 1.5
In addition to the incubation studies, the peak current, ip, was
measured as a function of the voltage scan rate for electrodes
9
J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9595

A R T I C L E S
Wei et al.
Figure 4. Panel A shows voltammograms for the imidazole films in which the surface has been exposed to cytochrome c and has not been exposed to
cytochrome c. Panel B shows the linear dependence of the peak current on the voltage scan rate (imidazole is circles and pyridine is squares). The filled
symbols are for the reduction wave, and the empty symbols are for the oxidation wave.
( 0.1 × 1012 cm-2 (or 2.5 ( 0.3 × 10-12 mol/cm2). The method
for determining the electrode areas is described in the Experi-
mental Section. The surface coverage of cytochrome was also
determined by integrating the oxidation and reduction peaks of
the voltammograms. This procedure generated coverages of 3.0
( 0.23 × 10-12 mol/cm2 for the imidazole system and 2.4 (
0.22 × 10-12 mol/cm2 for the pyridine system. The coverage
calculated from the ip dependence of the scan rate is consistent
with the value obtained from the total charge transferred. Using
the coverage of 1.5 × 1012 cm-2 cytochrome c on the pyridine-
terminated monolayers, one calculates an average area per
cytochrome c molecule of 67 nm2, which is ∼10 times the cross-
sectional area of a cytochrome c molecule. Although these data
do not quantify the homogeneity of the protein’s distribution
on the surface, they indicate that the average distance between
protein molecules is high.
The average coverage obtained from the electrochemical
measurements, average of 10%, is significantly larger than that
obtained from the STM image in Figure 3A (2.5%). Other
regions of the surface were also imaged, and many of these
had larger concentrations of protein features. In part, the
difference between these two coverage measures can be ac-
counted for by the limited sampling in a single STM image. In
addition, it may be possible that the features do not all represent
a single protein, but rather, two or more proteins might be
clustered in regions of high pyridine concentration, and these
clusters are not well-resolved. Other differences could arise from
differences in the preparation and incubation of the monolayer
films on the STM samples, as opposed to those on the Au ball
electrodes (see Experimental Section).
c immobilized on the pyridine-terminated mixed monolayers
determined from cyclic voltammetry (2.4 × 10-12 mol/cm2) is
about one-tenth of this value, or 1 cytochrome c for every 10
pyridines.
The voltammograms of the imidazole and pyridine mixed
films at some selected voltage scan rates are presented in Figure
5. These data display well-defined peaks and show a small shift
of the peak (oxidation)-to-peak (reduction) separation with the
scan rate. This dependence can be used to analyze the electron-
transfer rate constant (see below). It is evident that the noise
level for the pyridine-terminated films (panel B) is higher than
for the imidazole-terminated films (panel A). To obtain better
defined peaks for the pyridine films at the slower scan rates, a
filter (the time constant of the filter is 590 Hz) was used in the
data collection. The use of a filter accounts for the difference
in noise level between the slower scan rate curves and the higher
scan rate curves, for which no filter was employed (in panel
B). Table 1 reports the full width at half-maximum (∆E1/2) of
the reduction peak for the adsorbed cytochrome, and it is close
to the ideal value of 91 mV for a fully reversible response. The
peak widths for the mixed films are similar to that for dilute
films of cytochrome c on carboxylic acid-terminated alkanethiols
and indicate a high degree of homogeneity for the mixed
systems. By contrast, an earlier study, which immobilized
cytochrome c on pure layers of pyridine terminated alkanethiols,
displayed significant broadening of the voltammograms and a
large asymmetry between the oxidation and reduction re-
sponses5. For the electrodes where the cytochrome is freely
diffusing, the difference between the voltammogram’s peak
potential and the half-peak potential is reported in Table 1. In
this latter case, the ideal value should be 56 mV. The
voltammograms for the nitrile films were significantly noisier
than those shown here, and for this reason, the rest of the study
focuses on the pyridine and imidazole mixed films.
It is possible to estimate the relative composition of the
monolayers (e.g., percent pyridine-terminated alkanethiols, the
active component, and alkanethiol, the diluent) from the STM
data. The ratio of active alkanethiol to diluent in the solution
used to prepare the mixed monolayer is 1:9. From Figure 3B,
one determines ∼3.3% for the coverage of pyridine by integrat-
ing the high points on the surface. Others have characterized
the coverage of alkanethiol monolayer on polycrystalline gold
and found it to be ∼7 × 10-10 mol/cm2.17 Combining these
values gives a surface concentration of ∼2.3 × 10-11 mol/cm2
for the pyridine-terminated thiol. The coverage of cytochrome
Table 1 also provides data on the apparent formal potentials
for a number of different systems. For S(CH2)2-Py monolayers
and hydroxyl-terminated monolayers to which the cytochrome
does not adsorb, the reported formal potentials are 5 mV and
44 mV versus Ag/AgCl, respectively. For carboxylic acid-
terminated monolayers to which the cytochrome is immobilized
by electrostatic binding to the protein exterior, the apparent
formal potential is 12 mV versus Ag/AgCl, intermediate between
those found for the nonadsorbed protein. In contrast, for the
(17) Finklea, H. O. In Encyclopedia of Analytical Chemistry; Meyers, R. A.,
Ed.; John Wiley & Sons Ltd: Chichester, 2000.
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9596 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002

Direct Wiring of Cytochrome c Heme Unit, Electrode
A R T I C L E S
Figure 5. Voltammograms are shown for cytochrome c immobilized on the surface of mixed monolayer films containing imidazole functionalities (panel
A) and pyridine functionalities (panel B). The scan rates for these voltammograms are 20 15, 10, and 6 V/s.
Table 1. Electrochemical Parameters for Different Electrode/
Cytochrome Systems
0′
E
∆E
∆E
scan rate
(V/s)
1/2
(mV)a
p/2
(mV)b
system
(mV)
HOC(CH2)6Sc
44 ( 2
5
12 ( 3
58
56
0.2
0.2
0.6
1.0
1.0
8.0
PyCO2(CH2)2Sc
HOOC(CH2)10S
99
108
117
132
PyCO2(CH2)12S/C11H21S
Im(CH2)11S/C8H15S
NC(CH2)11S/C8H15S
-172 ( 10
-346 ( 20
-415 ( 20
Figure 6. The dependence of the peak potential on the scan rate is shown
for the imidazole system (panel A) and the pyridine system (panel B). The
symbols follow the convention of Figure 4. Fits of the data to Marcus theory
predictions are also shown for two different reorganization energies (0.8
eV is the solid line and 0.9 eV is the dashed line).
a ∆E1/2 is full width at half max (fwhm) of a peak (that for the reduction
peak is shown here). b ∆Ep/2 is the difference between the peak potential
and the half-peak potential (see 21). c In this system the cytochrome c is
not immobilized on the electrode surface but is in solution at a concentration
of 50 µM.
pyridine and imidazole terminated films. Figure 6 shows a plot
of the peak shift from apparent formal potential (Ep-E0′) versus
the voltage scan rate for each system, along with the best fit to
the classical Marcus theory for the electron-transfer rate constant.
The theoretical curves are shown for two different reorganization
energies, 0.8 and 0.9 eV.4 This procedure provides standard rate
constants (k0) of 780 s-1 for the pyridine-terminated layer
(electron transfer through a C12 chain) and 850 s-1 for the
imidazole-terminated layer (electron transfer through a C11
chain). The similar rates found for oxidation and reduction
suggest a symmetry factor of 0.5 for the reaction.21 These rate
constants are quite high (peak voltage shifts are small), and one
must be concerned about possible contributions from iR drop
to the observed peak shifts.7,21 To this end, the impedance of
the electrochemical cell was measured to have a resistance of
300-500 Ω, which leads to a shift of <2 mV at the highest
currents.
mixed monolayer films, which are composed of pyridine,
imidazole, and nitrile functionalities that can interact with the
cytochrome’s heme, a significant negative shift of the redox
potential is observed, ranging from -172 mV for the pyridine
system to -415 mV for the nitrile system. This shift in the
redox potential is consistent with those found in homogeneous
solution studies of cytochrome c when the ligands pyridine,
imidazole, and nitrile are present.18 Spectroscopic studies have
shown that the pyridine, imidazole, and nitrile functionalities
can bind to the redox center of the cytochrome in free solution.19
Consequently, the negative shift in redox potential indicates an
interaction between the terminal functionality of the layer and
the cytochrome’s heme.
The dependence of the reduction (or oxidation) peak’s
position on the voltage scan rate can be used to characterize
the electron-transfer rate constant.9,20 This method was used to
determine rate constants for cytochrome c immobilized on the
Association Strength. The strength of association and
stability of the adsorbed cytochrome c films was assessed by
monitoring the desorption kinetics. In this procedure, the coated
(18) See Table 1 of Fedurco, M. Coord. Chem. ReV. 2000, 209, 263, and
references therein.
(19) (a) Spiro, T. G.; Li, X. Y. Resonance Raman Spectra of Heme and
Metalloproteins in Biological Applications of Raman Spectroscopy;
Wiley: New York, 1988 Vol. 3, p 1. (b) Hirota, S.; Ogura, T.; Shingawa,
I. K.; Yoshikawa, S.; Kitagawa, T. J. Phys. Chem. 1996, 100, 15274. (c)
Yeh, S. R.; Rousseau, D. L. J. Biol. Chem. 1999, 274, 17853. (d) Smith,
M.; McLendon, G. J. Am. Chem. Soc. 1981, 103, 4912. (e) Ferrer, J. C.;
Gullemette, J. G.; Bogumil, R.; Inglis, S. C.; Smith, M.; Mauk, A. G. J.
Am. Chem. Soc. 1993, 115, 7507. (f) Liu, G.; Chen, Y. Tang. W. J. Chem.
Soc., Dalton Trans. 1997, 795.
(20) (a) Tender, L.; Carter, M. T.; Murray, R. W. Anal. Chem. 1994, 66, 3173.
(b) Weber, K.; Creager, S. E. Anal. Chem. 1994, 66, 3166. (c) Honeychurch,
M. J. Langmuir 1999, 15, 5158.
(21) (a) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New
York, 1980. (b) Bockris, J. O’M.; Reddy, A. K. N. Modern Electrochem-
istry; Plenum/Rosetta: New York, 1970.
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A R T I C L E S
Wei et al.
Discussion and Conclusion
The immobilization of the cytochrome c on functionalized
monolayer films is demonstrated through a combination of
electrochemical and structural probes. By combining ω-termi-
nated pyridine or imidazole alkanethiols with an alkanethiol
diluent, it is possible to immobilize cytochrome c onto electrode
surfaces through the interaction of the pyridine or imidazole
with the heme of the cytochrome. The immobilization is
demonstrated by the electrochemical observation that the peak
current of the voltammogram grows linearly with the voltage
scan rate and that the faradaic response is observed for the
cytochrome c treated electrodes when they are immersed in the
buffer solution. Complimentary studies used scanning tunneling
microscopy to observe the presence of nanometer-scale objects
on the surface of the monolayer film after they were treated in
a solution containing cytochrome c. The lateral scale of the
objects is similar to that expected for protein adsorption.
Figure 7. Time profiles for the surface concentration of immobilized
cytochrome c are shown for both the pyridine-terminated films and the
imidazole-terminated films. The symbol convention is the same as Figure
4.
film was placed in the solution, and within a few seconds (<10
s), a voltammogram was initiated with a scan rate of 20 V/s.
Voltammograms were run at subsequent time points until the
peak current was found to stabilize. Because the peak current
is proportional to the amount of cytochrome adsorbed on the
surface, this procedure generates a profile of the adsorbed
species concentration as a function of time. Figure 7 shows these
concentration profiles for both the imidazole and the pyridine-
terminated films. The desorption kinetics can be modeled by
considering that the system evolves toward an equilibrium of
the following type
Previous work reported the immobilization of cytochrome c
on the surface of pure monolayers of pyridinalalkanethiol. That
work demonstrated the immobilization in a similar manner;
however, the electrochemistry was not representative of a
homogeneous distribution of redox sites. The pure monolayer
films contained relatively broad peak widths (160-190 mV)
and displayed asymmetric redox kinetics. In particular, the
oxidation was found to be much faster than the rate constant
for reduction. This observation was believed to reflect a change
in the redox active sites after oxidation, associated with the
degree, or strength, of interaction between the cytochrome and
the pyridine. In contrast, the mixed monolayer systems show
much narrower widths for the redox peaks (see Table 1) and
yield similar rate constants for the reduction and oxidation
waves. This indicates a much more uniform distribution of sites
on the surface and not profound changes in binding geometry
upon electron transfer.
S-H2O + Cyt.C {k′
\k} H2O + S-Cyt.C
where S-H2O represents a solvated surface binding site, Cyt.C
represents a cytochrome c molecule in solution, and S-Cyt.C
represents a surface-bound cytochrome c. The rate constant k′
characterizes the binding to surface sites, and the rate constant
k characterizes the dissociation of the cytochrome from the
binding site. Under the initial condition that all the cytochrome
c is bound to the surface, one finds that the concentration of
the surface adsorbed cytochrome θ(t) evolves according to
The immobilization strategy utilized here is different from
that used to immobilize the cytochrome c on the surfaces of
-COOH-terminated alkanethiol monolayers. In that case, one
observes similar widths for the voltammetric peaks, but the
redox potentials observed on the COOH layers are much more
positive than those found for the mixed monolayer films (see
Table 1). The large difference in observed redox potential
indicates that the nature of the immobilization is different in
the two cases. The pyridine- and imidazole-terminated films
have redox potentials that are shifted negatively from that found
for cytochrome c in free solution and are similar to the shift
observed when cytochrome c/pyridine complexes are studied
in free solution. In contrast, the COOH-terminated layer has a
redox potential that is similar to that observed for cytochrome
c in solution. These findings are consistent with the immobiliza-
tion of cytochrome c on COOH-terminated films by adsorption
on the protein’s periphery, whereas the pyridine- and imidazole-
terminated layers interact with the heme of the cytochrome.
θ(t)
) f + (1 - f) exp( - (k + κ′)t)
θ(0)
where κ′ is given by k′[Cyt.C], θ(t) is the coverage at time t,
and f is the ratio κ′/(θ(0)(κ′ + k)). For the pyridine-terminated
layer, the decay constant is 2.5 × 10-3 s-1, and f is 0.14 (
0.03, whereas the imidazole-terminated layer has a decay
constant of 1.0 × 10-3 s-1 and f of 0.35 ( 0.04. The faster
decay rate and the lower final value for the pyridine film
indicates that its adsorption constant is smaller than that of the
imidazole film; that is, the imidazole has a stronger association
with the cytochrome than does the pyridine. If one assumes
that θ(0) ) 1, the fitting parameters give rate constants of k )
2.2 × 10-3 s-1 and κ′ ) 3.5 × 10-4 s-1 for pyridine, and k )
6.5 × 10-4 s-1 and κ′ ) 3.5 × 10-4 s-1 for imidazole. The
effective association rate constant κ′ appears to be the same for
both systems, suggesting that the association is diffusion-limited.
In contrast, the dissociation rate constants are different from
one another, with the imidazole system being almost three times
smaller than that of the pyridine system.
The desorption of cytochrome c from the layer was monitored
voltammetrically for both the pyridine-terminated layers and
the imidazole-terminated layers. It was found that the dissocia-
tion of the cytochrome from the imidazole films is ∼3 times
slower than that from the pyridine terminated films. This finding
is consistent with a stronger interaction between the heme and
the imidazole than with the heme and the pyridine.18
9
9598 J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002

Direct Wiring of Cytochrome c Heme Unit, Electrode
A R T I C L E S
Finally, the electron-transfer rate constants were measured
for the cytochrome c adsorbed on the film’s surface. For the
imidazole system, the rate constant was found to be 850 s-1
through a C11 methylene chain and 780 s-1 through a C12
methylene chain. These rate constants are comparable to rate
constants observed for electron transfer through C6 methylene
chains of carboxylic acid-terminated layers. A number of
explanations are possible for this observation, such as changes
in the reorganization energy, the electronic coupling, or the
importance of local dipole fields. Currently, we are exploring
these different issues, and our preliminary findings indicate that
differences in the electronic coupling are likely to play a
significant role in the final explanation. Detailed studies of the
carboxylic acid-terminated films have identified an electron-
transfer rate constant that is independent of distance for
methylene chain lengths of C6 and shorter. The difference in
length associated with C6 and C11 can be converted to a
distance. If one takes the C-C bond length to be 1.5 Å and the
alkane chains to be tilted at 30° from the surface normal, one
finds a distance of 5.5 Å. This value is in reasonable agreement
with the distance from the end of the COOH-terminated layer
through the cytochrome c outer surface to the redox active heme,
a tunneling distance of 5-6 Å, which is consistent with the
COOH binding electrostatically to the cytochrome’s outer
surface. For the COOH-terminated monolayers, it has been
reported that the cytochrome displays a tunneling dependence
for methylene chain lengths of C9 and longer.2,3 For the current
system, one might expect that the range of distances, for which
the electron-transfer rate constant is distance-independent, would
be extended if the recognition element (pyridine, imidazole)
binds to the heme of the protein rather than its outer surface.
For longer methylene chains, one observes an exponential
distance dependence for the electron-transfer rate.22
The ability to adsorb the redox active cytochrome c to the
surface of SAM-coated gold electrodes in a restricted geometry
has been demonstrated. These systems provide a model system
to investigate aspects of electron-transfer dynamics between
biomolecules and metal electrodes.
Acknowledgment. We acknowledge support in part from the
U.S.-Israel BSF, the NSF-REU program at the University of
Pittsburgh, and the Department of Energy. We thank D.
Khoshtariya and E. Borguet for useful discussions.
JA025518C
(22) Wei, J.; Liu, H.; Khoshtariya, D.; Yamamoto, H.; Dick, A.; Waldeck, D.
H. Angew. Chem., submitted.
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J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9599