Electrochemical Response of Cytochrome c Immobilized on Smooth

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Electrochemical Response of Cytochrome c Immobilized on Smooth and Roughened Silver and Gold Surfaces Chemically Modified with 11-Mercaptounodecanoic Acid Diego Millo,† Antonio Ranieri,‡ Peter Gross,† Hoang K. Ly,§ Marco Borsari,‡ Peter Hildebrandt,§ Gijs J. L. Wuite,† Cees Gooijer,† and Gert van der Zwan*,† Departments of Analytical Chemistry and Applied Spectroscopy and Physics of Complex Systems, Laser Centre, Vrije UniVersiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy, and Technische UniVersita¨t Berlin, Institut fu¨r Chemie, Sekr. PC14, Strasse des 17, Juni 135, D-10623 Berlin, Germany ReceiVed: September 4, 2008; ReVised Manuscript ReceiVed: December 19, 2008

Cyclic voltammetry was employed to determine the formal reduction potential and heterogeneous electrontransfer rate constant of cytochrome c immobilized on three different metal substrates chemically modified with 11-mercaptoundoecanoic acid. The metal substrates include smooth gold and silver electrodes as well as nanoscopically rough silver electrodes obtained via an oxidation-reduction cycle. Electrode roughening followed a protocol typically employed to prepare surface-enhanced Raman active surfaces such that the electrochemical results can be compared with those determined by surface-enhanced resonance Raman spectroscopy of cytochrome c. The roughness of the surfaces was estimated by means of atomic force microscopy. For all systems midpoint potentials were found to be -0.068 V (vs SCE), although for rough silver electrode the midpoint potential slightly shifted in time from -0.051 V to -0.068 V within 24 h. The heterogeneous electron-transfer rate constants differ for the various metal substrates and were found to be smaller by a factor of 2.5 for the rough and smooth Ag substrates compared to Au electrodes. These findings imply that it is primarily the kind of metal rather than its surface morphology that controls the thermodynamics and kinetics of interfacial redox processes of immobilized cytochrome c. The present paper reconciles the partly conflicting results obtained by electrochemical methods, usually done on Au, and surface-enhanced resonance Raman spectroscopic techniques which are usually performed on Ag electrodes. 1. Introduction The strong interest in characterizing the electron-transfer (ET) properties of redox proteins on electrodes is, on one hand, derived from the increasing potential of bioelectronic hybrid systems in which metalloproteins or metalloenzymes are attached to metal substrates as building blocks for biosensors or biocatalysts in biotechnological applications.1,2 On the other hand, the electrochemical interfaces bear some similarities with biological membranes where most of the natural biological ET reactions occur.3 Analyzing the interfacial processes of redox proteins in an electrochemical environment may, therefore, contribute both to the understanding the physiological processes of redox proteins and to the design of protein-based bioelectronic devices. There is a large body of experimental data on the thermodynamics and kinetics of the redox processes of electrontransferring proteins on electrodes.4-29 Whereas in early studies that were employed on bare electrodes protein denaturation was usually a serious drawback,3 the use of biocompatible electrode coatings substantially improved the structural stability of the immobilized proteins and turned out to be a crucial factor for ensuring a reversible electrochemical response.4-29 Among these coatings, self-assembled monolayers (SAMs) of ω-functionalized mercaptanes were found to be particularly versatile since * To whom correspondence should be addressed. Phone +31(0)205987635. Fax +31(0)205987543. E-mail [email protected]. † Vrije Universiteit. ‡ University of Modena and Reggio Emilia. § Technische Universita¨t Berlin.

they allow for a specific binding of proteins depending on the choice of the terminal functional group. SAM-coated electrodes thus have been employed to analyze in detail the interfacial redox processes of simple test proteins such as the heme protein cytochrome c (cyt-c) or the copper protein azurin.4-9,11-16,18-29 Inspection of the results obtained in these studies, however, reveal a surprising scattering of the data for both the formal reduction potential (E°′) and the heterogeneous electron-transfer rate constant (ks). For example, for cyt-c immobilized on electrodes coated with 11-mercaptounodecanoic acid (MUA) values between 0 and -60 mV (vs saturated calomel electrode [SCE]) have been reported for E°′, 5,6,11,13,15,23-25 and comparably large variations were found for ks (20-100 s-1).7-9,11,14,16,24,25,29 These studies were based on electrochemical and spectroelectrochemical methods requiring specific properties of the metal substrates. Cyclic voltammetry (CV), for instance, was performed, in most cases, on smooth gold substrates with singlecrystal or polished polycrystalline surfaces.5-9,11,15,16,23-25,30,31 Surface-enhanced resonance Raman (SERR) and surfaceenhanced infrared absorption (SEIRA) spectroscopy, however, require metals of nanoscale surface roughness which is a prerequisite for enhancement of the oscillating electric field of the radiation in the near-field of the metal.32-35 Systematic studies of redox proteins by means of these techniques are more demanding and time consuming than CV measurements but allow probing the molecular structure changes of the redox center (SERR) and the protein (SEIRA) during the redox process.36 The advantages of SERR spectroscopy, however, can only be exploited when the excitation line is in resonance with both the surface plasmons of the metal and an electronic

10.1021/jp807855y CCC: $40.75  2009 American Chemical Society Published on Web 01/26/2009

2862 J. Phys. Chem. C, Vol. 113, No. 7, 2009 transition of the redox center. These conditions are generally only fulfilled for silver substrates upon excitation in the visible and near-UV spectral region,34,37-39 whereas for SEIRA spectroscopy rough gold electrodes are the optimum metal substrates.35 Thus, various electrochemical and spectroscopic techniques are typically carried out under different experimental conditions and discrepancies between redox properties obtained by SERR and SEIRA spectroscopy as well as by electrochemical methods might be due to the effect of the metal and its surface roughness. In fact, the mean of the E°′ values reported for cyt-c on MUA-coated smooth gold electrodes is ca. -45 mV 5,6,24,25 compared to the ca. +5 mV observed on gold surfaces with nanoscale roughness.29 For rough silver surfaces, however, the formal reduction potential was found to be very similar to that for smooth gold electrodes,13,25 whereas values for smooth silver electrodes have not been reported. On the other hand, the scattering of the heterogeneous electron-transfer rate constants cannot be directly related to the kind of the metal and its surface morphology. To understand these partially conflicting results and identify the parameters that are essential for controlling the thermodynamics and kinetics of interfacial redox processes we analyzed the electrochemical response of cyt-c on various types of electrodes coated with a SAM of MUA. The formal reduction potentials and heterogeneous electron-transfer rate constants have been determined by cyclic voltammetry using smooth gold as well as smooth and rough silver electrodes. Rough silver electrodes were prepared according to a protocol used for SERR spectroscopy. For such electrodes we already showed that the E°′ values obtained with CV and SERR spectroscopy are similar.25,40 To achieve a sound comparison the experiments with the various electrodes were performed under identical conditions, specifically for preparation of the self-assembled monolayer (SAM), protein immobilization, and electrochemical and spectroscopic measurements. 2. Experimental Section 2.1. Chemicals. Bovine heart cytochrome c (cyt-c) was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Ten millimolar sodium phosphate buffer (NPB) solutions at pH 7.0 were prepared from Na2HPO4 and NaH2PO4 (J. T. Baker, Deventer, The Netherlands). KCl, and 11-mercaptounodecanoic acid (MUA) were purchased from Sigma-Aldrich. Ethanol and H2SO4 were purchased from Baker. Purified Milli-Q water (Millipore, Massachusetts) was used for all preparations and procedures. All chemicals were of reagent grade. 2.2. Electrochemical Instrumentation. Electrochemical measurements were performed in a glass cell with a filling volume of approximately 2 mL. The working electrodes (WE) were silver disk electrodes of 2 mm diameter (geometric area of 0.0314 cm2) purchased from IJ Cambria Scientific, Carms, U.K., and a 1 mm diameter polycrystalline gold wire;41 a platinum coil and a saturated calomel electrode (AMEL Instruments, Milano, Italy) were used as counter (CE) and reference electrodes (RE), respectively. The RE was placed in the working solution (10 mM NPB). The RE and cell were kept at constant room temperature (20 ( 0.1 °C) during all experiments. The three-electrode system was controlled with a µAutolab potentiostat (Eco Chemie, Utrech, The Netherlands). All potentials were calibrated against the methylviologen (MV) MV2+/MV+ couple and referred to the saturated calomel electrode (SCE). 2.3. Preparation of the Electrodes. Three types of electrodes systems were prepared on the basis of smooth Ag and

Millo et al. Au electrodes (AgS/MUA/cyt-c; Au/MUA/cyt-c) and electrochemically roughened Ag electrodes (AgR/MUA/cyt-c). AgS/MUA/cyt-c. Silver electrodes were polished with water on aluminum oxide lapping film sheets (261X and 262X, 3MTH, USA) from 5 to 1 µm grain size until a mirror-like appearance of the surfaces was obtained.42 After polishing the smooth silver electrodes were electrochemically pretreated (5 min at -1.0 V in 0.1 M KCl) and then immediately immersed in the 2 mM MUA solution in ethanol for 24 h without rinsing with water. AgR/MUA/cyt-c. Prior to roughening the polished silver electrodes were subjected to an electrochemical pretreatment (5 min at -1.0 V in 0.1 M KCl solution; vide supra). Subsequently, four oxidation-reduction cycles (ORCs) were applied (30 s at +0.4 V and 30 s at -0.4 V), resulting in a charge density of ca. 0.67 C/cm2 for each ORC.40 The electrochemical pretreatments and ORCs were performed in a glass electrochemical cell (5 mL) containing a 0.1 M KCl solution. After the ORCs the roughened silver electrodes were coated with MUA as described above. Au/MUA/cyt-c. Gold electrodes were kept in warm 1.0 M H2SO4 for 1 h. Subsequently, 10 voltammetric scans in 1 M H2SO4 were applied (see Supporting Information). Then the electrodes were rinsed with water and ethanol and finally coated with MUA as described above. After SAM formation all electrodes were rinsed with ethanol and dried under a gentle stream of nitrogen. Cyt-c was then adsorbed by immersing the coated electrodes into a 30 µM solution of cyt-c (10 mM NPB, pH 7.0) for 1 h at 4 °C. 2.4. AFM Measurements. Electrode topography was characterized via an Autoprobe CP AFM (Park Scientific Instuments) operating in contact mode. We used a commercial Si3N4 cantilever (Nanosensors, Wetzlar-Blankenfeld) with a spring constant of 0.03 N/m. The nominal tip radius of 20 nm corresponds to the lower limit of spatial resolution. The samples were characterized by nonfiltered 512 × 512 pixel images of areas ranging from 5 µm × 5 to 25 µm × 25 µm. Data analysis of the surfaces was done with SPIP (Denmark) software. The particle size distribution and root-mean-square values were determined after plane correction. 2.5. Raman Measurements with 413 nm Excitation. Spectroscopic measurements were carried out using a home-built Raman microscope in a backscattering configuration. A Zeiss microscope (D-7082 with a 40× objective, NA 0.60, working distance 2 mm) was coupled to a Andor Shamrock SR-303i-A single monochromator (Andor Technologies DV-420OE, Belfast, N. Ireland) with a 2400 g/mm holographic grating and an Andor Newton DU970N CCD camera (Andor Technologies DV-420OE, Belfast, N. Ireland). The 413 nm line of a continuous wave Kr ion laser (Coherent Innova 300c, California) was used for excitation. The Rayleigh scattered light was removed using a third Millenium edge long pass filter. A laser power of 5 mW at the sample and an accumulation time of 60 s were used for all experiments. The monochromator slit was set to 120 µm, yielding a resolution of approximately 4 cm-1, with an increment per data point of approximately 0.8 cm-1. Potential-controlled SERRS experiments were done using the spectroelectrochemical cell described elsewhere.43 2.6. Raman Measurements with 514 nm Excitation. SERR spectra were measured in a backscattering geometry using a confocal microscope coupled to a single-stage spectrograph (Jobin Yvon, LabRam 800 HR) equipped with a 2400 L/mm grating and liquid nitrogen cooled back-illuminated CCD detector. Elastic scattering was rejected by a Notch filter. The 514 nm line of a continuous wave Ar ion laser (Coherent Innova

Cytochrome c at Gold and Silver Electrodes

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Figure 2. CV plots of Au/MUA/cyt-c, AgR/MUA/cyt-c, and AgS/ MUA/cyt-c in 10 mM NPB (from darkest to lightest line). The scan rate was 0.2 V s-1. The data are displayed as current density j vs potential to facilitate comparison of signals obtained from electrodes with different surface areas. Areas of the smooth electrodes were taken to be the geometric ones (see section 2.2 and ref 41). The area of the roughened silver electrode was determined by the capacitance current method as shown in the Supporting Information.

Figure 1. AFM images of smooth (a) and roughened (b) silver electrodes: (a) 10 × 10 µm tile size and (b) 5 × 5 µm tile size. (c) Different dimensions of the surface features for the two profiles are compared.

70c) was focused onto the surface of the silver working electrode by means of a long working distance objective (20×; N.A. 0.35). 3. Results and Discussion 3.1. Morphological Characterization of Silver Electrodes. The roughness profile of the silver electrodes was examined via atomic force microscopy (AFM) measurements. Figure 1a shows the image of the smooth surface immediately after polishing. Despite the fact that the visual appearance of the surface is mirror-like, the AFM image reveals some distinct features, probably due to the mechanical treatment of the electrode. These features are, however, considerably smaller than those obtained after applying ORCs (Figure 1b). In fact, the presence of these features does not interfere significantly with the nanometers scale roughness observed on ORC-roughened surfaces (vide infra, Figure 1c). The AFM image of a silver surface after the ORC treatment shows the typical rough morphology already previously observed for SER-active surfaces.44 It includes globular structures of various sizes which are randomly distributed over the surface (Figure 1b). Visual inspection and data analysis of the Fourier spectrum do not reveal any preferential or regular distribution

of these features. Although a detailed characterization of surface roughness is beyond the purpose of this paper, an estimate of the average dimensions of the surface roughness may be instructive specifically in view of its relation to SER activity.37 According to the literature,45 the root-mean-square roughness (Rq) is an appropriate descriptor of the surface roughness for surface morphologies with a Gaussian height distribution. This is the case for ORC-roughened silver surfaces which exhibit irregular nanoscopic structures. The height distribution can be in fact adequately fitted with a Gaussian function (see Supporting Information). Assuming a hemispherical shape, Rq represents the diameter of such hemisphere.46 This quantity was determined to be 100 ( 6 nm, which is in the range of the values determined for nanostructured surfaces that exhibit SER activity.47 In fact, silver surfaces treated according to the ORC protocol used in this work afforded intense SERR and SER spectra upon excitation with 41340 (see Figure 3) and 514 nm, respectively.40,43 3.2. Spectrolectrochemical Characterization. The voltammetric responses of cyt-c immobilized on smooth and roughened electrodes, chemically modified with MUA, are shown in Figure 2. The CV signal of AgS/MUA/cyt-c is poor in comparison with that obtained under identical experimental conditions on gold surfaces. Well-defined voltammetric peaks are difficult to detect at scan rates below 0.1 V s-1 and above 10 V s-1, whereas the peaks of AgR/MUA/cyt-c and Au/MUA/ cyt-c were readily observed in a wider scan rate range from 0.02 to 10 V s-1 and from 0.02 to 18 V s-1, respectively. At a scan rate of 0.2 V s-1 the CV signal of AgS/MUA/cyt-c displays two current peaks attributable to the monoelectronic reduction and oxidation of cyt-c. Anodic and cathodic peak currents were found to be identical and proportional to the scan rate V, as expected for surface-confined redox species (see Supporting Information). Given the quasi-reversibility of the electrochemical process the midpoint potential E1/2 ) (Ep,c+Ep,a)/2 corresponds to the formal reduction potential E°′, which thus is determined to be -68 mV. This value, which was found to be constant over the entire scan rate range, is in agreement with those reported for cyt-c immobilized on MUA-coated electrodes using electrochemical methods.4,25 For Au/MUA/cyt-c as well as AgR/MUA/cyt-c at long equilibriation times (vide infra) the same values were determined, implying that the formal reduction potential of cyt-c is independent of the kind of metal and respective surface morphology (Table 1). The only difference refers to AgR/MUA/

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Figure 3. SERRS spectra of AgR/MUA/cyt-c measured at -0.2 V immediately after protein incubation (thick line) and after incubation of the electrode in buffer solution for 24 h (thin line). The spectra were obtained with 413 nm excitation. The inset shows the CV plots of AgR/ MUA/cyt-c immediately after protein incubation (thick line) and incubation of the electrode in buffer solution for 24 h (thin line). The scan rate was 0.1 V s-1. Further experimental details are given in the text.

TABLE 1: Electrochemical Properties of cyt-c at Different Electrode Systemsa system R

b

Ag /MUA/cyt-c AgS/MUA/cyt-c Au/MUA/cyt-c

E°′/mV

ks/s-1

∆Ep/mV

fwhm/mV

-68 (-51) -68 -67

16 (10) 13 33

20 (24) 49 18

99 (123) 121 108

a ∆Ep and fwhm were measured at V ) 0.2 V s-1. Average errors on E°′, ks, ∆Ep, and fwhm are (1 mV, (8%, (2 mV, and (4 mV, respectively. b The data refer to measurements after 24 h incubation in the buffer. The values obtained immediately after protein immobilization are given in parentheses.

cyt-c electrodes immediately after protein immobilization, which affords a CV with a formal reduction potential of - 51 mV, a peak separation of 24 mV, and a full-width at half-maximum (fwhm) value of 123 mV (Figure 3, inset). After 24 h of incubation in buffer solution at 4 °C the CV displayed a peak separation of 20 mV and a fwhm of 99 mV; the formal reduction potential shifted to -68 mV, i.e., to the same value observed for Au/MUA/cyt-c and AgS/MUA/cyt-c immediately after protein immobilization. This time dependence of the CV of the AgR/MUA/cyt-c electrode was found to be reproducible, and no further changes of the profile and fwhm and E°′ values were noted after incubation times longer than 24 h. Note that the value for E°′ obtained in SERR experiments on MUA-coated rough silver electrodes, carried out immediately after protein adsorption, is even more positive (-31 mV)13 than the initial value determined for AgR/MUA/cyt-c in the present CV experiments (-51 mV) (vide infra). The time dependence of the CV signals for AgR/MUA/cyt-c are reminiscent of previous findings by Clark et al.,6 who observed a decrease in peak broadening, peak separation, and formal reduction potential after a short time exposure (10 s) of the electrode to 1 M KCl solutions. These results could be reproduced in the present work by applying the same experimental procedure as described by Clark et al.6 These authors

ascribed the peak broadening in the CV recorded immediately after protein immobilization to a heterogeneous population of the adsorbed cyt-c. To check this hypothesis and identify the molecular nature of the heterogeneity we employed SERR spectroscopy to study the AgR/MUA/cyt-c electrode immediately after protein incubation and after 24 h incubation time. First, SERR spectra were measured in the potential range from +0.05 to -0.2 V using 413 nm excitation. In all spectra only the unique signature of the native forms of cyt-c (state B1) was observed, which in the reduced form displays the characteristic marker bands of the heme at 1361, 1492, 1591, and 1621 cm-1 corresponding to the ν4, ν3, ν2, and ν10 modes, respectively (Figure 3).48,49 The spectra do not show any extra bands or band broadening; so, there is no indication for structural heterogeneity on the level of the heme structure. This finding confirms the conclusions derived from previous SERR experiments.13 In fact, Clark et al.6 proposed that the heterogeneity may be related to protein-protein interactions between the adsorbed cyt-c molecules. Upon treatment with 1.0 M KCl solutions cyt-c partially desorbs, leading to a cyt-c submonolayer in which the proteins are not in direct contact with their neighbors. Accordingly, all proteins would experience a similar environment, giving rise to the homogeneous population as reflected by the quasi-ideal electrochemical behavior (∆Ep ) 20 mV; fwhm ) 99 mV). Such a rearrangement of the protein film on the electrode is not expected to involve structural changes of the heme site that would be reflected by a frequency shift of the vibrational modes. However, alterations of protein-protein interactions may cause changes in the average orientation of the immobilized proteins. Such effects can be probed by SERR spectroscopy under preresonance excitation using the 514 nm line.29 Under these conditions the orientation dependence of the SER enhancement is different for totally symmetric (A1g) and nontotally symmetric (B1g) modes such that changes of the intensity ratio of modes of different symmetry (e.g., ν10 and ν4) can be taken as a measure for the reorientation of cyt-c with respect to the surface. Thus, in a second series of experiments SERR spectra (514 nm excitation) of the AgR/MUA/cyt-c electrode were measured immediately after protein immobilization and subsequent to a 24 h incubation in the buffer. However, there are only small differences in the (potential-dependent) intensity ratios of the ν10 and ν4 modes which are hardly above the error margin of the data (see Supporting Information). Thus, protein reorientation can be neither excluded nor directly confirmed to be the origin for the time-dependent changes in the CV measurements. In contrast to the formal reduction potential, the formal heterogeneous ET rate constants ks are different for Au and Ag. These constants, which were determined on the basis of Laviron’s method,50,51 were found to be smaller by a factor of ca. 2.5-fold for both smooth and rough silver electrodes as compared to gold electrodes under otherwise the same experimental conditions (trumpet plots are shown in the Supporting Information). Thus, the origin for these differences must be related to the specific structural and electronic properties of MUA-coated gold and silver electrodes rather than to the surface morphology. Pretreatments of the bare metal surfaces, dedicated to remove chemical contaminants prior to SAM formation, are milder in the case of silver than for gold. Thus, these cleaning procedures may be less efficient for silver so that monolayer imperfections due to the presence of contaminants are more likely than for gold. However, even in the case of perfect monolayers the SAM films on the two metals will differ in thickness. The tilt angles of the aliphatic chains with respect to the surface normal have been determined to be ca. 30° on gold

Cytochrome c at Gold and Silver Electrodes and 10° on silver.52 Therefore, the through-space distance from the carboxyl group of the SAM surface to the electrode is estimated to be 19 and 17 Å for Ag/MUA and Au/MUA, respectively.13,53-55 Recent studies on cyt-c immobilized on SAM-coated gold electrodes7,24 have shown a decrease of ks upon increasing the number of methylene groups n in carboxylterminated mercaptanes, corresponding to a factor of 2.5-3 per methylene unit for SAMs with n g 10. This decrease is equivalent to a lowering of the ET rate constant by a factor of ca. 2 for an increase of the SAM thickness by 1 Å, which might account for the different ks values for MUA-coated silver and gold electrodes observed in this work. Alternatively, different structures or structural heterogeneities of the SAMs on gold and silver electrodes may cause different orientational distributions of the immobilized cyt-c which, in turn, would affect the average electron-tunneling rate and thus the measured rate constants. In addition, the different potentials of zero charge Epzc for coated gold and silver electrodes may affect electron tunneling of the immobilized protein. In fact, for SAM-coated silver Epzc is distinctly more negative than E°′, whereas in the case of gold Epzc and E°′ are similar.56,57 As a consequence, the local electric fields at the SAM/protein interfaces may differ in sign and magnitude for Au/MUA/cyt-c and Ag/MUA/cyt-c and thus either accelerate or slow down ET. On the basis of the present findings one may reconcile the results of this work and the kinetic data previously determined by CV as well as by SERR and SEIRA spectroscopy. For electrodes of nanoscale roughness a 2-times larger ET rate constant was observed for gold with SAM coatings of 16mercaptohexadecaonic acid as compared to silver.28,36 This increase is quite comparable to that found in the present work for MUA-coated electrodes. Also, the ET rate constant for rough MUA-coated gold electrodes determined by SEIRA spectroscopy and CV (ca. 40 s-1)28 agrees very well with the present results. A discrepancy, however, remains for the rate constants on SAM-coated Ag electrodes determined by CV in this work (16 s-1) and by time-resolved SERR spectroscopy reported previously (43 s-1).14 We cannot rule out that the origin lies in the different surface roughening procedure employed in the latter study. Under these conditions the silver surface exhibits distinctly higher surface roughness which, unlike rough silver surfaces prepared in this work, might affect the SAM structure and thus the ET rate. An alternative explanation is based upon the different techniques. Since the magnitude of the enhancement of the SERR signals depends on the surface morphology it is possible that the spectra originate predominantly from a small fraction of cyt-c molecules which are adsorbed close to specific sites of the metal electrode, providing a particularly strong enhancement. If due to a specific local SAM structure at these sites electron tunneling occurs with a different efficiency for this subpopulation of cyt-c, the ET rate constant derived from the time-resolved SERR spectra may not necessarily be identical to the average value that is probed by CV. This hypothesis is supported by the fact that the formal reduction potential determined by SERR spectroscopy is even more positive by 20 mV that the value obtained by CV immediately after protein immobilization (vide supra). Conclusions The formal reduction potentials of cyt-c immobilized on SAM-coated smooth silver and gold electrodes were found to be the same and thus independent of the metal. Electrochemically roughened silver supports display time-dependent changes

J. Phys. Chem. C, Vol. 113, No. 7, 2009 2865 of the CV corresponding to a cathodic shift of the reduction potential. The limiting value obtained after 24 h is identical to that obtained for smooth electrodes immediately after protein immobilization. These time-dependent changes might reflect a rearrangement of the SAM structure or a reorientation of the immobilized cyt-c. According to the present data there is thus no evidence for a dependence of the formal reduction potential on the surface morphology after sufficiently long equilibration times. The formal ET rate constant, ks, however, depends on the nature of the metal, most likely due to the different SAM thickness and possibly also due to different interfacial electric fields. Accordingly, most of the previously published kinetic results can be understood. Discrepancies between CV and SERR experiments might result from the fact electrochemical techniques probe all the electroactive adsorbed cyt-c molecules, which is not necessarily the case for surface-enhanced vibrational spectroscopies. Acknowledgment. We gratefully acknowledge Roald Boegschoten and Joost Buijs for technical support and Dr. Alois Bonifacio for helpful discussions. P.H. acknowledges support by the DFG (Cluster of Excellence “Unicat”). Supporting Information Available: Presentation of the electrochemical treatment of gold electrodes; Gaussian distribution of surface feature dimensions on roughened silver electrodes; linear plot of peak current vs scan rate for AgS/MUA/ cyt-c; SERR spectra to probe the heme reorientation; trumpet plots; determination of the surface area of roughened silver electrodes. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Katz, E.; Willner, E. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (2) Katz, E.; Willner, E. Angew. Chem., Int. Ed. 2000, 39, 1180–1218. (3) ComprehensiVe treatise of electrochemistry; Srinivasan, S.; Chiznadzhiev, Y. A.; Bockris, J. O. M.; Conway B. E. Yeager E., Eds.; Plenum Press: New York, 1985; Chapter 5. (4) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247–1250. (5) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564–6572. (6) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559–565. (7) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367–1370. (8) Arnold, S.; Feng, Z. Q.; Kakiuchi, T.; Knoll, W.; Niki, K J. Electroanal. Chem. 1997, 438, 91–97. (9) El Kasmi, A.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225–226. (10) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623– 2645. (11) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759–2766. (12) Chi, Q. J.; Zhang, J. D.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669–4679. (13) Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2001, 105, 1578– 1586. (14) Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062– 4068. (15) Chen, X.; Ferrigno, R.; Yang, J.; Whitesides, G. Langmuir 2002, 18, 7009–7015. (16) Niki, K.; Sprinkle, J. R.; Margoliash, E. Bioelectrochemistry 2002, 55, 37–40. (17) Leger, C.; Elliott, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones, A. K.; Armstrong, F. A. Biochemistry 2003, 42, 8653–8662. (18) Khoshtariya, D. E.; Wei, J. J.; Liu, H. Y.; Yue, H. J.; Waldeck, D. H. J. Am. Chem. Soc. 2003, 125, 7704–7714. (19) Ulstrup, J.; Zhang, J. D.; Hansen, A. G.; Wackerbarth, H.; Christensen, H. E. M. J. Inorg. Biochem. 2003, 96, 28. (20) Wei, J. J.; Liu, H. Y.; Niki, K.; Margoliash, E.; Waldeck, D. H. J. Phys. Chem. B 2004, 108, 16912–16917. (21) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 9445–9457.

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