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Langmuir 1998, 14, 7298-7305
Electron Transfer between Surface-Confined Cytochrome c and an N-Acetylcysteine-Modified Gold Electrode Tautgirdas Ruzgas, Lance Wong, Adolfas K. Gaigalas,* and Vincent L. Vilker Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001 Received July 9, 1998. In Final Form: August 27, 1998 Cytochrome c (cyt c) was adsorbed on N-acetylcysteine (NAC)-modified gold electrodes via electrostatic interaction. The cyt c layer exhibited reversible and stable electrochemical redox transformation in 0.01 M phosphate butter, pH 7.4, where the heterogeneous electron transfer (ET) constant k′het was measured by three techniques: cyclic voltammetry at high sweep rates (CV), electrochemical impedance (EI), and electroreflectance (ER) spectroscopy. In addition, k′het was also determined from combining sets of simultaneous electrochemical impedance (EI) and electroreflectance (ER) measurements in a new impedance model in which a constant-phase element was used. The negligible shift (-0.023 mV) in the formal potential from the solution value, and the close agreement of the measured distribution around the CV peaks (full-width voltage at half-peak-height, Efwhh ) 97 mV) with the theoretical value of 90.6 mV, suggested that the immobilized cyt c is retained at the electrode in the native state. Apparent k′het values, as determined by each method separately, were as follows: (k′CV ) 920 ( 280 s-1 by CV, k′EI ) 660 ( 200 s-1 by EI, and k′ER ) 2100 ( 300 s-1 by ER as interpreted using previously published methods.14,15,30 In the combined EI/ER measurements, k′ER was found to increase linearly with the frequency of the ac modulating current and spanned a range from about 400 to 800 s-1, which is close to the interprotein electron-transfer rate constant (800 s-1) measured between cyt c and one of its natural redox partners, cytochrome c peroxidase.31 It is concluded that further attempts to reconcile these discrepancies in k′het determinations will require more detailed descriptions of the interfacial elements in the impedance models.
Introduction Reduction and oxidation of biological macromolecules at electrodes usually requires modification of the electrode surface with compounds that attract the macromolecule to the surface and facilitate electron transfer (ET) between them.1-3 These interfaces are complex, and various electrochemical and optical techniques such as cyclic voltammetry (CV), electrochemical impedance (EI), surface enhanced Raman spectroscopy (SERS), and electroreflectance (ER) spectroscopy have been used to characterize them. In most previous studies one or another of the above techniques was used to extract the ET rate constant and no attempt was made toward a consistent interpretation of measurements obtained by several techniques (e.g. electrochemical and ER measurements). While CV (or EI) measures the current response when the protein’s oxidation state changes, ER measures the change in concentration of oxidized (or reduced) protein that was “in the neighborhood” of the electrode surface. Hence the two techniques are independent measurements of the same process, and a combined analysis would be a more stringent test of a model used in describing the ET process. In a previous study of electron transfer between a surface-modified silver electrode and the P450 redox protein putidaredoxin (Pdx), we found that the heterogeneous electron-transfer rate constant determined by ER and CV would be consistent only if the Pdx was in solution. Analysis with a model of immobilized Pdx gave rates which differed by almost 2 orders of magnitude.4 * Corresponding author. Address: 222/A353 NIST, Gaithersburg, MD 20899-0001. Phone: (301) 975 2873. Fax: (301) 975-5449. E-mail:
[email protected]. (1) Armstrong, F. A. Struct. Bond. 1990, 72, 137. (2) Hill, H. A. O.; Hunt, N. I. In Methods in Enzymology: Metallobiochemistry; Riordan, J. F., Vallee, B. L., Eds.; Academic Press: New York, 1993; p 501. (3) Bond, A. M. Inorg. Chim. Acta 1994, 226, 293.
10.1021/la9808519
Since Pdx gave a poor signal-to-noise current response during CV measurements, it was difficult to determine the degree of reversibility, the extent of diffusion control, and the magnitude of the heterogeneous electron-transfer rate constant. In the present study, CV, EI, and ER measurements were undertaken using cytochrome c (cyt c) and an electrode modification procedure which will be shown to confine the protein to the electrode surface for a time that is long relative to the time scale of electron transfer. We used high-sweep-rate CV, EI, and ER methods on the same electrodes in order to construct a self-consistent interpretation of all the hetero-ET rate measurements. The combined analysis leads to a frequency dependent rate constant which suggests that the model needs further modification. The redox behavior of cyt c at gold, silver, platinum, carbon, and metal oxide electrodes has been studied extensively, in part, because the oxidation/reduction of the heme redox center, which is partially exposed to the solution/electrode interface, gives a strong signal in electrochemical and spectroscopic measurements. The electron transfer can be reversible or irreversible, depending on various treatments and modifications. Shifts in the measured formal potential for a surface-confined cyt c species, relative to the solution formal potential E°′ ) +0.258 V (vs NHE) measured by titration techniques,5 also vary with the electrode material and the nature of the modifier. Bare metal electrodes,6-8 or metal electrodes coated with small bipyridyl molecules,6,7,9 give downshifts (4) Gaigalas, A. K.; Reipa, V.; Vilker, V. L. J. Colloid Interface Sci. 1997, 186, 339. (5) Hawkridge, F. M.; Kuwana, T. Anal. Chem. 1973, 45, 1021. (6) Szucs, A.; Hitchens, G. D.; Bockris, J. O’M. Electrochim. Acta 1992, 37, 403. (7) Hinnen, C.; Niki, K. J. Electroanal. Chem. 1989, 264, 157. (8) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710. (9) Hobara, D.; Niki, K.; Zhou, C.; Chumanov, G.; Cotton, T. M. Colloids Surf. 1994, 93, 241.
This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 11/19/1998
Electron Transfer from Surface-Confined Cytochrome c
of up to -0.5 V from the solution value. Raman spectroscopy has been used to show that these shifts may be associated with conformational changes away from the native state.8-10 Metal oxide electrodes11,12 and metal electrodes coated with long chain aliphatic hydrocarbons13-17 tend to show small or negligible shifts of the formal potential. Heterogeneous electron-transfer rates for surfaceconfined cyt c species have been measured by CV, EI, or ER methods13-17 using gold electrodes that have been modified by coating with alkanethiols, HS(CH2)nCOOH with n ) 2-16. For n > 10, all measurement methods give approximate agreement for the value of the hetero-ET rate constant (k′het ≈ 0.1-1.0 s-1 for n ) 15 and k′het ≈ 10-100 s-1 for n ) 11; k′het is any measured electrontransfer rate coeffcient with the oxidation/reduction reaction treated as an electrode surface reaction (s-1)). Also, there is agreement that k′het decreases as n increases. However, for n < 10, slow-scanning CV techniques are reported not to give reliable measures of the hetero-ET rate constant.17 In these cases, ER methods have been used to determine values of k′het up to 1300 s-1, although analysis of ER and EI measurements to isolate and identify k′het is dependent on the model used for the electrode/ modifier/protein/solution interface.18 Cooper et al. used CV techniques to measure ET on a gold electrode modified with the nonaliphatic compound N-acetyl-L-cysteine
to which the protein had been fixed using the water soluble condensing agent 1-ethyl-3-(3,3-dimethylaminopropyl)carbodiimide (EDC).19 As in the case of alkanethiol modification, the value of E°′ was only slightly affected (downshifted) when cyt c was surface confined by this procedure. However, the value of k′CV ) 3.4 s-1 for surfaceconfined cyt c was almost 3 orders of magnitude lower than the value determined by ER for short chain alkanethiol-modified electrodes.17 Differences between these two reports include method of cyt c surface confinement and the method of measuring electron transfer, as described above. In the present study, we chose to modify the gold electrodes with N-acetyl-L-cysteine (NAC) in order to achieve ET between the heme center and the electrode that is not dominated by the exponential decay seen with the alkanethiol modifiers, while avoiding the complications that are introduced with carbodiimide cross-linking.20 We (10) Niaura, G.; Gaigalas, A. K.; Vilker, V. L. J. Electroanal. Chem. 1996, 416, 167. (11) Willit, J. L.; Bowden, E. F. J. Phys. Chem. 1990, 94, 8241. (12) Daido, T.; Akaike, T. J. Electroanal. Chem. 1993, 344, 91. (13) Song, S. A.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564. (14) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electroanal. Chem. 1995, 394, 149. (15) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electroanal. Chem. 1996, 408, 15. (16) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559. (17) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367. (18) Gaigalas, A. K.; Niaura, G. J. Colloid Interface Sci. 1997, 193, 60. (19) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. J. Electroanal. Chem. 1993, 347, 267. (20) Peerey, L. M.; Kostic, N. M. Biochemistry 1989, 28, 1861.
Langmuir, Vol. 14, No. 25, 1998 7299
Figure 1. Electrochemical cell used for cyclic voltammetry, electrochemical impedance, and electroreflectance spectroscopy measurements. The gold working electrode is mounted in a Teflon sleeve that gives an exposed area of about 0.32 cm2 (6.35 mm diameter); the platinum sheet counter electrode area is about 30 cm2; the overall height of the working chamber is 4.5 cm; working and reference electrodes are fixed in place by tight press-fit; in electroreflectance measurements, illumination is done with a 100-W xenon-arc light source directed at 15° from the normal to the working electrode. The remaining parts of the ER apparatus are described in ref 4. All potentials measured with this device are reported relative to the saturated Ag/AgCl reference electrode potential.
used high-sweep-rate CV, along with EI and ER methods on the same electrodes, in order to construct self-consistent interpretations of the hetero-ET rates. Experimental Section Materials.21
Horse heart cytochrome c (Sigma, type VI) was purified using CM-32 Sephadex cation-exchange chromatography. The most intensively red fraction was collected and dialyzed against 10 mM sodium phosphate buffer (pH 7.4). Aliquots of 2.4 mM cyt c were placed into plastic vials and stored at -20 °C until use. N-Acetyl-L-cysteine (NAC; CAS # 616-91-1) was obtained from Sigma (Product #A8199) and used without further purification. Gold electrodes were made from polycrystalline 99.99% purity gold rod purchased from Alpha (Ward Hill, MA). Potassium dicyanoaurate (I) from Aldrich (Milwaukee, WI) was 98% pure. Water for all experiments was purified on a Milli-Q system (Millipore). Instrumentation. All electrochemical and spectroelectrochemical measurements were performed using the quartz halfcylindrical cell shown in Figure 1. The three electrodes mounted into the cell were a saturated Ag/AgCl reference electrode (Abtech, Yardley, PA), a platinum sheet counter electrode with total area of more than 30 cm2, and a gold rod working electrode fitted into a Teflon holder with a geometric area of 0.32 cm2. Working electrode potentials for linear ramp, direct, and alternating current were controlled by an EG&G potentiostat (21) Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment is necessarily the best available for the purpose.
7300 Langmuir, Vol. 14, No. 25, 1998 model 263A (Princeton, NJ) during cyclic voltammetry, electrochemical impedance, and electroreflectance measurements. All potentials mentioned in the paper are relative to the saturated Ag/AgCl reference electrode potential. The setup for electroreflectance measurements has been previously described.4 In some experiments, electroreflectance and electrochemical impedance measurements were performed simultaneously and an additional EG&G lock-in amplifier model 5301 was connected to the current output on the potentiostat. The ac reference potential was the same for electroreflectance and electrochemical impedance data. For the EI and ER measurements, signal filtering and IR compensation were turned off on the potentiostat. Electrode Preparation. Gold electrode surfaces were polished initially with 5-mm alumina powder and finished with 0.05 mm to produce mirror-like surfaces. The electrodes were ultrasonicated in water for 5 min to remove bound alumina and then electrochemically cleaned by potential cycling between 1.7 and -0.35 V at 0.7 V/s (30 cycles in total) in 1 M sulfuric acid. Following another water wash, a fresh gold layer was galvanostatically deposited by immersion of an electrode in a buffer solution of 0.2 g/mL potassium dicyanoaurate (I) in 0.2 M phosphate buffer, pH 7.4, and application of an about -0.9 V reduction potential. The electrodeposition current density was maintained constant at 0.63 mA/cm-2 for 4 min. Next, the electrode was washed with water and electrochemically cleaned by potential cycling at the same conditions described prior to electrodeposition of the fresh gold layer. After the freshly prepared electrode was washed with water, it was modified with thiol by immersion for 30 min into a solution of about 5 mM NAC in 0.2 M phosphate buffer, pH 7. After extensive water washing, a 30 mL aliquot of 0.5 mM cytochrome c solution in 0.01 M phosphate buffer was placed on the gold surface for 15 min at +4 °C. Another water wash was followed with placement of the electrode into the electrochemical cell shown in Figure 1. Measurement Procedures. All electrochemical and electroreflectance measurements were performed in 0.01 M phosphate buffer (pH 7.4, ca. 20 °C) which was deoxygenated by continuous argon bubbling before and during measurements. Prior to determining heterogeneous electron-transfer rate parameters by cyclic voltammetry, the solution resistance was evaluated by using the measurement procedure available with the EG&G potentiostat in IR compensation mode. This solution resistance measurement was repeated 7-10 times, resulting in mean values ranging from 260 to 680 W, depending on the distance between reference and working electrodes. For a single electrode, these values were within 5% of the solution resistance that was seen at the high-frequency limit in electrochemical impedance measurements (as seen in Figure 3b). These measurements were used for IR compensation in CV measurements (but not in the EI and ER measurements) and for evaluating the phase correction in ER measurements. All default signal-filtering procedures that are usually in place during CV experiments were turned off in order to eliminate the effect of those filters on peak separation in the cyclic voltammograms of cyt c-modified electrodes. For each specific electrode, three successive cyclic voltammograms were recorded by cycling the potential between 0.25 and -0.25 V, starting at the positive potential. The last (third) cyclic voltammogram was used for calculating the electrontransfer parameters. For each electrode, four to seven different sweep rates ranging from 7 to 20 V/s were used to establish peak separation (Ea - Ec) (Ea and Ec are the CV anodic and cathodic peak voltages, respectively). The apparent heterogeneous electrontransfer rate constant k′CV was determined from peak separation measurements using Laviron’s diffusionless model.22 Only data where (Ea - Ec) g 10 mV were used to interpret the rate constant. To reduce the uncertainty associated with small peak separation and the noise of unfiltered signals, peak potentials were determined by fitting portions of the recorded cyclic voltammograms to the sum of a Gaussian function and a linear function that accounted for background charging current and its dependence on dc potential. The amount of electrochemically active, surface-confined cyt c was calculated by integrating the charge passed during cyt c (22) Laviron, E. J. Electroanal. Chem. 1979, 101, 19.
Ruzgas et al.
Figure 2. Cyclic voltammograms for the Au/NAC/cyt c electrode in 0.01 M phosphate buffer, pH 7.4. Potential sweep rates of 7, 8.5, 10, and 11.5 V/s were recorded with instrument IR compensation enabled and signal filtration procedures disabled. Solution resistance was estimated to be equal to 293 ( 4 W. Values for the anodic Ea and cathodic Ec peak potentials, and their respective currents ia and ic, were determined by nonlinear least-squares fitting (SigmaPlot V. 4.0, Jandel Scientific, San Rafael, CA) of portions of the recorded cyclic voltammograms to a sum of a linear function and a Gaussian function (a + bECV(t) + Gaussian), where ECV(t) is the time dependent dc voltage imposed on the electrode during cyclic voltammetry (V) and the constants a and b account for the background charging current and its dependence on dc potential, respectively. The insert shows the linear dependence of the peak currents on the potential sweep rate. redox conversion: cyt c3+ + e- S cyt c2+. This calculation corresponds to measuring the area under the CV peaks (Gaussian function) after subtraction of background current (linear function). Following CV determinations, electrochemical impedance (EI), electroreflectance (ER), or combined EI/ER measurements were carried out on the same electrode/electrochemical cell setup with the applied electrode voltage Eappl ) Edc + ∆Eac sin(ωt), where Edc is a dc potential, ∆EAC is the amplitude of the modulation voltage (0.02 V rms, 0.0566 V peak-to-peak), and ω is the radial frequency of the modulation voltage. Edc was stepped in 0.01 V increments from -0.2 to 0.2, and at each increment, the electrode current (impedance data) and the output from the photodiode (electroreflectance data) were simultaneously measured by two lock-in amplifiers and stored electronically. The ac component ∆Eac sin(ωt) was used as a reference signal for both lock-in amplifiers. The current response iresp ()∆Iac cos(ωt + φ)) consisted of in-phase and out-of-phase components, Iiφ and Ioφ, respectively. The electrochemical impedance was calculated using Z ) ∆Eac/ (Iiφ + jIoφ) and the measured quantities ∆Eac, Iiφ, and Ioφ. The in-phase and out-of-phase components of the photodiode signal were normalized by the dc component of the reflected light to give in-phase and out-of-phase electroreflectance responses, (∆R/ R)iφ and (∆R/R)oφ, respectively. Simultaneous current and electroreflectance measurements were repeated at several modulation frequencies (ω/2π) over the range 20-150 Hz. The ER measurements were made with 550 nm irradiation using a 100 W xenon arc lamp and a monochromator. The angle of light incidence measured relative to the normal of the electrode surface was about 15° or less. Reflected light from the electrode was measured with a photodiode, and the output signal was separated into ac and dc components with appropriate electronic filters. The minimal detection limit was ∆R/R e 10-6.
Results Cyclic Voltammetry Measurements. Typical cyclic voltammograms of cyt c adsorbed on gold modified with N-acetyl-L-cysteine are shown in Figure 2. Measurements were performed in deoxygenated 0.01 M phosphate buffer, pH 7.4, at high sweep rates from 1 to 11.5 V/s. Signal filtration procedures were disabled, since it was noticed
Electron Transfer from Surface-Confined Cytochrome c
Figure 3. Impedance measurements for the Au/NAC/cyt c electrode in 0.01 M phosphate buffer, pH 7.4. (A) In-phase Iiφ and out-of-phase Ioφ components of the alternating current response (iresp) at different dc potentials (Edc) which were obtained for potential modulation frequency (ω/2π) ) 21 Hz and amplitude ∆Eac ) 20 mV rms. The arrows indicate values of current (Iiφ and Ioφ) used to calculate the in-phase and outof-phase components of the impedance for the total cell {ZT,iφ, ZT,oφ at Edc ) E°′} and the values I′iφ and I′oφ for impedance in the absence of cyt c faradaic processes {Z′T,iφ, Z′T,oφ}. (B) Inphase and out-of-phase components of electrochemical impedance at 0.013 V (versus Ag/AgClsat) and different modulation frequencies: {(3) ZT,oφ, (1) ZT,iφ} and {(O) Z′T,oφ, (b) Z′T,iφ}. The curves in part B are simulations using the equivalent circuit presented in the inset and circuit element values given in line 1 of Table 1.
that the EG&G potentiostat processing filters (even at the highest cutoff frequency of 590 Hz) caused an increase of the peak separation in voltammograms. Therefore, high noise levels are seen in the voltammograms. Voltammograms were persistent; no more than a 10% decrease in the redox peaks was found during 3-4 h of CV, EI, and ER measurements. The electrode was kept at open circuit potential when between measurements. No systematic study was made of stability during continuous cycling or maintenance at a constant potential. Redox peaks decreased to about 30% of the initial value when the molarity of the phosphate buffer was raised to 0.05 M. The peaks completely vanished when the phosphate, sodium perchlorate, or sodium chloride concentration was raised to 0.2 M. At this high ionic strength, it was not possible to observe redox signals even with much more sensitive techniques such as square wave or electroreflectance voltammetry. The sensitivity of redox peak attenuation to increases in ionic strength is taken as an indication that cyt c is confined to the electrode surface through electrostatic immobilization mediated by the NAC-modifying layer.
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Further characteristics of the cyt c layer include the small oxidation peak-to-reduction peak separation (Ea - Ec), the linear dependence of peak currents on sweep rate (see Figure 2 inset), the narrowness of the potential distribution about Ea and Ec, and the closeness of the formal potential of the surface confined cyt c to its value in solution. Peak separations approached zero at the sweep rates ( 0.9. An alternative procedure for performing phase correction is the interface impedance model14,15,30 generalized so that any electrode interface impedance element can be connected in series with the solution resistance. In the context of this impedance model, the phase difference is due to the uncompensated solution resistance, which is significant when working with low-molarity buffer solutions (0.01 M in our case). Then Efar ) Eappl - (irespRsol) is representative of the interfacial electron-transfer process. The electrode current (iresp) was recorded simultaneously with ER measurements, and Rsol was determined (away from E°′) independently from these current measurements. Since the ET process is in series with the solution resistance Rsol, it was possible to use the vectorial diagram in Figure 5 to calculate the phase difference (ψ) between the applied potential Eappl and Efar. The uncorrected and corrected in-phase and out-of-phase ER signal components are presented in Figure 5 by circles and solid curves, respectively. An alternate phase correction procedure18 using background ER response (ER for dc potentials far from E°′) as the reference leads to similar results. The ET rate constants k′ER evaluated by this method for five differently prepared electrodes show dependence on potential modulation frequency, as indicated in Figure 6. For one of these electrodes, k′ER ranged from 405 s-1 at the lowest frequency (21 Hz) to 742 s-1 at 110 Hz.
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Table 2. Summary of Heterogeneous Electron-Transfer Rate Constants Measured by Cyclic Voltammetry (k′CV), Electrochemical Impedance (k′EI), and Electroreflectance Voltammetry (k′ER) for Cytochrome c Electrostatically Immobilized on Gold Electrodes Modified with N-Acetyl-L-cysteinea electroreflectance spectroscopy
electrode
cyclic voltammetryb k′CV, s-1
electrochemical impedancec k′EI, s-1
1 2 3
710 ( 140 750 ( 120 720 ( 160
860 ( 200 690 ( 220 960 ( 200
combined ER/EI + phase Figure 3 @ 21 Hz Figure 3 @ 110 Hz k′ER, s-1 k′ER, s-1 370 410 310
700 740 780
(-ω cot φ versus -ω2) per refs 15 and 30 k′ER, s-1 1860 ( 480 2170 ( 230 2360 ( 350
a Measurements were made in 0.01 M phosphate buffer, pH 7.4, with all cyt c electrostatically immobilized on the Au/NAC electrode surface. b CV measurements were made at sweep rates of 7-20 V/s with instrument signal filtering disabled; k′CV is calculated from data where peak separation (Ea - Ec) g 10 mV by the Laviron method (see ref 22, Table 1). c EI measurements.
Discussion The three measurement techniques gave relatively consistent values for the heterogeneous ET rate constant k′het. Table 2 summarizes a series of measurements performed on each of three different electrodes that were prepared by identical procedures using common stock reagent solutions. Values of k′CV, k′EI, and k′ER that were analyzed independent of one another are presented with estimates of standard errors in columns 2, 3, and 6, respectively, while the values obtained by combined ER and EI measurements are given only at the specific frequencies shown in columns 4 and 5. The combined EI/ ER measurements show that the extracted values of k′ER depend on frequency. The divergent values of k′ER shown in columns 4-6 are indicative of the sensitivity to the choice of impedance model for data analysis. In the following, we briefly discuss the nature of the interaction of cyt c that is confined at the Au/NAC/protein interface and then discuss features that were unique to our three measurement techniques. Special emphasis is placed on the implications of the frequency dependence of the combined EI/ER measurement of k′ER. Cytochrome c Interactions with NAC-Modified Gold Electrodes. The hydrophobic-electrostatic interaction between cyt c and electrodes modified with long chain self-assembled monolayers has previously been shown to lead to electrochemical responses that suggest the protein is surface confined and in its native solution state.9,31,32 The electrochemical responses we obtained with cyt c on NAC-modified electrodes are similar, although the mechanism of immobilization is mainly electrostatic with a minor contribution from hydrophobic interaction. Electrostatic attraction between cyt c and the NACmodified electrodes is suggested by the stability of the electrochemical responses in low-ionic-strength buffers. The disappearance of the cyt c redox signal in 0.2 M phosphate buffer suggests desorption of the protein from the electrode. Redox electrochemistry could be recovered on the NAC-modified electrodes that had been exposed to high ionic strength if they were again exposed to cyt c in a low-molarity buffer. Retention of its native state, when cyt c is electrostatically immobilized on the NAC-modified electrodes, is suggested by the negligible shift in the formal potential, E°′, and by the close agreement of the measured potential distribution around the CV peaks (full-width voltage at half-peak-height, Efwhh) with the theoretical value of 90.6 mV from Laviron’s model.22 Our measured value of the formal potential, E°′ ) +0.235 ( 0.005 V (versus NHE), is only slightly downshifted from the solution formal potential +0.258 V (versus NHE) measured by titration techniques.5 Metal oxide electrodes11,12 (31) Hobara, D.; Niki, K.; Cotton, T. M. Denki Kagaku 1993, 61, 776. (32) Maeda, Y.; Yamamoto, H.; Kitano, H. J. Phys. Chem. 1995, 99, 4837.
and metal electrodes coated with long chain aliphatic hydrocarbons13-17 show similar small or negligible shifts of the formal potential. Dispersion about the formal potential beyond the theoretical limit of 90.6 mV is attributed to heterogeneity in the population of immobilized cyt c molecules due to a distribution of (i) interfacial environments, (ii) adsorption energies, and/or (iii) charge-transfer-induced alterations of formal potential.16 Compared with relatively larger values measured on SAM-modified electrodes (Efwhh ) 104 mV for Au/ mercaptopropionic acid/cyt c,14 110-120 and 125-152 mV for Au/mercaptohexanoic acid/cyt c,13,16 101-104 and 115-135 mV for Au/mercaptoundecanoic acid/cyt c,14,16 and 140-170 mV for Au/mercaptohexadecanoic acid/ cyt c16), our measured Efwhh ) 97 mV suggests that the Au/NAC/cyt c electrode surface is a molecularly homogeneous population with the protein close to its native conformation. Heterogeneous Electron-Transfer Rate Constants: k′CV, k′EI, and k′ER. The heterogeneous ET rate constant for cyt c at the Au/NAC electrode ranged from 300 s-1 to over 2000 s-1, inclusive of all methods of measurement and analysis. As far as we know, this is the first attempt to compare k′het values obtained with the three different measurement techniques with the interpretation based on the same kinetic and impedance models. In the following, we review our approach using each method and compare it to previously reported results. The electron-transfer rate that we measured by cyclic voltammetry, k′CV ≈ 900 s-1, is one of the highest reported for heterogeneous protein ET measured by this technique. Our use of high sweep rates was the major difference from other CV applications where much slower ET rates were measured. These higher sweep rates require IR compensation, a feature which is available with any modern potentiostat. The drawback to IR compensation is that the solution resistance has to be estimated with high precision. For sweep rates g 10 V/s, an uncertainty of 3-5% in the solution resistance leads to substantial uncertainty in the peak separation, from which k′CV is determined by the method of Laviron.22 Analog signal filtering cannot be used during these measurements because the filter introduces a large systematic error in the resulting value of the rate constant. In fact, the extracted rate constant can reflect the characteristics of the electronic filters and not the actual faradaic process. In lieu of the analog filters, digital filtering can be performed after data collection. The range for ET rate constants measured by electrochemical impedance spectroscopy (k′EI ) 400-1000 s-1) over many different Au/NAC/cyt c electrode preparations includes the range of our k′CV results and represents much higher values than we find reported in the literature by this technique. Quantifying the frequency dependence of
Electron Transfer from Surface-Confined Cytochrome c
the current response is the usual approach for extracting k′EI from values of the circuit elements comprising the total impedance. Differences in the circuit elements that we used from those used by others13-15 to measure much lower rates of protein heterogeneous ET were described above. The most important of these is our use of a constant phase element to account for a distributed double-layer capacitance. Column 6 of Table 2 gives the ET rate constant determined by our electroreflectance measurements when they are analyzed from the frequency dependence of the ER response15,30 (slope -ω cot φ versus -ω2). These values (k′ER ) 1800-2400 s-1), which are 2-3 times higher than those found for the same electrodes by the CV or EI method, or by the combined EI/ER method (columns 4 and 5, Table 2), are close to previous reports for cyt c immobilized on Au electrodes coated with short chain (n ) 2) alkanethiols.17 The lower values are in the range of the interprotein electron-transfer rate constant (800 s-1) measured between cyt c and one of its natural redox partners, cytochrome c peroxidase.33 We are not the first to observe the discrepancy in values of k′het determined by the three methods. Literature reports generally show that electroreflectance measurements give higher ET rate constants compared with those measured by cyclic voltammetry or electrochemical impedance spectroscopy. In one of the earliest applications of ER to heterogeneous electron transfer, the rate coefficients for Nile Blue A adsorbed on pyrolytic graphite29 were determined to be k′CV ) 5 s-1 and k′ER ) 63-78 s-1. Measurements of heterogeneous ET rate constants for cyt c immobilized on gold electrodes coated with long chain alkanethiols HS(CH2)nCOOH have been given: k′EI ) 23 s-1 and k′ER ) 186 s-1 for n ) 11,32 and k′CV ) 20-30 s-1 and k′ER ) 320 s-1 for n ) 9.17 Feng et al.17 were careful to point out that CV measurements (under their conditions) cannot track rapid ET rates, and the value of approximately 20-30 s-1 is our interpretation of their reported CV data. In our lab, we have previously reported a similar large discrepancy in ET rate constants (k′CV ) 4-12 s-1 and k′ER ) 50-200 s-1) for azurin immobilized on 1-hexanethiol-modified gold electrodes.18 Because the ET rate constant extracted from the frequency dependence of the ER response (-ω cot φ versus -ω2 slope) is extremely sensitive to the selection of the impedance model that describes the interfacial effects on the current response, discrepancies between the heterogeneous ET rate constants evaluated from electrochemical and ER measurements can be anticipated. This is especially true if the interfacial elements show distributed (33) Pappa, H. S.; Tajbaksh, S.; Saunders, A. J.; Pielak, G. J.; Poulos, T. L. Biochemistry 1996, 35, 4837.
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character (e.g. distributed character of double-layer capacitance). In previous reports of ER response analysis, it was assumed that all impedance elements are ideal resistors and capacitors.14 However, the double-layer capacitance should not be represented as an ideal impedance element,24 especially in low-molarity buffer solutions and at relatively low frequencies (f < 300 Hz). Dependence of k′ER on Modulation Frequency. In Table 2 columns 4 and 5, results for k′ER are reported for our measurements of an ER response that takes advantage of the high signal-to-noise ratio of our instrument and for a new analysis model that allows direct estimation of a reference phase for the faradaic potential driving the cyt c ET. The circuit element parameter values were subject to the dual constraints of matching simultaneous impedance and electroreflectance signal responses. The phase correction technique is not sensitive to the details of the impedance model but assumes that the ET process is in series with the solution resistance. The combined analysis of the EI and ER data allows calculation of the ET rate constant assuming that the ET process is in series with the solution resistance; all other electrochemical processes which are parallel to the ET process do not need to be specified. The ET rate coefficient k′ER is given in Table 2 columns 4 and 5 for the three separately prepared electrodes measured at modulation frequencies of 21 and 110 Hz, respectively. These results are in closer agreement with values determined by CV and EI but show a systematic increase with increasing frequency (Figure 6). This frequency dependence indicates that the simple impedance model, in which the elements describing ET of cyt c are connected between Rsol and the electrode, is not an adequate model. A more accurate impedance model, based on recent improvements in understanding the behavior of double-layer charging in low-ionic-strength buffers,35-38 is needed. Such an extended analysis of the combined EI and ER measurements is currently under investigation by our group. Acknowledgment. We are grateful to Vytas Reipa, Martin Mayhew, and Gediminas Niaura for their invaluable support and assistance in the execution of the research reported here. LA9808519 (34) Burris, S. C.; Bowden, E. F. Abstract ANYL-24, 212th National ACS Meeting, Orlando, FL, 1996. (35) Smith, C. P.; White, H. S. Anal. Chem. 1992, 64, 2398. (36) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1993, 97, 6233. (37) Fawcett, W. R. J. Electroanal. Chem. 1994, 378, 117. (38) Creager, S. E.; Rowe, G. K. J. Electroanal. Chem. 1997, 420, 291.
