Electron Transfer between Surface-Confined Cytochrome cand 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 electrostaticinteraction. The cyt c layer exhibited reversible and stable electrochemical redox transformation in 0.01M phosphate butter, pH 7.4, where the heterogeneous electron transfer (ET) constant k′het was measuredby three techniques: cyclic voltammetry at high sweep rates (CV), electrochemical impedance (EI), andelectroreflectance (ER) spectroscopy. In addition, k′het was also determined from combining sets ofsimultaneous electrochemical impedance (EI) and electroreflectance (ER) measurements in a new impedancemodel in which a constant-phase element was used. The negligible shift (-0.023 mV) in the formal potentialfrom 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, suggestedthat the immobilized cyt c is retained at the electrode in the native state. Apparentk′het values, as determinedby each method separately, were as follows: (k′CV ) 920 ( 280 s-1 by CV, k′EI ) 660 ( 200 s-1 by EI, andk′ER ) 2100 ( 300 s-1 by ER as interpreted using previously published methods.14,15,30 In the combinedEI/ER measurements, k′ER was found to increase linearly with the frequency of the ac modulating currentand spanned a range from about 400 to 800 s-1, which is close to the interprotein electron-transfer rateconstant (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 requiremore detailed descriptions of the interfacial elements in the impedance models.

IntroductionReduction and oxidation of biological macromolecules

at electrodes usually requires modification of the electrodesurface with compounds that attract the macromoleculeto the surface and facilitate electron transfer (ET) betweenthem.1-3 These interfaces are complex, and variouselectrochemical and optical techniques such as cyclicvoltammetry (CV), electrochemical impedance (EI), sur-face enhanced Raman spectroscopy (SERS), and elec-troreflectance (ER) spectroscopy have been used tocharacterize them. In most previous studies one or anotherof the above techniques was used to extract the ET rateconstant and no attempt was made toward a consistentinterpretation of measurements obtained by severaltechniques (e.g. electrochemical and ER measurements).While CV (or EI) measures the current response whenthe protein’s oxidation state changes, ER measures thechange in concentration of oxidized (or reduced) proteinthat was “in the neighborhood” of the electrode surface.Hence the two techniques are independent measurementsof the same process, and a combined analysis would be amore stringent test of a model used in describing the ETprocess. In a previous study of electron transfer betweena surface-modified silver electrode and the P450 redoxprotein putidaredoxin (Pdx), we found that the hetero-geneous electron-transfer rate constant determined byER and CV would be consistent only if the Pdx was insolution. Analysis with a model of immobilized Pdx gaverates which differed by almost 2 orders of magnitude.4

Since Pdx gave a poor signal-to-noise current responseduring CV measurements, it was difficult to determinethe degree of reversibility, the extent of diffusion control,and the magnitude of the heterogeneous electron-transferrate constant. In the present study, CV, EI, and ERmeasurements were undertaken using cytochrome c (cytc) and an electrode modification procedure which will beshown to confine the protein to the electrode surface fora time that is long relative to the time scale of electrontransfer. We used high-sweep-rate CV, EI, and ERmethods on the same electrodes in order to construct aself-consistent interpretation of all the hetero-ET ratemeasurements. The combined analysis leads to a fre-quency dependent rate constant which suggests that themodel needs further modification.

The redox behavior of cyt c at gold, silver, platinum,carbon, and metal oxide electrodes has been studiedextensively, in part, because the oxidation/reduction ofthe heme redox center, which is partially exposed to thesolution/electrode interface, gives a strong signal inelectrochemical and spectroscopic measurements. Theelectron transfer can be reversible or irreversible, de-pending on various treatments and modifications. Shiftsin the measured formal potential for a surface-confinedcyt c species, relative to the solution formal potential E°′) +0.258 V (vs NHE) measured by titration techniques,5also vary with the electrode material and the nature ofthe modifier. Bare metal electrodes,6-8 or metal electrodescoated with small bipyridyl molecules,6,7,9 give downshifts

* Correspondingauthor.Address: 222/A353NIST,Gaithersburg,MD 20899-0001. Phone: (301) 975 2873. Fax: (301) 975-5449.E-mail: adolfas.gaigalas@nist.gov.

(1) Armstrong, F. A. Struct. Bond. 1990, 72, 137.(2) Hill, H. A. O.; Hunt, N. I. In Methods in Enzymology: Metallo-

biochemistry; Riordan, J. F., Vallee, B. L., Eds.; Academic Press: NewYork, 1993; p 501.

(3) Bond, A. M. Inorg. Chim. Acta 1994, 226, 293.

(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.

7298 Langmuir 1998, 14, 7298-7305

10.1021/la9808519 This article not subject to U.S. Copyright. Published 1998 by the American Chemical SocietyPublished on Web 11/19/1998

of up to -0.5 V from the solution value. Raman spec-troscopy has been used to show that these shifts may beassociated with conformational changes away from thenative state.8-10 Metal oxide electrodes11,12 and metalelectrodes coated with long chain aliphatic hydrocar-bons13-17 tend to show small or negligible shifts of theformal potential.

Heterogeneous electron-transfer rates for surface-confined cyt c species have been measured by CV, EI, orER methods13-17 using gold electrodes that have beenmodified by coating with alkanethiols, HS(CH2)nCOOHwith n ) 2-16. For n > 10, all measurement methods giveapproximate agreement for the value of the hetero-ETrate 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 electron-transfer rate coeffcient with the oxidation/reductionreaction 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 arereported not to give reliable measures of the hetero-ETrate constant.17 In these cases, ER methods have beenused to determine values of k′het up to 1300 s-1, althoughanalysis of ER and EI measurements to isolate and identifyk′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 agold electrode modified with the nonaliphatic compoundN-acetyl-L-cysteine

to which the protein had been fixed using the water solublecondensing agent 1-ethyl-3-(3,3-dimethylaminopropyl)-carbodiimide (EDC).19 As in the case of alkanethiolmodification, the value of E°′ was only slightly affected(downshifted) when cyt c was surface confined by thisprocedure. However, the value of k′CV ) 3.4 s-1 for surface-confined cyt c was almost 3 orders of magnitude lowerthan the value determined by ER for short chain al-kanethiol-modified electrodes.17 Differences between thesetwo reports include method of cyt c surface confinementand the method of measuring electron transfer, asdescribed above.

In the present study, we chose to modify the goldelectrodes with N-acetyl-L-cysteine (NAC) in order toachieve ET between the heme center and the electrodethat is not dominated by the exponential decay seen withthealkanethiolmodifiers,whileavoiding thecomplicationsthat are introduced with carbodiimide cross-linking.20 We

used high-sweep-rate CV, along with EI and ER methodson the same electrodes, in order to construct self-consistentinterpretations of the hetero-ET rates.

Experimental SectionMaterials.21 Horse heart cytochrome c (Sigma, type VI) was

purified using CM-32 Sephadex cation-exchange chromatogra-phy. The most intensively red fraction was collected and dialyzedagainst 10 mM sodium phosphate buffer (pH 7.4). Aliquots of 2.4mM cyt c were placed into plastic vials and stored at -20 °C untiluse. N-Acetyl-L-cysteine (NAC; CAS # 616-91-1) was obtainedfrom Sigma (Product #A8199) and used without further purifica-tion. Gold electrodes were made from polycrystalline 99.99%purity gold rod purchased from Alpha (Ward Hill, MA). Potassiumdicyanoaurate (I) from Aldrich (Milwaukee, WI) was 98% pure.Water for all experiments was purified on a Milli-Q system(Millipore).

Instrumentation. All electrochemical and spectroelectro-chemical measurements were performed using the quartz half-cylindrical cell shown in Figure 1. The three electrodes mountedinto the cell were a saturated Ag/AgCl reference electrode (Abtech,Yardley, PA), a platinum sheet counter electrode with total areaof more than 30 cm2, and a gold rod working electrode fitted intoa Teflon holder with a geometric area of 0.32 cm2.

Working electrode potentials for linear ramp, direct, andalternating current were controlled by an EG&G potentiostat

(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.

(21) Certain commercial equipment, instruments, and materials areidentified in this paper to specify adequately the experimental procedure.In no case does such identification imply recommendation or endorse-ment by the National Institute of Standards and Technology, nor doesit imply that the material or equipment is necessarily the best availablefor the purpose.

Figure 1. Electrochemical cell used for cyclic voltammetry,electrochemical impedance, and electroreflectance spectroscopymeasurements. The gold working electrode is mounted in aTeflon sleeve that gives an exposed area of about 0.32 cm2 (6.35mm diameter); the platinum sheet counter electrode area isabout 30 cm2; the overall height of the working chamber is 4.5cm; working and reference electrodes are fixed in place by tightpress-fit; in electroreflectance measurements, illumination isdone with a 100-W xenon-arc light source directed at 15° fromthe normal to the working electrode. The remaining parts ofthe ER apparatus are described in ref 4. All potentials measuredwith this device are reported relative to the saturated Ag/AgClreference electrode potential.

Electron Transfer from Surface-Confined Cytochrome c Langmuir, Vol. 14, No. 25, 1998 7299

model 263A (Princeton, NJ) during cyclic voltammetry, electro-chemical impedance, and electroreflectance measurements. Allpotentials mentioned in the paper are relative to the saturatedAg/AgCl reference electrode potential. The setup for electrore-flectance measurements has been previously described.4 In someexperiments, electroreflectance and electrochemical impedancemeasurements were performed simultaneously and an additionalEG&G lock-in amplifier model 5301 was connected to the currentoutput on the potentiostat. The ac reference potential was thesame for electroreflectance and electrochemical impedance data.For the EI and ER measurements, signal filtering and IRcompensation were turned off on the potentiostat.

Electrode Preparation. Gold electrode surfaces were pol-ished initially with 5-mm alumina powder and finished with0.05 mm to produce mirror-like surfaces. The electrodes wereultrasonicated in water for 5 min to remove bound alumina andthen electrochemically cleaned by potential cycling between 1.7and -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 galvano-statically deposited by immersion of an electrode in a buffersolution of 0.2 g/mL potassium dicyanoaurate (I) in 0.2 Mphosphate buffer, pH 7.4, and application of an about -0.9 Vreduction potential. The electrodeposition current density wasmaintained constant at 0.63 mA/cm-2 for 4 min. Next, theelectrode was washed with water and electrochemically cleanedby potential cycling at the same conditions described prior toelectrodeposition of the fresh gold layer. After the freshly preparedelectrode was washed with water, it was modified with thiol byimmersion for 30 min into a solution of about 5 mM NAC in 0.2M phosphate buffer, pH 7. After extensive water washing, a 30mL aliquot of 0.5 mM cytochrome c solution in 0.01 M phosphatebuffer was placed on the gold surface for 15 min at +4 °C. Anotherwater wash was followed with placement of the electrode intothe electrochemical cell shown in Figure 1.

Measurement Procedures. All electrochemical and elec-troreflectance measurements were performed in 0.01 M phos-phate buffer (pH 7.4, ca. 20 °C) which was deoxygenated bycontinuous argon bubbling before and during measurements.

Prior to determining heterogeneous electron-transfer rateparameters by cyclic voltammetry, the solution resistance wasevaluated by using the measurement procedure available withthe EG&G potentiostat in IR compensation mode. This solutionresistance measurement was repeated 7-10 times, resulting inmean values ranging from 260 to 680 W, depending on thedistance between reference and working electrodes. For a singleelectrode, these values were within 5% of the solution resistancethat was seen at the high-frequency limit in electrochemicalimpedance measurements (as seen in Figure 3b). These mea-surements were used for IR compensation in CV measurements(but not in the EI and ER measurements) and for evaluating thephase correction in ER measurements. All default signal-filteringprocedures that are usually in place during CV experiments wereturned off in order to eliminate the effect of those filters on peakseparation in the cyclic voltammograms of cyt c-modifiedelectrodes.

For each specific electrode, three successive cyclic voltam-mograms were recorded by cycling the potential between 0.25and -0.25 V, starting at the positive potential. The last (third)cyclic voltammogram was used for calculating the electron-transfer parameters. For each electrode, four to seven differentsweep rates ranging from 7 to 20 V/s were used to establish peakseparation (Ea - Ec) (Ea and Ec are the CV anodic and cathodicpeak voltages, respectively). The apparent heterogeneous electron-transfer rate constant k′CV was determined from peak separationmeasurements using Laviron’s diffusionless model.22 Only datawhere (Ea - Ec) g 10 mV were used to interpret the rate constant.To reduce the uncertainty associated with small peak separationand the noise of unfiltered signals, peak potentials weredetermined by fitting portions of the recorded cyclic voltammo-grams to the sum of a Gaussian function and a linear functionthat accounted for background charging current and its depen-dence on dc potential.

The amount of electrochemically active, surface-confined cytc was calculated by integrating the charge passed during cyt c

redox conversion: cyt c3+ + e- S cyt c2+. This calculationcorresponds to measuring the area under the CV peaks (Gaussianfunction) after subtraction of background current (linear func-tion).

Following CV determinations, electrochemical impedance (EI),electroreflectance (ER), or combined EI/ER measurements werecarried out on the same electrode/electrochemical cell setup withthe applied electrode voltage Eappl ) Edc + ∆Eac sin(ωt), whereEdc is a dc potential, ∆EAC is the amplitude of the modulationvoltage (0.02 V rms, 0.0566 V peak-to-peak), and ω is the radialfrequency of the modulation voltage. Edc was stepped in 0.01 Vincrements from -0.2 to 0.2, and at each increment, the electrodecurrent (impedance data) and the output from the photodiode(electroreflectance data) were simultaneously measured by twolock-in amplifiers and stored electronically. The ac component∆Eac sin(ωt) was used as a reference signal for both lock-inamplifiers. The current response iresp ()∆Iac cos(ωt + φ)) consistedof 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. Thein-phase and out-of-phase components of the photodiode signalwere normalized by the dc component of the reflected light togive in-phase and out-of-phase electroreflectance responses, (∆R/R)iφ and (∆R/R)oφ, respectively. Simultaneous current and elec-troreflectance measurements were repeated at several modu-lation frequencies (ω/2π) over the range 20-150 Hz.

The ER measurements were made with 550 nm irradiationusing a 100 W xenon arc lamp and a monochromator. The angleof light incidence measured relative to the normal of the electrodesurface was about 15° or less. Reflected light from the electrodewas measured with a photodiode, and the output signal wasseparated into ac and dc components with appropriate electronicfilters. The minimal detection limit was ∆R/R e 10-6.

ResultsCyclic Voltammetry Measurements. Typical cyclic

voltammograms of cyt c adsorbed on gold modified withN-acetyl-L-cysteine are shown in Figure 2. Measurementswere performed in deoxygenated 0.01 M phosphate buffer,pH 7.4, at high sweep rates from 1 to 11.5 V/s. Signalfiltration procedures were disabled, since it was noticed(22) Laviron, E. J. Electroanal. Chem. 1979, 101, 19.

Figure 2. Cyclic voltammograms for the Au/NAC/cyt celectrode in 0.01 M phosphate buffer, pH 7.4. Potential sweeprates of 7, 8.5, 10, and 11.5 V/s were recorded with instrumentIR compensation enabled and signal filtration proceduresdisabled. 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 bynonlinear least-squares fitting (SigmaPlot V. 4.0, JandelScientific, San Rafael, CA) of portions of the recorded cyclicvoltammograms to a sum of a linear function and a Gaussianfunction (a + bECV(t) + Gaussian), where ECV(t) is the timedependent dc voltage imposed on the electrode during cyclicvoltammetry (V) and the constants a and b account for thebackgroundcharging current and its dependenceon dc potential,respectively. The insert shows the linear dependence of thepeak currents on the potential sweep rate.

7300 Langmuir, Vol. 14, No. 25, 1998 Ruzgas et al.

that the EG&G potentiostat processing filters (even atthe highest cutoff frequency of 590 Hz) caused an increaseof the peak separation in voltammograms. Therefore, highnoise levels are seen in the voltammograms. Voltammo-grams were persistent; no more than a 10% decrease inthe redox peaks was found during 3-4 h of CV, EI, andER measurements. The electrode was kept at open circuitpotential when between measurements. No systematicstudy was made of stability during continuous cycling ormaintenance at a constant potential. Redox peaks de-creased to about 30% of the initial value when the molarityof the phosphate buffer was raised to 0.05 M. The peakscompletely vanished when the phosphate, sodium per-chlorate, or sodium chloride concentration was raised to0.2 M. At this high ionic strength, it was not possible toobserve redox signals even with much more sensitivetechniques such as square wave or electroreflectancevoltammetry.

The sensitivity of redox peak attenuation to increasesin ionic strength is taken as an indication that cyt c isconfined to the electrode surface through electrostaticimmobilization mediated by the NAC-modifying layer.

Further characteristics of the cyt c layer include the smalloxidation peak-to-reduction peak separation (Ea - Ec),the linear dependence of peak currents on sweep rate (seeFigure 2 inset), the narrowness of the potential distribu-tion about Ea and Ec, and the closeness of the formalpotential of the surface confined cyt c to its value insolution. Peak separations approached zero at the sweeprates (<1 V/s) that are conventionally used in bioelec-trochemical studies. For the high sweep rates used here,peak separations were in the range 10-30 mV with thedispersion about the peak potentials having full-width-at-half-height (fwhh) values ranging from 82 to 113 mVand a mean value of 97 mV. The formal potential of thesurface-confined cyt c was calculated as the mean valueof the peak potentials and was found to be 13 ( 5 mVversus saturated Ag/AgCl for all experiments.

The amount of electrochemically active cyt c wasestimated from integration of the redox peaks and wasfound to differ for individual electrodes in the range from0.30 to 0.59 fractional monolayer coverage. The theoreticalmonolayer coverage was estimated using crystallographiccyt c dimensions (3.0 × 3.4 × 3.4 nm3),23 so that onemolecule with the long axis parallel to the electrode surfacewould occupy 3.4 × 3.4 ) 11.6 nm2, thereby leading to asurface concentration of about 14.3 pmol/cm2 at fullmonolayer coverage. For the data presented in Figure 2,electrode coverage is 0.45 ( 0.03 of a monolayer.

The heterogeneous ET rate constant of cyt c, k′CV ) 920( 280 s-1, was determined from measurements of peakseparations on nine separate, similarly prepared elec-trodes.

Electrochemical Impedance Measurements. Thedependences of the in-phase and out-of-phase currentresponses on the dc potential (Edc) are presented in Figure3a. Both Iiφ and Ioφ increase when Edc approaches E°′ forcyt c. At the potential where the response is a maximum(13 mV vertical line in Figure 3a), Iiφ and Ioφ are used tocalculate the 21 Hz impedance data shown in Figure 3b.The total cell impedance, as related by ZT ) ∆EAC/(Iiφ +jIoφ), was determined using these measurements of∆EAC, Iiφ, and Ioφ at Edc ) Eo′. The total cell impedance,in the absence of a faradaic response of cyt c, was calcu-lated from Z′T ) ∆EAC/(I′iφ + jI′oφ), where the values forI′iφ and I′oφ were determined by extrapolation, as shownin Figure 3a. The impedance values (ZT and Z′T) at otherfrequencies were similarly extracted from initial in-phaseand out-of-phase current measurements using modulatingpotential frequencies ranging from 21 to 111 Hz (pointsin Figure 3b).

The equivalent circuits shown in Figure 3b were usedto interpret the EI data. In these circuits, Rsol representsan uncompensated solution resistance, Rct and Ca are thecharge transfer (faradaic) resistance and the immobilized-layer pseudocapacitance, respectively, that describe theET properties of the surface-confined NAC/cyt c, and Qdlis a constant-phase element that describes the distributed-double-layer capacitance. The element Qdl is often usedinstead of an ideal double-layer capacitance when surfaceroughness and/or ion adsorption/desorption kinetics leadto the dependence of interfacial capacitance on potentialmodulation frequency.24

First, Rsol and Qdl were evaluated by fitting the frequencydependence of Z′T,iφ and Z′T,oφ data to the equivalent circuitZ′T (see Figure 3b) using the commercial programEQUIVALENT CIRCUIT version 3.96 (EG&G, Princeton,

(23) Dickerson, R. E.; Takano, T.; Eisenberg, D.; Kallai, O. B.; Samson,L.; Cooper, A.; Margoliash, E. J. Biol. Chem. 1971, 246, 1511.

(24) Pajkossy, T. J. Electroanal. Chem. 1994, 364, 111.

Figure 3. Impedance measurements for the Au/NAC/cyt celectrode in 0.01 M phosphate buffer, pH 7.4. (A) In-phase Iiφand out-of-phase Ioφ components of the alternating currentresponse (iresp) at different dc potentials (Edc) which wereobtained for potential modulation frequency (ω/2π) ) 21 Hzand amplitude ∆Eac ) 20 mV rms. The arrows indicate valuesof current (Iiφ and Ioφ) used to calculate the in-phase and out-of-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 inthe absence of cyt c faradaic processes Z′T,iφ, Z′T,oφ. (B) In-phase and out-of-phase components of electrochemical imped-ance at 0.013 V (versus Ag/AgClsat) and different modulationfrequencies: (3) ZT,oφ, (1) ZT,iφ and (O) Z′T,oφ, (b) Z′T,iφ. Thecurves in part B are simulations using the equivalent circuitpresented in the inset and circuit element values given in line1 of Table 1.

Electron Transfer from Surface-Confined Cytochrome c Langmuir, Vol. 14, No. 25, 1998 7301

NJ). Later, these Rsol and Qdl values were used to calculateRct and Ca by fitting the frequency dependence of the ZT,iφand ZT,oφ data to the equivalent circuit ZT (see Figure 3b).From Laviron,25 the parameters Rct and Ca are related tothe heterogeneous ET rate constant (k′EI) and the amountof adsorbed cyt c (Γ) through eqs 1 and 2, which applywhen the potential Eappl ) E°′:

where R is the universal gas constant, T is the absolutetemperature, n is 1 for the number of electrons transferredduring redox transformation of cyt c, F is Faraday’sconstant, and A is the geometric area of the electrode.

The values for equivalent circuit elements, ET rateconstants, and cyt c surface concentration Γ are sum-marized for six equivalently prepared electrodes in Table1. The average cyt c surface concentration for these sixelectrodes was Γ ) 9.0 ( 2.2 pmol/cm2, corresponding toa 0.63 ( 0.15 theoretical monolayer, which is a littlegreater than the coverage deduced from CV measure-ments. The heterogeneous ET rate constant for the sixtrial electrodes was k′EI ) 660 ( 200 s-1.

Electroreflectance Measurements. An ER spectrumintensity function was calculated from (∆R/R)iφ and (∆R/R)oφ measurements to show the relationship of the ERsignal to solution cyt c absorbance spectra when theelectrode was set near the formal potential of cyt c. Thisintensity function, which combines the amplitude of bothin-phase and out-of-phase responses with the lead termused to make the sign the same as that of the in-phasecomponent, is

This ER intensity function is shown in Figure 4 as afunction of incident light wavelength with Edc ) 10 mV(versus Ag/AgClsat) in 0.01 M phosphate buffer, pH 7.4.R would correspond to the true in-phase signal at lowfrequencies, where electron transfer keeps up with thechanges in potential and where the lock-in reference isequal to the actual potential driving electron transfer.The features of the ER spectrum are qualitatively similarto those of the absorbance difference spectrum calculatedfrom published spectra26 for cyt c3+ and cyt c2+, especially

with respect to the strong transitions around the Soretabsorption band (410 nm) and the R- and â-absorptionbands (500-550 nm). Discrepancies such as the red-shiftfor the zero point of R relative to the absorbance zero atabout 408 nm most probably originate from the uncom-pensated background signal at the NAC-modified goldelectrode. However, the features of the cyt c ER spectrumwhich are caused mainly by the modulation of the protein’soxidation state are not thought to be significantly affectedby interactions with the organic layers adsorbed on thepolycrystalline gold electrodes.27 The inset of Figure 4shows deterioration of ER response when using incidentlight irradiation that is near the Soret absorption wave-length (408-420 nm). All ER signal (as well as CVresponse) was lost when the electrode was continuouslyirradiated with 420 nm monochromatic light. When theirradiation wavelength was near the R- and â-absorptionbands (500-550 nm), the ER signal was initially about30% weaker but much more stable. The reason for thisdifference in ER signal stability between these irradiationwavelengths is not clear, but we have previously observedphotodegradation of heme protein Raman signals incytochrome proteins at high laser power (more than 1mW).28 Accordingly, we used 550 nm light for the electrodeirradiation in all subsequent ER voltammetry experimentsreported here.

The measured ER voltammograms, (∆R/R)iæ and (∆R/R)oæ versus Edc, are shown as the points in Figure 5. Sagaraet al.29 derived the relationship between the expectedelectroreflectance response and the ET rate constant:

Equation 4 was obtained from the linearized kineticequations describing ET between the electrode and cyt c;

(25) Laviron, E. J. Electroanal. Chem. 1979, 97, 135.(26) Cartling, B. In Biological Applications of Raman Spectroscopy.

Vol. 3 Resonance Raman Spectra of Heme and Metalloproteins; Spiro,T. J., Ed.; John Wiley & Sons: New York, 1988; Chapter 5.

(27) Hinnen, C.; Parsons, R.; Niki, K. J. Electroanal. Chem. 1983,147, 329.

(28) Niaura, G.; Gaigalas, A. K.; Vilker, V. L. J. Raman Spectrosc.1997, 28, 1009.

(29) Sagara, T.; Igarashi, S.; Sato, H.; Niki, K. Langmuir 1991, 7,1005.

Table 1. Impedance Parameters for Cyt c on a GoldElectrode Modified with N-Acetylcysteine (Also See

Figure 3)

electrodeaRsol,

ΩQdl,µF n

Rct,Ω

Ca,µF

Γ, pmol cm-2

(θ, monolayerfraction)b

k′EI,s-1

1 277 16.5 0.9088 240 2.43 7.9 (0.55) 8572 279 12.1 0.8981 187 3.90 13 (0.91) 6863 309 12.0 0.8556 490 2.39 7.8 (0.55) 4274 306 11.8 0.8605 330 3.11 10 (0.69) 4875 289 12.8 0.8445 372 2.55 8.3 (0.58) 5276 196 11.8 0.8620 249 2.09 6.8 (0.47) 962

a Separate electrodes prepared in a similar fashion. Impedanceand electroreflectance data were acquired simultaneously in 0.01M phosphate buffer, pH 7.4. b θ is the fraction of monolayer coverageby cyt c based on an electrode geometric area of 0.32 cm2 and thetheoretical monolayer concentration of 14.3 pmol/cm2.

k′EI ) (2RctCa)

-1 (1)

Γ ) 4RTCa/n2F2A (2)

R ) [(∆R/R)iφ/|∆R/R|iφ]x(∆R/R)iφ2 + (∆R/R)oφ

2 (3)

Figure 4. (connected circles) Electroreflectance intensityspectrum (see eq 3) determined for the Au/NAC/cyt c electrodein 0.01 M phosphate buffer, pH 7.4. The electrode was subjectedto an applied potential (versus Ag/AgClsat): Eappl ) 10 mV + (20mV rms) sin(ωt), where ω/2π ) 77 Hz. (Curve) Absorbancedifference spectrum between oxidized and reduced cyt ccalculated from cyt c3+ and cyt c2+ published solution spectra.26

The absorbance difference spectrum was scaled to fit the Rscale. (Inset) Stability of R for 550 or 420 nm irradiation. Onemeasurement is approximately equal to a 10 min data collectionperiod with Edc ) 10 mV and ∆Eac ) 20 mV rms.

k′ER ) ω2 [|∆R/R|iφ

|∆R/R|oφ]

Edc)E°′(4)

7302 Langmuir, Vol. 14, No. 25, 1998 Ruzgas et al.

therefore, theresultapplieswhenthemodulatingpotentialis small. In the present case, the amplitude of themodulation potential is small enough (20 mV rms) so thatthe linearized kinetic equations describing ET betweenthe electrode and cyt c, which lead to eq 4, are appropriate.In our previous studies on electron transfer to putidare-doxin4 and azurin,18 the larger modulation amplitudes(∼100 mV) required numerical solution of the kineticequations, and eq 4 could not be used to find k′ER. The ERresponse that has to be used in eq 4 is relative to thephase of the modulating potential actually driving the ET(and ER) process (faradaic potential Efar). The measuredvalues of ER ((∆R/R)iæ and (∆R/R)oæ in Figure 5) areobtained from a lock-in amplifier whose reference is theapplied modulating potential Eappl. Since Eappl and thefaradaic potential Efar may have different phases, the useof eq 4 with measured ER values is not appropriate. Theissue of the phase difference between Eappl and Efar is ofcentral importance, since prior to using the measuredvalues (∆R/R)iæ and (∆R/R)oæ in eq 4 it is necessary toconvert them to values which would be obtained if Efarwas used as the reference of the lock-in amplifier. Atpresent, the only method for obtaining Efar is to assumean electrical impedance model for the interface.

Feng et al. presented a mathematical procedure toaccount for this phase difference by invoking an idealequivalent impedance circuit, consisting of solution re-sistance in series with double-layer capacitance.14,15,30 InFeng’s approach, the charge-transfer impedance (consist-ing of a resistance in series with a pseudocapacitance)was connected in parallel with the double-layer capaci-tance. The main limitations of Feng’s equivalent circuitwere its assumptions of an ideal double-layer capacitance(which is very often found to be better described as aconstant-phase element24) and the neglect of parasiticfaradaic processes that may contribute to the electrodecurrent (iresp). In Feng’s analysis, the amplitudes of theuncorrected bell-shaped (∆R/R)iæ and (∆R/R)oæ componentswere used to calculate the cot φ parameter shown in theinset of Figure 6, and the dependence of this parameteron the frequency of the modulating potential was used toinfer the rate constant. When the data in this study were

analyzed by this method,14,15,30 (-ω cot φ) was seen to belinearly dependent (correlation coefficient 0.985) on thesquare of the modulation frequency, as shown in the insetin Figure 6, yielding an ET rate constant of 2170 s-1 fromthe slope and intercept of this plot. The values of solutionresistance and double-layer capacitance used in thecalculation of -ω cotφwere 293 Ω and 11.5 µF, respectively.The high degree of linearity in the plot of -ω cot f versus-ω2 was not observed for all five of the electrodes that areshown in Figure 6; the corresponding correlation coef-ficients were 0.379, 0.808, 0.840, 0.983, and 0.985. Theresults summarized in Table 2 are only for the threeelectrodes on which all three measurements (CV, EI, ER)were obtained and for which the plot of -ω cot φ versus-ω2 gave a linear-fit correlation coefficient > 0.9.

An alternative procedure for performing phase correc-tion is the interface impedance model14,15,30 generalizedso that any electrode interface impedance element can beconnected in series with the solution resistance. In thecontext of this impedance model, the phase difference isdue to the uncompensated solution resistance, which issignificant when working with low-molarity buffer solu-tions (0.01 M in our case). Then Efar ) Eappl - (irespRsol) isrepresentative of the interfacial electron-transfer process.The electrode current (iresp) was recorded simultaneouslywith ER measurements, and Rsol was determined (awayfrom E°′) independently from these current measure-ments. Since the ET process is in series with the solutionresistance Rsol, it was possible to use the vectorial diagramin Figure 5 to calculate the phase difference (ψ) betweenthe applied potential Eappl and Efar. The uncorrected andcorrected in-phase and out-of-phase ER signal componentsare presented in Figure 5 by circles and solid curves,respectively. An alternate phase correction procedure18

using background ER response (ER for dc potentials farfrom E°′) as the reference leads to similar results. The ETrate constants k′ER evaluated by this method for fivedifferently prepared electrodes show dependence onpotential modulation frequency, as indicated in Figure 6.For one of these electrodes, k′ER ranged from 405 s-1 atthe lowest frequency (21 Hz) to 742 s-1 at 110 Hz.(30) Feng, Z. Q.; Sagara, T.; Niki, K. Anal. Chem. 1995, 67, 3564.

Figure 5. Electroreflectance voltammogram for the Au/NAC/cyt c system in 0.01 M phosphate buffer, pH 7.4. Appliedpotential: Eappl ) Edc + (20 mV rms) sin(ωt). Points representin-phase (∆R/R)iφ and out-of-phase (∆R/R)oφ components of theelectroreflectance signal relative to the phase of the ac potentialapplied on the electrode. Curves are mathematically correctedin-phase and out-of-phase electroreflectance components rela-tive to the phase of Efar, which is the potential computed aftervectorial subtraction of the solution IR-drop (irespRsol) from Eappl.

Figure 6. Electroreflectance ET rate constant, as a functionof modulating potential frequency, for cytochrome c on fivedifferent Au/NAC/cyt c electrodes in 0.01 M phosphate buffer,pH 7.4, using simultaneous and combined electroreflectanceand electrochemical impedance data. The inset is a representa-tion of the electroreflectance data for one of the electrodes fromwhich the slope and intercept can be used to calculate k′ER (seeTable 2, Electrode 2) when only electroreflectance data areused:14,30 ω is the angular potential modulation frequency, andcot φ represents the ratio of the amplitudes of the directlymeasured (uncorrected) bell-shaped (∆R/R)iæ and (∆R/R)oæcomponents shown as points in Figure 5.

Electron Transfer from Surface-Confined Cytochrome c Langmuir, Vol. 14, No. 25, 1998 7303

DiscussionThe three measurement techniques gave relatively

consistent values for the heterogeneous ET rate constantk′het. Table 2 summarizes a series of measurementsperformed on each of three different electrodes that wereprepared by identical procedures using common stockreagent solutions. Values of k′CV, k′EI, and k′ER that wereanalyzed independent of one another are presented withestimates of standard errors in columns 2, 3, and 6,respectively, while the values obtained by combined ERand EI measurements are given only at the specificfrequencies shown in columns 4 and 5. The combined EI/ER measurements show that the extracted values of k′ERdepend on frequency. The divergent values of k′ER shownin columns 4-6 are indicative of the sensitivity to thechoice of impedance model for data analysis. In thefollowing, we briefly discuss the nature of the interactionof cyt c that is confined at the Au/NAC/protein interfaceand then discuss features that were unique to our threemeasurement techniques. Special emphasis is placed onthe implications of the frequency dependence of thecombined EI/ER measurement of k′ER.

Cytochrome c Interactions with NAC-ModifiedGold Electrodes. The hydrophobic-electrostatic inter-action between cyt c and electrodes modified with longchain self-assembled monolayers has previously beenshown to lead to electrochemical responses that suggestthe protein is surface confined and in its native solutionstate.9,31,32 The electrochemical responses we obtained withcyt c on NAC-modified electrodes are similar, althoughthe mechanism of immobilization is mainly electrostaticwith a minor contribution from hydrophobic interaction.

Electrostatic attraction between cyt c and the NAC-modified electrodes is suggested by the stability of theelectrochemical responses in low-ionic-strength buffers.The disappearance of the cyt c redox signal in 0.2 Mphosphate buffer suggests desorption of the protein fromthe electrode. Redox electrochemistry could be recoveredon the NAC-modified electrodes that had been exposed tohigh ionic strength if they were again exposed to cyt c ina low-molarity buffer. Retention of its native state, whencyt c is electrostatically immobilized on the NAC-modifiedelectrodes, is suggested by the negligible shift in the formalpotential, E°′, and by the close agreement of the measuredpotential distribution around the CV peaks (full-widthvoltage at half-peak-height, Efwhh) with the theoreticalvalue of 90.6 mV from Laviron’s model.22 Our measuredvalue of the formal potential, E°′ ) +0.235 ( 0.005 V(versus NHE), is only slightly downshifted from thesolution formal potential +0.258 V (versus NHE) mea-sured by titration techniques.5 Metal oxide electrodes11,12

and metal electrodes coated with long chain aliphatichydrocarbons13-17 show similar small or negligible shiftsof the formal potential. Dispersion about the formalpotential beyond the theoretical limit of 90.6 mV isattributed to heterogeneity in the population of im-mobilized cyt c molecules due to a distribution of (i)interfacial environments, (ii) adsorption energies, and/or(iii) charge-transfer-induced alterations of formal poten-tial.16 Compared with relatively larger values measuredon SAM-modified electrodes (Efwhh ) 104 mV for Au/mercaptopropionic acid/cyt c,14 110-120 and 125-152mV for Au/mercaptohexanoic acid/cyt c,13,16 101-104and 115-135 mV for Au/mercaptoundecanoic acid/cytc,14,16 and 140-170 mV for Au/mercaptohexadecanoic acid/cyt c16), our measured Efwhh ) 97 mV suggests that theAu/NAC/cyt c electrode surface is a molecularly homo-geneous population with the protein close to its nativeconformation.

Heterogeneous Electron-Transfer Rate Con-stants: k′CV, k′EI, and k′ER. The heterogeneous ET rateconstant for cyt c at the Au/NAC electrode ranged from300 s-1 to over 2000 s-1, inclusive of all methods ofmeasurement and analysis. As far as we know, this is thefirst attempt to compare k′het values obtained with thethree different measurement techniques with the inter-pretation based on the same kinetic and impedancemodels. In the following, we review our approach usingeach method and compare it to previously reported results.

The electron-transfer rate that we measured by cyclicvoltammetry, k′CV ≈ 900 s-1, is one of the highest reportedfor heterogeneous protein ET measured by this technique.Our use of high sweep rates was the major difference fromother CV applications where much slower ET rates weremeasured. These higher sweep rates require IR compen-sation, a feature which is available with any modernpotentiostat. The drawback to IR compensation is thatthe solution resistance has to be estimated with highprecision. For sweep rates g 10 V/s, an uncertainty of3-5% in the solution resistance leads to substantialuncertainty in the peak separation, from which k′CV isdetermined by the method of Laviron.22 Analog signalfiltering cannot be used during these measurementsbecause the filter introduces a large systematic error inthe resulting value of the rate constant. In fact, theextracted rate constant can reflect the characteristics ofthe electronic filters and not the actual faradaic process.In lieu of the analog filters, digital filtering can be per-formed after data collection.

The range for ET rate constants measured by electro-chemical impedance spectroscopy (k′EI ) 400-1000 s-1)over many different Au/NAC/cyt c electrode preparationsincludes the range of our k′CV results and represents muchhigher values than we find reported in the literature bythis technique. Quantifying the frequency dependence of

(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.

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

combined ER/EI + phase

electrode

cyclicvoltammetryb

k′CV, s-1

electrochemicalimpedancec

k′EI, s-1Figure 3 @ 21 Hz

k′ER, s-1Figure 3 @ 110 Hz

k′ER, s-1

(-ω cot φ versus -ω2)per refs 15 and 30

k′ER, s-1

1 710 ( 140 860 ( 200 370 700 1860 ( 4802 750 ( 120 690 ( 220 410 740 2170 ( 2303 720 ( 160 960 ( 200 310 780 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 electrodesurface. b CV measurements were made at sweep rates of 7-20 V/s with instrument signal filtering disabled; k′CV is calculated from datawhere peak separation (Ea - Ec) g 10 mV by the Laviron method (see ref 22, Table 1). c EI measurements.

7304 Langmuir, Vol. 14, No. 25, 1998 Ruzgas et al.

the current response is the usual approach for extractingk′EI from values of the circuit elements comprising thetotal impedance. Differences in the circuit elements thatwe used from those used by others13-15 to measure muchlower rates of protein heterogeneous ET were describedabove. The most important of these is our use of a constantphase element to account for a distributed double-layercapacitance.

Column 6 of Table 2 gives the ET rate constantdetermined by our electroreflectance measurements whenthey are analyzed from the frequency dependence of theER response15,30 (slope -ω cot φ versus -ω2). These values(k′ER ) 1800-2400 s-1), which are 2-3 times higher thanthose 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 immobilizedon Au electrodes coated with short chain (n ) 2) al-kanethiols.17 The lower values are in the range of theinterprotein electron-transfer rate constant (800 s-1)measured between cyt c and one of its natural redoxpartners, cytochrome c peroxidase.33

We are not the first to observe the discrepancy in valuesof k′het determined by the three methods. Literature reportsgenerally show that electroreflectance measurements givehigher ET rate constants compared with those measuredby cyclic voltammetry or electrochemical impedancespectroscopy. In one of the earliest applications of ER toheterogeneous electron transfer, the rate coefficients forNile Blue A adsorbed on pyrolytic graphite29 were deter-mined to be k′CV ) 5 s-1 and k′ER ) 63-78 s-1. Measure-ments of heterogeneous ET rate constants for cyt cimmobilized on gold electrodes coated with long chainalkanethiols HS(CH2)nCOOH have been given: k′EI ) 23s-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 carefulto point out that CV measurements (under their condi-tions) cannot track rapid ET rates, and the value ofapproximately 20-30 s-1 is our interpretation of theirreported CV data. In our lab, we have previously reporteda similar large discrepancy in ET rate constants (k′CV )4-12 s-1 and k′ER ) 50-200 s-1) for azurin immobilizedon 1-hexanethiol-modified gold electrodes.18

Because the ET rate constant extracted from thefrequency dependence of the ER response (-ω cot φ versus-ω2 slope) is extremely sensitive to the selection of theimpedance model that describes the interfacial effects onthe current response, discrepancies between the hetero-geneous ET rate constants evaluated from electrochemicaland ER measurements can be anticipated. This is espe-cially true if the interfacial elements show distributed

character (e.g. distributed character of double-layercapacitance). In previous reports of ER response analysis,it was assumed that all impedance elements are idealresistors and capacitors.14 However, the double-layercapacitance should not be represented as an ideal imped-ance element,24 especially in low-molarity buffer solutionsand at relatively low frequencies (f < 300 Hz).

Dependence of k′ER on Modulation Frequency. InTable 2 columns 4 and 5, results for k′ER are reported forour measurements of an ER response that takes advantageof the high signal-to-noise ratio of our instrument and fora new analysis model that allows direct estimation of areference phase for the faradaic potential driving the cytc ET. The circuit element parameter values were subjectto the dual constraints of matching simultaneous imped-ance and electroreflectance signal responses. The phasecorrection technique is not sensitive to the details of theimpedance model but assumes that the ET process is inseries with the solution resistance. The combined analysisof the EI and ER data allows calculation of the ET rateconstant assuming that the ET process is in series withthe solution resistance; all other electrochemical processeswhich are parallel to the ET process do not need to bespecified.

The ET rate coefficient k′ER is given in Table 2 columns4 and 5 for the three separately prepared electrodesmeasured at modulation frequencies of 21 and 110 Hz,respectively. These results are in closer agreement withvalues determined by CV and EI but show a systematicincrease with increasing frequency (Figure 6). Thisfrequency dependence indicates that the simple impedancemodel, in which the elements describing ET of cyt c areconnected between Rsol and the electrode, is not anadequate model. A more accurate impedance model, basedon recent improvements in understanding the behaviorof double-layer charging in low-ionic-strength buffers,35-38

is needed. Such an extended analysis of the combined EIand ER measurements is currently under investigationby our group.

Acknowledgment. We are grateful to Vytas Reipa,Martin Mayhew, and Gediminas Niaura for their invalu-able support and assistance in the execution of the researchreported here.

LA9808519

(33) Pappa, H. S.; Tajbaksh, S.; Saunders, A. J.; Pielak, G. J.; Poulos,T. L. Biochemistry 1996, 35, 4837.

(34) Burris, S. C.; Bowden, E. F. Abstract ANYL-24, 212th NationalACS 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.

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