Direct and through-film dissolution of Ni during anodic polarization in aqueous HF media

Journal of Electroanalytzcal

Chemzstry, 369 (1994) 79-86

79

Direct electrochemical reactions of cytochrome c at iodide-modified electrodes Tlanhong Laboratory

Lu, Xlupan of Electroanalytzcal

Yu and Shaojun

Chengh

Zhou,

Department

of Chemzstry and Ames Laboratory,

(Received

12 Aprd

Dong

Chemzstry, Changchun Znstztute of Applzed Chemzstry, Academza

Shuyu Ye and Therese 1993, m revised

M. Cotton

Sznzca, Changchun,

130022 (Chzna)

l

Iowa State Unzuerszty, Ames, ZA 50011 (USA)

form 5 August

1993)

Abstract Quasi-reversible and direct electron transfer was observed between an lodlde-modified Au electrode and cytochrome c, as well as between cytochrome c m an lodlde-contammg solution and a bare Au electrode The results suggest that an electrostatic mteractlon between cytochrome c and the lodlde-modified electrode surface plays an Important role m the electrochemical response A mechamsm IS proposed which mvolves adsorptlon of cytochrome c on the surface of the lodlde-modlfled electrode, followed by rapld desorptlon after the electron transfer process

1. Introduction

Cytochrome c, an electron carrier between cytochrome c reductase and cytochrome c oxldase, 1s widely distributed m hvmg organisms Its physlologlcal redox partners are bound to the inner membrane of mltochondna, whereas cytochrome c itself resides m the cytosol between the inner and outer membranes [l] and is subJect to very high electric fields (lo’-lo8 V m-‘) near the surface of the membrane [2] A field of similar magnitude exists at an electrode I solution mterface, and consequently the electrochemical behavior of cytochrome c 1s of considerable mterest However, early studies have shown that the electrochemical reactions of cytochrome c at metal electrodes, such as Hg, Pt, Au, Ag, etc , are n-reversible and sometimes not detectable 131despite the fact that cytochrome 1s readily oxldlzed or reduced by chemical reagents (e g NaNO,, Na,SO, etc 1 For this reason mediators have often been employed m electrochemical studies of cytochrome c In 1977 quasi-reversible direct electron transfer was observed between cytochrome c and tm-

l

To whom correspondence

0022-0728/94/$7 00 SSDZ 0022-0728(93)03115-6

should

be addressed

doped mdmm oxide and tm oxide electrodes [4] and at 4,4’-blpyndme-modified electrodes 151 In the latter system, 4,4’-blpyndme was termed a “promoter” of the electron transfer process because it 1s not redox active wlthm the potential range used to reduce or oxidize cytochrome c Followmg the mltlal observations of Hill and coworkers, an extensive research effort has been devoted to the study of the direct electrochemical reaction between cytochrome c and promoter-modified electrodes [5-311 To date, most of the promoters that have been studied are organic compounds such as 4,4’-blpyndme [53, 1,2-bls-(4-pyndyljethylene [5], bls(4-pyrldyl)dlsulphlde [12] and purme [16] Only a few morgamc promoters, such as S and As adatoms [26] and heteropolytungstates [30], have been reported Au electrodes were used most often m these studies, but quasi-reversible electron transfer behavior between cytochrome c and other promoter-modified metal electrodes, such as Pt [91 and Ag [14], has also been reported With respect to other blologlcal compounds, Lane and Hubbard 1321reported a study of the electrochemical behavior of catecholammes which showed that modlflcatlon of Pt electrodes with iodide ion produced a surface which 1s inert with respect to electro0

1994 - Elsevler

Sequoia

All rights reserved

80

T Lu et al / Electrochenucal reactions of cytochrome c

chemical and chemical interference over the potential range of interest Adsorbed iodide prevented the formation of surface oxides on the Pt as well as adsorption of blologlcal compounds Detection of the dopamme was possible at the iodide-modified electrode, whereas no current was dlscermble at bare Pt owmg to the high background current as well as the irreversible nature of the electron transfer process The mechanism of the heterogeneous electron transfer process between cytochrome c and promotermodified electrodes 1s not well understood It has been shown that cytochrome c 1s structurally altered (1 e unfolded or flattened) when adsorbed directly on metal electrodes, and under these condltlons it undergoes irreversible electron transfer [33-361 It may be that promoters somehow prevent structural changes m cytochrome c at the electrode surface The maJorlty of promoters studied so far are blfunctlonal One functional group binds to the electrode surface and the second 1s believed to interact with lysme-NH: groups of cytochrome c through hydrogen bondmg or salt bridging [17,21,25] When bound to the promoter-modified electrodes, cytochrome c mamtams its native structure and undergoes quasi-reversible electron transfer [35] However, Nlkl and coworkers [35,36] have shown that three types of mteractlons can occur between cytochrome c and organic modifiers, and the role of the surface modifier 1s not as simple as onglnally proposed Hawkridge and coworkers [37,38] have made extensive mvestlgatlons of cytochrome c electrochemistry They found that, after purlflcatlon of cytochrome c and m the absence of lyophlhzatlon, a quasi-reversible electron transfer process can be observed at bare metal electrodes [39,40] These authors suggested that the lyophlhzatlon process produces small amounts of ohgomerlc and polymerized cytochrome c and that these forms adsorb strongly onto the metal electrode surfaces Electron transfer between bulk cytochrome c m solution and the electrode 1s irreversible under these condltlons [39] The heterogeneous electron transfer rates for small metalloprotems, such as cytochrome c553 [40] are greater than those for larger metalloprotems such as cytochrome c Thus it was concluded that the size of the blologlcal molecule (1 e the distance of closest approach between the heme edge and the electrode surface) controls the rates of the heterogeneous electron transfer of blologlcal molecules at electrode surfaces [39-411 Electrostatic mteractlons between cytochrome c and its redox partners 1s known to play an important role m homogeneous electron transfer reactions [42,431 Several studies have emphasized similar electrostatic mteractions between the metalloprotem and an electrode

surface [44-461 Surface-enhanced resonance Raman scattering @ERRS) studies of cytochrome c also provide support for protein adsorption on Ag electrodes 147,481 Moreover, Hlldebrandt and Stockburger [48] have shown that the spm state marker bands m the SERRS spectra are sensitive to structural perturbations resulting from the interaction of the protein with the metal The high spm form of the protein was found to exhibit a more negative redox potential than the low spin form ( - 0 35 V versus 0 0 V) It was proposed that the orientation of the protein at the electrode surface changed with the adsorption potential as a result of electrostatic mteractlons between the charged ammo acid groups on the surface of the protein and the electrode In the work reported m this paper, quasi-reversible direct electron transfer 1s demonstrated to occur between cytochrome c and an iodide-modified Au electrode or between cytochrome c m an iodide-contammg solution and a bare Au electrode Direct evidence for adsorption of the protein on an iodide-modified Ag electrode was obtained from SERRS These results support the role of electrostatic mteractlons m the electrochemistry of cytochrome c A mechanism for the electrochemical reaction of cytochrome c at the iodide-modified electrode 1s proposed 2. ExperImental Horse heart cytochrome c (type VI, Sigma Chemical Co ) was used without further purlflcatlon or was punfled according to the published procedures [49] All other chemicals were reagent grade A BAS 100 electrochemical analyzer and conventional three-electrode electrochemical cell were used for the electrochemical measurements The working electrode was constructed from a Au rod which was sealed mto glass tubing with a Torr seal (Vanan) The exposed area was approximately 5 mm2 A Pt wire was used as the auxlhary electrode A saturated calomel electrode (SCE) served as the reference electrode and all the potentials are reported with respect to it The modified electrodes were prepared using the followmg procedure The workmg electrode was sequentially polished with 5, 0 3 and 0 05 pm alumma + water slurries until a shmy mirror-like fmlsh was obtamed The electrode was then somcated m delomzed water and washed thoroughly with deionized water Surface modlflcatlon of the Au electrode was carried out by dipping the freshly polished Au electrode into 0 1 M KI solution for 2 mm, followed by rinsing twice with deionized water The electrochemical studies of cytochrome c were carried out at a freshly polished Au electrode contam-

T Lu et al / Electrochemical

mg JSI or at an iodide-modified Au electrode m 0 38 mM cytochrome c solution The electrolyte solution contained 0 025 M phosphate buffer (pH 7 0) and 0 1 M sodium perchlorate Oxygen was purged from solution by bubbling with prepurlfled nitrogen for 10 mm prior to the electrochemical measurement The mtrogen atmosphere was maintained durmg the measurements by passing nitrogen gas over the solution SERRS spectra were obtained from cytochrome adsorbed on a roughened Ag electrode The electrode was constructed from Ag wire and roughened by a double-potential-step oxldatlon-reduction cycle (ORC) as described previously [13] Followmg the ORC, the electrode was dipped mto a cytochrome c solution for 15 mm The electrode was removed and excess solution was shaken from its surface, and it was placed m a 0 025 M phosphate buffer solution (pH 7 0) In the case of iodide modlflcatlon, the roughened Ag electrode was dipped mto 0 1 M KI for 2 mm, removed from the solution and rinsed with delomzed water Cytochrome c was then adsorbed as described above for the bare Ag electrode The SERRS spectra were recorded at room temperature and open-circuit potential The 413 1 nm lme of a Krf laser (Coherent, Innova 100) was used as the excitation wavelength, the power was 1 mW at the sample The scattered light was collected m a backscattermg geometry and focused on the slit of a monochromator-spectrograph (Spex Trlplemate 1377) equipped with a 1200 lmes mm-’ grating The detector was an mtenslfled diode array (model 1420, OMA III, EG&G Inc ) 3. Results Cytochrome c does not undergo reversible electron transfer at a freshly polished Au electrode (Fig l(a)) In contrast, a well-defined peak 1s obtained at an

E/L&s

SCE)

Fig 1 Cychc voltammograms of 038 mM cytochrome c at (a) a freshly pohshed Au electrode, (b) an lodlde-amon-modified Au electrode m phosphate buffer solution (pH 6 97) contammg 0 1 M NaClO, Scan rate, 0 050 V s-l, mltlal potential, + 0 20 V/SCE

reactions of cytochrome

c

81

f

2~tA 1

E /V(vs SCE) Fig 2 Cychc voltammograms of 0 38 mM cytochrome c at an lodlde-amon-modlfled Au electrode m phosphate buffer solution (pH 6 97) contammg 0 1 M NaClO, Scan rates/V s-l (a) 0 010, (b) 0 025, (c) -0 050, (d) 0 100, (e) 0 200, (f) 0 500 V s-l, Imtlal potential, + 0 20 V

iodide-modified electrode (Fig l(b)) This current 1s entirely due to cytochrome c, iodide anions exhibit no electrochemical response m the potential range from -0 2 V to +0 2 V The separation between the cathodic and anodlc peak potentials 1s about 0 070 V, which 1s rather larger than that for a fully reversible one-electron transfer reaction The mldpomt between the cathodic and anodlc peak potentials 1s approxlmately +0 01 V, which is close to the formal potential of cytochrome c [50] The cathodic peak current 1s almost the same as the anodlc peak current The cathodic and anodlc peak currents are proportional to the square root of the scan rate m the range 0 010-O 500 V s-l (Fig 21, showing that the reactlon 1s diffusion controlled From the slope of the plot of the cathodic peak current ZP versus the square root u112 of the scan rate, the calculated diffusion coefficient of cytochrome c 1s 109 x 10e6 cm2 s-l which 1s m good agreement with that obtained at Au electrodes modified with 4,4’-blpyrldme [5] or bls(4-pyndyl)dlsulphlde [121 Using Nicholson’s method [51], the heterogeneous electron transfer rate constant k, was determined to be ca (6 2 k 0 1) X lop3 cm s-l from the scan rate dependence (0 010-O 100 V s-l) of the peak separation This value 1s larger than that obtained at a purme-modified Au electrode (1 x 10e3 cm s-l) [16], but less than that obtained at 4,4’-blpyndme-modified Au electrodes ((1 4-l 9) X lop2 cm s-l> [71 All the above characterlstlcs are mdlcatlve of a quasi-reversible direct electron transfer reaction between cytochrome c and the iodide-modlfled Au electrode Moreover, the performance of the iodideamon-modified Au electrode was very stable After several days, the cychc voltammetrlc response was almost the same as that observed mltlally

T Lu et al / Electrochemtcal reactlou of cytochrome c

82

E /V(vs SCE) Fig 3 Cychc voltammograms of 0 38 mM cytochrome c at a freshly polished Au electrode III phosphate buffer solution (pH 6 97) contamrng 0 1 M NaCIO, and (a> 0 38, (b) 0 76 and (c) 3 8 mM ICI Scan rate, 0 050 V s-l,

mitial potential,

+ 0 20 V

The electrochemical behavior of cytochrome c was also studied at a freshly pohshed Au electrode m a cytochrome c solution contammg varying concentrations of KI The cychc voltammograms were measured immediately after placing the freshly polished bare Au electrode mto the cytochrome c solution It was found that, under these condltlons, the mltlal response depended upon the KI concentration Figures 3(a), 3(b) and 3(c) show the responses at a freshly pohshed Au electrode for cytochrome solutions contammg KI concentrations of 3 8 X 10d4 M, 7 2 X 10e4 M and 3 8 x 1O-3 M respectively It can be seen that peak current increases and peak separation decreases with mcreasmg KI concentration Figure 4 shows the relationship between the peak separation and a series of KI concentrations The peak separation decreases sharply to a hmltmg value with mcreasmg KI concentration It was also noted that at low KI concentrations the peak separation decreased with time For example, m the case of a 0 1 mM KI solution, the peak separation stabilized after approxtmately 1 h, whereas m 1 mM solutions only 15 mm was reqmred At the highest KI concentrations (10 mM) the peak separation reached a stable value wlthm the time required to record a cychc voltammogram The peak current also increased with addition of KI, as shown m Fig 3 These results suggest that the iodide ion is adsorbed from solution onto the electrode surface and forms a stable modified electrode m the presence of cytochrome c Any cytochrome c that 1s mltlally adsorbed 1s displaced by the iodide ion which 1s irreversibly adsorbed The higher the iodide concentration, the more rapldly the cytochrome 1s displaced In the absence of cytochrome c, the time required for adsorption of iodide from solution 1s also concentration dependent SERRS was used to determine whether cytochrome 1s adsorbed on iodide-modified electrodes In order to obtain surface enhancement on Au substrates it is

necessary to excite at wavelengths near 600 nm At this wavelength cytochrome 1s not resonantly enhanced, however, and both resonance and surface enhancement are necessary to obtain sufflclent scattering mtenslty from cytochrome at the lodlde modlfled electrode For this reason, a Ag electrode was used because strong resonance and surface enhancement occurs at 413 nm The electrochemical response of cytochrome c at iodide-modified Ag was slmllar to that at iodide-modified Au, although the modlflcatlon was not as stable as m the case of Au For comparison, the SERRS spectrum of cytochrome c adsorbed directly on a roughened Ag electrode (m the absence of iodide ion) was also measured The position of the oxldatlonstate-sensitive band at 1372 cm-’ shows that cytochrome adsorbed on the bare Ag electrode 1s m the oxidized state (Fig 5B) [48] Bands at 1502 cm-’ and 1491 cm- ’ are spm state marker bands and are mdlcatlve of a mvrture of low and high spm states respectively [48] Figure 5A shows the SERRS spectra of cytochrome adsorbed on an iodide-modified electrode, and m this case it 1s clear from the posltlon of the oxldatlon state marker band at 1358 cm-’ that the cytochrome 1s reduced Also, the spm state marker bands at 1578 cm-’ and 1486 cm-’ are similar to those of the low spm form of reduced cytochrome m solution (1583 cm-’ and 1493 cm-‘) [48] Reduction of the cytochrome can be attributed to the shift m the rest

60 t

I 00

1

40

20

60

cKL/mM Fig 4 Plot concentration

of the peak separation E, as a function Experimental condltlons as in Fig 3

of the KI

T Lu et al / Electrochemrcal

-r

w 3

1800

h

(b)

600

Fig 5 SERRS spectra of cytochrome c adsorbed on a Ag electrode m 2.5 mM phosphate buffer solution (pH 7 0) (A) cytochrome adsorbed on lodlde-modified Ag electrode, (B) cytochrome c adsorbed on bare Ag electrode from 1 X 10e5 M solution ExperImental condltlons 413 nm excltatlon wavelength, laser power, 1 mW, exposure time, 50 s per scan, 20 scans

potential of the Iodide-modified electrode from + 0 100 to -0 05 V, as determmed experrmentally Small drfferences (5-7 cm-‘) between the spm state bands m the resonance Raman spectrum of the protein m solution compared with those at the surface may be partially due to the strong electric field near the electrode surface (electrochemrcal Stark effect) as well as to a slightly different structure of the protein m the vrcmrty of the heme 4. Discussion The above results show that adsorptron of a simple anion, such as iodide, at a Au electrode can facrhtate the heterogeneous electron transfer process between cytochrome c and a metal electrode The electrostatic interaction between cytochrome c and the electrode surface appears to play a role m rts electrochemrcal response The sign and magnitude of the excess charge density at a metal electrode surface depends on the electrode potential relative to the potentral of zero charge (pzc) and the differential capacitance The pzc for a Au electrode m the absence of specrfrc ion

reac ttons of cytochrome

c

83

adsorption 1s near -0 05 V [52] which 1s slightly more negative than that of the rest potential of the Au electrode m cytochrome c solutron (+O 09 VI Therefore a slight excess posrtrve charge exrsts at the Au electrode surface at the rest potential It has been reported that iodide anions adsorb strongly and rrreverstbly on the Au surface [53,54] The forces responstble for the strong interaction involve not only the simple coulombrc attraction or solvent structure-breaking factors, but also covalent bonding between the iodide amon and the electrode surface, as shown by the surface-enhanced Raman scattering mvestrgatron of Gao and Weaver [54] In our experiments, the iodide-modified Au surface 1s negatively charged at the rest potential ( - 0 050 V, as verified by direct measurement) The change m the rest potential with adsorption of iodide ran leads to reduction of the adsorbed cytochrome c The possrbrhty that iodide ions are adsorbed at specific sites on the cytochrome c surface and that this 1s responsrble for the enhanced electron transfer kmetICS should also be considered It 1s known that cytochromes bmd a number of different anions, and the ion-bmdmg properties vary widely with species [55] Ion bmdmg has been shown to have an effect on the redox potentials of cytochromes from different species, and the shift m potential has been analyzed m terms of the bmdmg constants [56] Assuming that rodrde also bmds to the protein, rt 1s concervable that this could influence the protein mteractron with the electrode surface and hence the electron transfer kinetics However, experimental evidence suggests that the direct interaction of the iodide ion with the electrode surface plays a more Important role Ex-situ modrfrcatron of the electrode results m a stable surface which, when transferred to an iodide-free solutron of cytochrome c, exhibits quasi-reversible electron transfer kinetics In view of the results of Gao and Weaver [54], rt 1s not likely that under these condttrons the iodide ion desorbs from the electrode and associates with the cytochrome m solution to an appreciable extent These authors have shown that the iodide ion remained bound to Au throughout the potential range from +0 100 V to -09oov A consrderatron of the structure of cytochrome c suggests the possible mteractrons that may occur between the ammo acid groups on its surface and a charged electrode Gytochrome c 1s a highly ionic protem with a net charge of +9 m the oxrdrzed state at pH 7 5 The posrtrvely charged residues are fairly homogeneously distributed on the protem surface However, the drstrrbutron of the negative surface charges 1s asymmetrrc, with nearly all the negatively charged residues located m the small area on the back surface

84

T Lu et al / Electrochemzcal reactlons of cytochrome c

of cytochrome c This results m a large dipole moment (325 D and 308 D for the oxldlzed and reduced forms respectively) The dipole axis through the positive and negative centers crosses the cytochrome c surface at phenylalamne-82 (front surface) and asparagme-103 (back surface) respectively The angle between the heme plane and the dipole axis of cytochrome c 1s 33” Thus phenylalanme-82 1s located near the solventaccessible heme edge 157,581 The heme group, the plane of which 1s approximately perpendicular to the protein surface, sits m a crevice surrounded by the polypeptlde chain of 104 ammo acids The solvent-exposed surface of the heme corresponds to a very small proportion (0 06%) of the total molecular surface, and its edge 1s located approximately 0 3 nm below the molecular surface [59] This region of the protein surface 1s surrounded by posltlvely charged lysmes and constitutes an electron transfer domain for interaction with cytochrome c oxldase or reductase [60] Both these mltochondrlal reaction partners of cytochrome c are negatively charged, so that the molecules are electrostatlcally oriented as they approach one other Every colhslon 1s productive and the electron transfer rates between cytochrome c and its physlologlcal reactants are close to diffusion controlled even though the surface area of the heme edge 1s only 0 6% of the total surface of cytochrome c [61] Based upon the above properties of cytochrome c, a mechanism for the electrochemical reaction at a Au electrode can be proposed For a freshly polished Au electrode, the surface charge 1s posltlve The cytochrome molecules will tend to orient with the negatively charged back surface proximal to the electrode surface and the posltlvely charged surface near the heme crevice distal to the electrode surface In the extreme case, the cytochrome interacts at the negatively charged patch of ammo acids and the heme 1s quite distant from the electrode surface as shown m Fig 6A Under these condltlons efficient electron transfer to the heme 1s prevented When the Au electrode 1s modified with iodide, the surface becomes negatively charged Cytochrome c 1s adsorbed with the posltlvely charged region of the protein surface closest to the electrode (Fig 6B) The heme group 1s proximal to the electrode surface and quasi-reversible electron transfer 1s observed The SERRS results support this interpretation In the presence of adsorbed iodide ion, the heme extsts m the normal low spm six-coordinate state, whereas at the bare Ag electrode both high spm five-coordinate and low spm six-coordinate forms are present Adsorbed cytochrome c 1s m the oxldlzed state at bare Ag, as indicated by the posltlon of the oxldatlon state marker band It 1s reduced at the iodide modified

0 0 0

T?TxnF7 (0)

Au

Fig 6 Schematic representation of the orientation of cytochrome c adsorbed on a Au electrode surface m (A) the absence and (B) the presence of lodlde anions The large circles represent cytochrome c molecules, + and - signs m the circles represent the posltlve and negative centers of the dipole moment of cytochrome c, the lme wlthm the circle represents the heme plane

electrode because of the shift m the rest potential from + 0 080 V to -0 040 V This latter fact also indicates that the E”’ value of cytochrome c at the iodide-modified electrode 1s closer to that of the protein in solution It is known from previous potential dependent SERRS studies of cytochrome a at bare Ag electrodes that the redox potential 1s shifted by ca - 0 400 V owing to changes m the hydrophoblclty of the heme environment and alterations of the protein structure near the heme [48] Thus it 1s apparent that m the presence of iodide these parameters are not affected to any slgmflcant extent These results are also m agreement with the model proposed by Hlldebrandt and Stockburger [48] based upon their observation of two different cytochrome conformers When cytochrome c was adsorbed at negative potentials ( < - 0 2 V/SCE) the heme was m the normal low spm six-coordmate state, whereas when It was adsorbed at more posltlve potentials (> -0 2 V>, the heme was m the high spm five-coordinate state They suggested that cytochrome c 1s attached to the metal via different groups of ammo acids m the two conformers In state II the electrostatic mteractlons are sufflclently strong to modify the coordmatlon shell of the heme iron, whereas m state I the interactions are much weaker and the heme 1s maintained m its normal coordmatlon and spm state These authors also noted an increase m the low spm six-coordinate form of state II m the presence of ClAlbery et al [8] have studled the kinetics of the electron transfer reaction of the cytochrome c at a 4,4’-blpyndme-modified Au electrode They concluded that rapid adsorption and desorptlon of cytochrome c 1s necessary for fast electron transfer Conslderatlon of the amon bmdmg properties of cytochrome c m solution may provide some insight regarding its adsorptlon and desorptlon behavior at the iodide-modified electrode Two factors must be considered the number of

T Lu et al / Electrochermcal

ions bound as a function of the redox state and the strength of ion bmdmg In one study the amon bmdmg behavior was shown to be species dependent Horse heart and bovine cytochrome were found to bmd two chloride ions m the ferrl form and three m the ferro form, whereas m the case of tuna cytochrome the situation was reversed [56] However, the bmdmg strength for chloride was greater for the oxldlzed form of horse cytochrome than for the reduced form In another study, it was concluded that certain anions (1 e those which are impermeable to the mltochondrlal membrane) bmd more strongly to the oxidized form of cytochrome c than to the reduced form based upon the electrophoretlc moblhtles of cytochrome [55] It may be that at an electrode surface specific bmdmg sites on the cytochrome mteract act directly with the surfaceadsorbed iodide and its adsorption/ desorptlon behavior 1s affected by the changes m bmdmg strength with redox state Further experiments are needed to determme these parameters However, the SERRS data obtained for cytochrome on lodlde-modlfled Ag electrodes does indicate that oxldlzed cytochrome c molecules are adsorbed more strongly than the reduced species The SERRS spectrum of adsorbed cytochrome was stable with time at posltlve potentials, whereas the overall intensity decreased with cycling of the potential to negative potentials or when the potential was maintained at sufficiently negative values to reduce the cytochrome Acknowledgments

The authors are grateful for the fmanclal support of the National Institutes of Health (GM 35108) and the National Nature Science Foundation of China References 1 Y Huang and T Imura, Blochemlstry, 23 (1984) 2231 2 S McLaughhn, m F Bronner and A KIelwleller, (Eds ), Current TOPICS m Membrane Transport, Academic Press, New York, 1972, Vol 9, p 71 3 C Hmnen, R Parsons and K Nlkl, J Electroanal Chem, 147 (1983) 329, and references cited therem 4 P Yeh and T Kuwana, Chem Lett , (1977) 1145 5 M J Eddowes and HA 0 Hill, J Chem Sot , Chem Commun, (1977) 771 6 M J Eddowes and HA0 Hill, J Am Chem Sot, 101 (1979) 4461 7 M J Eddowes, H A 0 Hill and K Uosakl, J Electroanal Chem, 116 (1980) 527 8 W J Albery, M J Eddowes, HA 0 Hdl and AR Hdlman, J Am Chem Sot, 103 (1981) 3904 9 I Tamguchl, T Murakaml, K Toyosawa, H Yamaguchl and K Yasukouchl, J Electroanal Chem, 131 (1982) 397 10 M J Eddowes and H A 0 Hill, Adv Chem Ser ,201 (1982) 173 11 J Haladjian, R Pdard, P Blanc0 and PA Serre, Bioelectrochem Bloenerg , 9 (1982) 91

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and K Yasukouchl, J 12 I Tdmguchi, K Toyosawa, H Yamaguchl Chem Sot , Chem Commun , 18 (1982) 1032 Chem , 13 R Dhesl, T M Cotton and R Tlmkovlch, J Electroanal 154 (1983) 129 14 TM Cotton, D Kaddl and D Iorga, J Am Chem Sot, 154 (1983) 129 Chem , 164 15 V Razumas, A Samahus and J Kulys, J Electroanal (1984) 195 M Isekl, K Toyosawa, H Yamaguchl and K 16 I Tamguchl, Yasukouchl, J Electroanal Chem , 164 (1984) 385 17 M P Allen, H A 0 Hill and NJ Walton, J Electroanal Chem , 178 (1984) 68 18 HA 0 Hdl, D J Page, NJ Walton and D J Whitford, J Electroanal Chem , 187 (1985) 315 19 B A Kuznetsov, G P Shumakovlch and A A Mutuskm, Bloelectrochem Bloenerg , 14 (1985) 347 T Funatsu, K Umehta, H Yamaguchl and K 20 I Tamguchl, Yasukouchl, J Electroanal Chem , 199 (1985) 455 21 D Elliott, A Hamnett, 0 C Lettmgton, H A 0 Hdl and NJ Walton, J Electroanal Chem , 202 (1986) 303 22 I Tamguchl, N Hlgo, K Umeklta and K Yasukouchl, J Electroanal Chem , 206 (1986) 341 ~. ~~ 23 Y GUI and T Kuwana, J Electroanal Chem , 226 (1987) 199 24 K Nwa, M Furukawa and K Nlkl, J Electroanal Chem, 245 (1988) 275 HA 0 Hill and N L Walton, Act Chem Res , 25 F A Armstrong, 21 (1988) 407 26 M Shlbata and NJ Furuya, J Electroanal Chem, 250 (1988) 201 M P Sammartmo, P Stefanom and G Tranchlda, 27 L Campanella, Bloelectrochem Bloenerg , 21 (1989) 55 J Electroanal Chem , 261 (1989) 28 P N Bartlett and J Farmgton, 471 29 S Song, W Zhang and S Dong, J Inorg Blochem, 40 (1990) 189 Chem , 278 (1990) 387 30 G Chottard and D Lexa, J Electroanal Cotton, X Yu, T Lu and S Dong, J 31 C Zhou, S Ye, TM Electroanal Chem , 319 (1991) 71 32 R F Lane and AT Hubbard, Anal Chem, 48 (1976) 1287 Bloenerg , 4 (1977) 522 33 H Berg, Bloelectrochem 34 PA Serre, J Haladjlan and P Blanco, Blopolymers, 21 (1982) 1781 35 T Sagara, K Nlwa, A Sone, C Hmnen, and K Nlkl, Langmuir, 6 (1990) 254 S Igarahsl, H Sata, and K Nlkl, 36 T Sagara, H Murakaml, Langmuir, 7 (1991) 3190 37 E F Bowden, FM HawkrIdge and H N Blount, J Electroanal Chem , 161 (1984) 355 Anal Chem , 59 (1987) 2334 38 D E Reed and F M Hawkndge, LH Richard and FM 39 S C Sun, D E Reed, J K Culhson, Hawkridge, Mlkrochlm Acta, 111 (1988) 97 40 K B Koller, F M Hawkridge, G Fague and J LeGall, Biochem Blophys Res Commun , 145 (1987) 619 41 B C Kmg and F M HawkrIdge, J Electroanal Chem , 237 (1987) 81 42 DC Rees, Proc Nat1 Acad Sa USA, 82 (1985) 3082 43 MA Cusanovlch, J T Hazzard, T M Meyer and G Tolhn, J Macromol SCI Chem , A26 (1989) 433 44 C Van DUk, J W Van Leeuwen and C Veeger, Bloelectrochem Bloenerg , 9 (1982) 743 45 F A Armstrong, HA 0 Hill, B N Oliver and D Whitford, J Am Chem Sot , 107 (1985) 1473 46 A Manjaoui, J Haladjian and P Blanco, Electrochlm Acta, 35 (1990) 177

86

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Cotton,

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al /

D

Chumanov, Raman Speccited therein Stockburger, Blochemlstry, 28 (1989)

trosc, 22 (1991) 729, and references

48 P Hddebrandt, and M 6710 49 D L Brautlgan, S F Mdler and E Margohash, Methods Enzymol , 53 (1978) 128 50 R W Henderson and W R Rawlmson, Blochemlstry, 62 (1956) 21 51 R S Ntcholson, Anal Chem , 37 (1965) 1351 52 J Clavdler and C N Van Huong, J Electroanal Chem , 80 (1977) 101 53 F C Anson, Act Chem Res , 8 (1975) 400 54 P Gao, and M J Weaver, J Phys Chem , 90 (1986) 4057

reac tlons

cytochrome

c

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