Anal. Chem.
1994,66,
3873-3881
This Research Contribution is in Commemoration of the Life and Science of I . M. Kolthoff (1894- 1993).
Electrochemistry of Adsorbed Cytochrome c3 on Mercury, Glassy Carbon, and Gold Electrodes Dongbo Zhang,t George S. Wilson,'vt and Katsumi Nlki* Department of Chemistry, University of Kansas, Lawrence, Kansas 66044, and Electrochemistry Laboratory, Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama, 240, Japan The electrochemical behavior of cytochrome c3 from Desulfovibrio vulgaris Hildenborough was studied on mercury, glassy carbon, and gold electrodes. The adsorbed cytochrome c3 molecules behave differently on mercury, glassy carbon, and gold electrodes, exhibiting decreasing adsorption on the respective electrodes. Three conformations of cytochrome c3 have been found on the surface of mercury. In conformation A, the adsorbed cytochrome c3 molecules are electroactive and conductive. The reduction and reoxidation of the adsorbed cytochrome c3 in this conformation are one-electron processes, and the redox potential is very close to that of the soluble cytochrome c3. The heterogeneous electron transfer reaction of the soluble cytochrome c3 on this electrode is a diffusioncontrolled process involving more than one electron. In conformation B, the adsorbed cytochrome c3 is neither electroactive nor conductive, and the heterogeneous electron transfer of the soluble cytochrome c3 is blocked by the cytochrome c3 film on the surface of the mercury electrode. The adsorbed cytochrome c3 in conformation Cis electroactive and undergoes multielectron transfer on the surface of mercury at a redox potential which is more negative than that of the soluble cytochrome c3. However, the electron transfer of the soluble cytochrome c3 cannot be mediated by the adsorbed cytochrome c3 film. The conformations of cytochrome q on the mercury surface depend on the applied potential. The conformational transformation is irreversibleand can take place only from conformation A to B, B to C, or A to C. No potentialcontrolled conformational transformation of adsorbed cytochrome c3 has been observed on glassy carbon and gold electrodes. A structural changing process takes place when cytochrome c3 is adsorbed on the surface of a glassy carbon electrode. As a result, the redox potential of adsorbed cytochrome c3 on a glassy carbon electrode is about 0.2 V less negative than that of soluble cytochrome c3. Only weak adsorption has been observed on gold surfaces, and no evidence of structural change induced by the adsorption has been found. Electron transfer in biological systems frequently occurs at charged interfaces. As a result, studies of heterogeneous electron transfer of redox proteins are of considerable interest. Some redox proteins are subjected to a large electric field under physiological conditions as the surface electric field of + University of Kansas. Yokohama National University.
*
0003-2700/94/0386-3873$04.50/0 0 1994 American Chemical Society
themembranecan reach 107-108V/m.' Similar electric fields also exist at electrode-solution interfaces. Therefore, studying the heterogeneous electron transfer between redox proteins and electrodes provides a powerful approach to understanding the physiological behavior of such proteins. In heterogeneous electron transfer processes, the nature of the electrode surface is a decisive factor for a rapid electron transfer between the protein and the electrode. Some C-type cytochromes are strongly adsorbed on electrode surfaces, so that the heterogeneous electron transfer reactions of the protein are highly dependent on the nature of the adsorbed layer. Adsorption of molecules on the surface of the electrode can influence the heterogeneous electron transfer of the soluble species inhibition, promotion, mediation, or manifesting no effect at all. In the first case, the heterogeneous electron transfer is blocked by the adsorbate. In the second case, the adsorbate can make the electron transfer of the soluble species easier or make the electron transfer possible for an otherwise electroinactive species. The electron transfer of the soluble species occurs at its own characteristic reversible potential. In the third case, the adsorbate acts as an electron carrier between the electrode and the substances in solution. The redox peak appears at the redox potential of the adsorbate. The electrode reaction of proteins is typically accompanied by the adsorption of protein molecules. When strong adsorption of protein occurs, two major models for the heterogeneous electron transfer have been proposed: the electron transfer occurs through the adsorbed p r ~ t e i nor ~ .it~occurs through the pores between the adsorbed protein molecules.M Kuznetsov and co-workers4g5proposed that adsorbed cytochrome c molecules are unfolded irreversibly on the electrode surface and that the electron transfer of the soluble protein is inhibited by the adsorbed layer. A modifier on the surface of the electrode can prevent the irreversible unfolding of the protein molecule to promote the electron transfer of the protein molecules. Extensive studies have been performed using 4,4'-bipyridyl and other adsorbable molecules modifying the electrode surface to achieve a reversible electrode reaction of cytochrome c.73 (1) Pilla, A. A. Bioelectrocfiem. Bioenerg. 1974, I , 227. (2) Scheller, F. Bioelecfrochem. Bioenerg. 1977, 4, 490.
(3) Scheller, F.; Priimke, H. J. J. Electroonul. Cfiem. 1976, 70, 219. (4) Kuznetsov, B. A.; Shumakovich, G. P.; Mestechkina, N. M. Bioelectrocfiem. Bioenerg. 1977, 4, 512. ( 5 ) Kuznetsov, B. A.; Mestechkina, N. M.; Shumakovich, G. P. Bioelectrocfiem. Bioenerg. 1977, 4 , 1. (6) Biichi, F. N.; Bond, A. J . Elecfroanal. Cfiem. 1991, 314, 191. (7) Eddowes, M. J.; Hill, H. A. 0. J . Cfiem.Soc., Cfiem. Commun. 1977,771.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994 3673
Cytochrome c3, a tetraheme protein, has been isolated from different species of sulfate-reducing bacteria. In these bacteria, cytochrome c3 acts a natural electron donor and acceptor for hydrogenase. The cytochrome c3 molecule from Desulfovibrio vulgaris Hildenborough consists of a protein chain of 107 amino acids and four heme groups which are covalently bonded to the polypeptidechain. The environments around the four heme groups are not identical. Positivecharges are predominant near heme 1. This might be the reason that heme 1 has the highest redox potential. The designation of the hemes used here follows the crystallographic n ~ t a t i o n . ~ The unique characteristic of this protein is that it can exhibit reversible electron transfer without the need for an exogenously added surface modifier.Io-l2 It was established that cytochrome c3 undergoes four successive one-electron transfers in s01ution.I~ Both macroscopic and microscopicredox potentials of cytochrome c3 were measured by lH NMR.14J5 The rate constants measured by N M R in aqueous solution for intraand intermolecular electron transfer were (1.5-7.8) X los s-l and 1 X lo4M-' s-I, respectively.I6 The heterogeneous electron transfer rate constant on a mercury electrode reaches 1.2 cm/ s.16 The role of adsorbed cytochrome c3 in the electrode reaction of cytochrome c3 has drawn a great deal of attention. The behavior of adsorbed cytochrome c3 has been studied on mercury,' 1~16,17gold,I8 ~ i l v e r , ' and ~ , ~g~r a ~ h i t e ~ surfaces. l-~~ The results of surface enhanced Raman scattering and electroreflectanceexperiments have suggested that cytochrome c3 molecules maintain their original conformation after adsorption on a silver or gold A very small conformational change coupled to the redox transition has been detected recently by FTIR spectroele~trochemistry.~~ These conformational changes were observed on the cytochrome c3 in the solution phase. However, the previous work failed to answer the questions about the properties of adsorbed cytochrome c3 films and their functions in the heterogeneous electron transfer of soluble cytochrome c3. The heterogeneous electron transfer between cytochrome c3 and a mercury electrode was reported to be fast, reversible, and diffusion controlled.' 1 9 1 6 , 1 7 The electrode reaction of ~~
~
~
~~
~~~~
(8) Albery, W. J.; Eddowes, M. J.; Hill, H . A. 0.;Hillman, A. R. J . Am. Chem. SOC.1981, 103, 3904. (9) Higuchi, Y.; Bando, S.; Kusunoki, M.; Matsuura, Y.; Yasuoka, N.; Kakudo, M.; Yamanaka, T.;Yagi, T.;Inokuchi, H. J. Biochem. 1981, 89, 1695. (10) (a) Niki, K.; Yagi, T.; Inokuchi, H.; Kimura, K. J. Elecfrochem. SOC.1977, 124, 1889. (b) Niki, K.; Yagi, T.; Inokuchi, H.; Kimura, K. J. Am. Chem. SOC.1979, 101, 3335. (11) Bianco, P.; Faugue, G.; Haladjian, I. J. Elecfroanal. Chem. 1979, 104, 385. (12) Sokol, W. F.; Evans, D. H.; Niki, K.; Yagi, T.J. Elecfroonal, Chem. 1980, 108, 107. (13) Niki, K.; Kobayashi, Y.; Matsuda, H. J. Elecfroanal.Chem. 1984,178, 333. (14) Fan, K.; Akutsu, H.; Niki, K.; Higuchi, N.; Yasuoka, Y. J. Chem. SOC.Jpn. 1988, 512. i15) Kimura, K.; Nakajima, S.;Niki, K.; Inokuchi, H. Bull. Chem. SOC.Jpn. 1985, 58, 1010. (16) Sagara, T.; Nakajima, S . ; Akutsu, H.; Niki, K.; Wilson, G. S. J . Elecfroanal. Chem. 1991, 297, 271. (11) Dijk, C .V.;Van Leeuwen, J. W.;Veeger, C.;Schreurs, J. P. G. M.; Barendrecht, E. Bioelecfrochem. Bioenerg. 1982, 9, 743. (18) Hinnen, C.; Parsons, R.; Niki, K. J. Elecfroonal. Chem. 1983, 147, 329. (19) Niki, K.; Kawasaki, Y.; Kimura, Y.; Higuchi, Y.; Yasuoka, N. k n g m u i r 1987, 3(6), 983. (20) Kitagawa, T.; Verma, A. L.; Kimura, K.; Yagi, T. Chem. Phys. Leu. 1989, 159, 189. (21) Brabec, V.; Bianco, P.; Haladjian, J. Gen. Physiol. Biophys. 1982, I , 269. (22) Bianco, P.; Manjaoui, A.; Haladjian, J.; Bruschi, M. J . Elecfroanal. Chem. 1988, 249, 241. (23) Haladjian, J.; Draoui, K.; Bianco, P. Elecrrochim. Acfa 1991, 36(9), 1423. (24) Schlereth, D. D.; Fernandez, V. M.; Mantele, W. Biochemistry 1993, 32, 9199.
3874
Analytlcal Chemistry, Vol. 66, No. 22, November 15, 1994
cytochrome c3 on a mercury surface has been shown to involve four consecutive reversible one-electron transfer steps.'3J4,25 However, we found in our experiments that the heterogeneous electron transfer of cytochrome c3 on mercury surface is more complicated than previously thought. In the present study, the electrochemical behavior of cytochrome c3 from D. vulgaris Hildenborough was studied on different surfaces. The behavior of adsorbed cytochrome c3 was dependent on the nature of the electrode surface and, in the case of mercury, was also dependent upon the applied potential.
EXPERIMENTAL SECTION Materials. Cytochrome c3 from D. vulgaris Hildenborough was provided by M. A. Cusanowich. The purity ofcytochrome c3 was determined by measuring the purity index, the ratio of A552(ferro-)/A280(ferri-)r which should have a maximum value of 3.0.26 The purity indexes of the samples we used were 2.95 or higher. Na1251was from ICN Biomedicals, Inc., Costa Mesa, CA. The triply distilled mercury was from D. F. Goldsmith Chemical & Metal Corp., Evanston, IL, and was filtered before use. Nanopure water (18 MQ) was used. Other chemicals wereof reagent grade and were used without further purification. All the experiments were carried out in 30 mM pH 7.00 phosphate buffer. Instrumentation. A three-electrode system with a platinum flag as counter electrode and a AgCl/Ag electrode (in saturated KCI solution) as reference electrode were used. The working electrode was one of the following: a static hanging mercury drop electrode (PARC Model 303A), a homemade Au disk electrode ( r = 1 mm), or a homemade glassy carbon disk electrode ( r = 1.5 mm). They were made by sealing the electrode material in glass tubing and heat shrink tubing with epoxy, respectively. The potentials given in this paper are versus AgCl/Ag (saturated KC1). An EG&G M273 potentiostat controlled by M270 electrochemical analysis software was used in the electrochemical measurements. A HewlettPackard 8452A diode array spectrophotometer was used to measure the concentration of the cytochrome c3. A Beckman GPR centrifuge was used to purify the radio labeled cytochrome c3. An LKB Wallac 1282 Compugamma CS universal y counter was used to measure the 35 KeV y radiation from 1251.
Pretreatment of Solid Electrodes. The gold electrode and the glassy carbon electrode were polished with 1.O, 0.3, and 0.05 pm alumina successively and then was cleaned in distilled water using an ultrasonic cleaner. Experimental Atmosphere. The electrochemical experiments were performed in an oxygen-free atmosphere unless specified that the solution was saturated with air. The oxygen was removed by purging Ar through the solution for 10 min, and then the solution was blanketed with Ar during the experimental period. Film Transfer Experiment. The working electrode was soaked in a solution containing the adsorbable molecules of interest for 5 min and was washed with distilled water and transferred into the buffer or cytochrome c3 solution. (25) Niki, K.; Kawasaski, Y.; Nishimura, N.; Higuchi. Y.;Yasuoka. Y.; Kakudo, M. J. Elecfroanal. Chem. 1984, 168. 275. (26) Niki, K.; Yagi, T.; Inokuchi, H. J. Elecfroanal. Chem. 1984, 178, 333.
Radioiodination of Cytochrome c3. A 20 pL portion of 0.5 mM IC1 was mixed with 2 pCi of NalZ51to form 1251Cl.Next, 100 p L of 189 pM cytochrome c3 was added to the mixture. Iodination was allowed to proceed at room temperature in dark for 5 min. The mixture was then applied to a column packed with AGl-X8 ion-exchange resin and eluted with phosphate buffer. After the fractions were collected and counted, the first radioactive peak was pooled for further purification. The sample was washed and concentrated by centrifuging the diluted solution against a Millipore UltrafreeCL filter with molecular weight cutoff of 5000. The purification steps were repeated until at least 99% of the radioactivity of the solution was contributed by the radiolabeled protein (TCA assayz7). It is extremely important that the 1251-labeledprotein be purified. If not, specific adsorption of free 1251can cause a substantial error in the estimation of total adsorbed protein. Surface Coverage Measurement. The mercury electrode was soaked in the solution of the labeled protein and then transferred into buffer solution and incubated at different potentials. Afterward, the drop was knocked off into a test tube, and its radioactivity was measured. Only the surfaces of the solid electrodes were contacted with the solution of the labeled protein for 5 min. The radioactivities of these electrodes were measured after the electrodes were rinsed with buffer solution. Adsorption of trace amounts of free lZ5Ion the electrode was corrected as follows: an electrode fully covered by nonlabeled cytochrome c3 was soaked in the solution of labeled cytochrome c3 and then taken out and rinsed. The radioactivity of this electrode was measured and taken as blank. The exchange of the cytochrome c3 molecules adsorbed on the electrode surface with the cytochrome c3 molecules in the bulk solution was not observed within 30 min.
RESULTS AND DISCUSSION I. Electrochemistry of Cytochrome q on Mercury Electrodes. 1. Influence of Concentrationof Cytochrome c3. The cyclic voltammograms of cytochrome c3 with different concentrations were obtained at different scan rates. The plot of the peakcurrent versus the concentration of cytochrome c3 at different scan rates is presented in Figure 1. The peak current-scan rate dependence was inspected in a scan rate range from 0.1 to 10 V/s. The peak currents were measured at more than 15 scan rates at each concentration. According to the peakcurrent-scan ratedependence, Figure 1 was divided into three zones. In zone I, the peak currents are proportional to the scan rate. This relation indicates that surface electron transfer is the dominant contributor to the peak current. In zone 11, slow interaction kinetics between soluble and adsorbed cytochrome c3 is presumed to dominate. The peak currents are proportional to the square root of the scan rate at slow scan rates (diffusion-controlled electron transfer) and are directly proportional to the scan rate at moderate to high scan rates (surface electron transfer). In zone 111,the peakcurrents are proportional to the square root of the scan rate at low to moderate scan rates and directly proportional to the scan rate at high scan rates. It was reported that the mercury electrode (27) Der-Balian, G. P. Anal. Eiochem. 1980, 106,411.
1 I
2.00
I
II
I
111
P e /
5-
-8.
1.00
0.00 0.00
10.00
20.00 30.00 40.00 Concentration( pM)
50.00
Flgure 1. Concentratlondependent peak current profile of cytochrome q solutions. Working electrode, static mercury drop electrode. A fresh drop was used for each data point. Scan rates: (a) 0.1; (b) 0.6; (c) 1; (d) 3; and (e) 5 V I S .
surface could be fully covered by a monolayer of cytochrome c3 in a drop time of 4 s when the concentration of the protein is 10 pM.2a Since the concentration of cytochrome c3 in zone I11 was higher than 10 pM, the electron transfer reaction involves a mixed diffusion-controlled and surface electron transfer process on an electrode fully covered by cytochrome c3 molecules. The current due to surface electron transfer remained constant when the protein concentration changed. From these observations it was assumed that there are two processes involved in the electrode reaction of cytochrome c3 on the mercury surface: the electrode reaction of the species adsorbed on the electrode surface and the heterogeneous electron transfer of cytochrome c3 from the bulk solution. The competition between the surface and diffusion-controlled electron transfer processes was observed in all concentration ranges. In zone 111, the apparent peak current can be expressed as29
Zp= 2.69 X 10'n13/2ADo'/2v'/2C + n:F2AvI'*/4RT
(1)
In eq l , I , is the apparent peak current, nl is the apparent number of electrons involved in the diffusion-controlled electron transfer process, 112 is the apparent number of electrons involved in the electrode reaction of the surface species, and *'I is the surface concentration of cytochrome c3 on the mercury electrode. The other symbols have their conventional meanings. The surface coverage of a monolayer (I'*) which was determined by radioactivity measurements (Experimental Section) was 1.1 X lo-" mol/cm2. This value corresponds to monolayer coverage calculated from the cross section of the cytochrome c3 molecule and is consistent with the value (0.92 X mol/cm2) previously reported.25 Since the surfaceof the mercury electrode was fully covered in zone 111, the apparent cathodic peak current came from the reduction (28) Niki, K.; Takizawa, Y.; Kumagai, H.; Fujiwara, R.; Yagi, T.; Inokuchi, H. Biochim. Biophys. Acta 1981, 636, 136. (29) Bard, A. J.; Faulkner, L. R. Electrochemical Methods Fundamentals and Applications; John Wiley & Sons: New York, 1980.
AnalytlcaIChemistty, Vol. 66, No. 22, November 15, 1994
3875
0.80
-
0.70
-
I
l ' l ' l ' l ' l ' l -0.20 -0.30 -0.40 -0.50 -0.60 -0.70 E(V) vs AgCllAg
I
Flgure 2. Muttisweep cyclic voltammogram of cytochrome q (2.6
KM)in pH 7.00 phosphate buffer. Working electrode, static mercury drop electrode. Scan rate, 0.1 VIS. (a) First sweep;(b) second sweep; (c) 10th sweep.
0.60 20.00
0.00
of cytochrome c3 both on the electrode surface and in the bulk solution. According to eq 1, the slopes of the straight lines in zone 111are K = 2.69 X 105n1312ADo1/2v112, and the intercepts of these straight lines are b = n22F2A~I'*/4RT. By plotting K versus v1I2and b versus v, tViO straight lines were obtained. The number of electrons per mole, nl and n2, calculated from the slopes of the two straight lines, were 2.9 and 0.9, respectively. Values of DO = 1.0 X 10" cm2/s, r* = 1.1 X lo-" mol/cm2,and A = 0.018 cm2wereusedinthecalculation. The surface area of the electrode was obtained as follows. First, the weight of 100 drops of mercury was measured. Second, the surface area of the electrode was calculated by assuming the shape of the electrode to be spherical. It is interesting to note that the number of electrons transferred in the electrode reaction of adsorbed cytochrome c3 on the mercury electrode surface is about 1. (This result will also be supported by the radiolabeling experiments discussed subsequently.) Meanwhile, the number of electrons transferred for the diffusion-controlled process was larger than 1, and both reduction and oxidation peaks were observed. This suggested that the heterogeneous electron transfer of cytochrome c3 in bulk solution was not inhibited by the adsorbed cytochrome c3. 2. Influence of Applied Potential. A low concentration of cytochrome c3, under conditions where the surface electron transfer process was dominant, was used in the potential dependence studies. The multisweep cyclic voltammogram of cytochrome c3 solution (Figure 2) was obtained on a single drop of mercury by continuously cycling the applied potential. The scan started 5 s after the drop was dispensed. One pair of peaks appeared at a potential around -0.52 V on the first sweep. In the subsequent sweeps, the peakcurrents decreased until they disappeared. The result suggested that a conformational change of cytochrome c3, Le., reorientation of the heme groups, occurred on the surface of the mercury electrode during the multiple sweep. The adsorbed cytochrome c3 in the new conformation was neither electroactive nor conductive, so that the electron transfer between the electrode and cytochrome c3 in bulk solution was blocked. Cytochrome c3 can adsorb on the electrode spontaneously at open circuit. The reaction commenced as soon as the electrode was immersed in the cytochrome c3 solution and reached a plateau at about 20 s. This was established by the 3876
AnalyticalChemistry, Vol. 66, No. 22, November 15, 1994
60.00
40.00 Tlme(s)
Figure 3. Dependence of the peak current on the equilibrium time. Working electrode, static mercury drop electrode. Cytochrome q (56 pM) in 0.03 M, pH 7.0 phosphate buffer. Scan rate, 0.5 V i s .
2
-
Q
0'41 A
0.20
%.--
0.00
I , 0.00
-0.40
-0.80
-1.20
E(V) vs AgCIIAg Flgure 4. Dependenceof the peak current on the incubationpotential. Working electrode, static mercury drop electrode. Scan rate, 1 V/s.
experiment in which cyclic voltammograms of cytochrome c3 were obtained after the electrode was incubated in cytochrome c3 solution at open circuit for different lengths of time. The peak currents versus the incubation time are plotted in Figure 3, There are two experimental variables which could influence the conformational changes: time and potential. If the incubation time is the cause of the conformational change, a decrease in peak current should be observed after the adsorption reached a plateau. The curve in Figure 3 suggested that this is not the case. The influence of applied potential was investigated in the following experiment. The cyclic voltammograms of cytochrome c3 were obtained on an electrode which was incubated for 2 min in cytochrome c3 solution at different potentials. The peak currents were measured and plotted versus the applied potentials. The results, presented in Figure 4, suggest that the conformational transformation of cytochrome c3 is defined by the potential applied to the electrode. There were three stableconformations of cytochrome c3 on the mercury surface corresponding to different applied potentials. Conformation A was obtained when the incubation potential was higher than -0.4 V (the potential of zero charge for mercury in phosphate buffer30). The adsorbed protein was electroactive. Conformation B was
1
o.60
1250nA
-0.3V 0.00 0.00
-0.7V I
1
40.00
80.00
I
T 120.00
I
I 160.00
' 200.00
Time (s) Flgure5. Incubationtimedependentpeak current profile of cytochrome q (2.6 pM)at different incubationpotentials. Working electrode, static mercury drop electrode. Scan rate, 1 V l s .
formed when a moderate incubation potential between -0.4 and -0.8 V was applied to the mercury electrode. The adsorbed protein became electroinactive and nonconductive. Conformation C was generated when the incubation potential was lower than -0.8 V. A new pair of peaks emerged at lower potentials than that of the cytochrome c3 in conformation A. The adsorbed layer became electroactive again. The conformational transformations were irreversible and could occur only from conformation A to B, B to C, or A to C. A time profile of the peak current at different incubation potentials is given in Figure 5 . Three typical incubation potentials were used to form the threedifferent conformations of cytochrome c3 on the mercury surface. A fresh drop of mercury was used for each data point. From the time profile, it was established that the conformational transformation of cytochrome c3 on a mercury surface occurred in a time scale of minutes. At -0.3 V, cytochrome c3 kept the same conformation as that at open circuit. Though these experimental observations can be interpreted as the conformational transformation of the adsorbed cytochrome c3 on a mercury surface, it is also possible that the phenomena observed were caused by the variable adsorbability of cytochrome c3 at different potentials or by the impurities in the protein solution. For this reason, the film transfer experiments were performed to support the hypothesis of conformational transformation. 3. Film Transfer Experiment. The voltammograms of the adsorbed cytochrome c3 film on a mercury surface in phosphate buffer are given in Figure 6. The experimental steps were as follows. First, the mercury electrode was soaked in cytochrome c3 solution for 4 min, and then the electrode was rinsed and transferred to phosphate buffer solution. Second, one cyclic voltammogram of the adsorbed cytochrome c3 was obtained after the cytochrome c3-adsorbed electrode was incubated at open circuit (-0.05 V) (Figure 6A), -0.7 V (Figure 6B), and -1.2 V (Figure 5C). As the same mercury electrode was used to obtain the three cyclic voltammograms and the adsorbed cytochrome c3 was the only source of electroactive species, the phenomena observed could only be caused by a property change of the adsorbed film. The results suggested that the adsorbed
I -0.20
I
I -0.40
I
I
I
-0.60
I -0.80
1
I
-1.oo
E(V) vs AgCIIAg Figure 6. Cyclic voltammogram of cytochrome q adsorbed on a mercurysurface. Scan rate, 1 V/s. Incubationtime;4 min. Incubation potentials: (A) open circuit, (0.05 V; (B) -0.7 V; (C) -1.2 V.
cytochrome c3 molecules remained on the electrode surface at different potentials but that their conformation was changed by the applied potential. In this experiment, the conformation of cytochrome c3 on the electrode surface transformed from conformation A to B and to C. Since the surface concentration of cytochrome c3 was fixed, the area under the peak reflects the number of electrons involved in the electrode reaction. The different peak areas in Figures 6 A and C implied that there were more electrons involved in the reduction of the film in form C than in form A. The conformational transformation was also established by an i m m u n ~ a s s a y . ~Two ~ monoclonal antibodies from mouse, which were selected for an epitope in the vicinity of heme 1, were used to detect the conformational changes of cytochrome c3 on the electrode surface. Conformational variations of cytochrome c3 would presumably change the apparent affinities of cytochrome c3 to the antibodies. Thus, the amount of antibody bound to the mercury drop covered with cytochrome c3 will be altered and can be detected by enzyme linked immunosorbent assay (ELISA). An enzymelabeled secondary antibody (goat anti-mouse) was used to detect the immobilized monoclonal antibodies of cytochrome c3 from a mouse. It was established that the antibodies bind strongly to the A form of the cytochrome c3 film on the mercury surface. A substantial decrease in apparent affinity of the antibody for the film in forms B and C was observed. This suggests that the conformations of the adsorbed cytochrome c3 in the B and C forms are different from that of the A form. For better understanding of the role that adsorbed cytochrome c3 plays in the subsequent electron transfer of diffusing species, film transfer experiments were performed by putting the mercury electrode with adsorbed film back into the cytochrome c3 solution. When a mercury electrode covered
(30) Bard, A. J. Encyclopedia of Electrochemistry of the Elements, IX, Marcel Dekker, Inc.: New York, 1982; Part A.
Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
3877
0.2pA
I -0.20
I
I
I
I
1
-0.40 -0.60 E(V) vs AgCllAg
Flgure 7. Cyclicvoltammogramof cytochrome c3(2.6@l)on a mercury electrode after incubation at -0.7 V. (a) The electrode was incubated in cytochrome c3 at -0.7 V for 4 min. (b) The mercury drop was enlarged after (a). Scan rate, 0.1 V/s.
with the B form of cytochrome c3 was transferred into cytochrome c3 solution, the electron transfer of the soluble cytochrome c3 was blocked by the adsorbed protein molecules (Figure 7a). The redox peaks of cytochrome c3 appeared again (Figure 7b) when the surface area of the same drop of mercury was enlarged by dispensing additional mercury from the static hanging mercury drop electrode. The peak area was proportional to the area of the newly exposed mercury surface. When an electrode modified with the film of the C form of cytochrome c3 was transferred to cytochrome c3 solution, the cyclic voltammogram in Figure 8b was obtained. Presumably, the electrode was covered with a grid of the C form of cytochrome c3. If the heterogeneous electron transfer of cytochrome c3 were mediated by the adsorbed cytochrome c3, then the cathodic peak of the cyclic voltammogram in Figure 8b should have appeared at the peak potential of the adsorbed cytochrome c3 (-0.58 V), and the mediation reaction would have significantly increased and decreased the peak height of the cathodic and anodic peaks, respectively. If the C form of cytochrome c3 can exchange the electrons only with the electrode but is incapable of exchanging electrons with soluble cytochrome c3 or the reaction is very slow, Le., the heterogeneous electron transfer is blocked by the C form of cytochrome c3, then the cyclic voltammogram obtained on an electrode covered by the C form of cytochrome c3 will contain two peaks. The first peak is from the reduction of the soluble cytochrome c3 on mercury surface which is not covered by the C form of cytochrome c3. The second peak is from the reduction of the adsorbed cytochrome c3. In Figure 8b, the cathodic peak observed at -0.54 V could be attributed to the reduction of cytochrome c3 from the solution (see Figure 8c). The shoulder at -0.58 V could be attributed to the reduction of the adsorbed cytochrome c3 in the C form (see Figure 8a). When Figure 8a is subtracted from Figure 8b, the resulting cyclic voltammogram will be similar to theone in Figure 8c, but with a smaller peakcurrent. Therefore, the cyclic voltammogram (Figure 8b) of soluble cytochrome c3 on a mercury electrode modified by the C form 3878
Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
-0.40
-0.60
-0.80
E(V) vs AgCllAg Flgure 8. Cyclic vottammogramsof cytochrome c3. (a)CV of adsorbed cytochrome c3 (C form) on a mercury surface in phosphate buffer.(b) CV of cytochrome q (20 pM) by using the same electrode as In (a). ( c )CV of cytochrome q (20 rM) on a fresh mercury surface. Scan rate, 0.4 VIS.
of cytochrome c3 can be attributed to the combination of the electron transfer of the adsorbed cytochrome c3 on the electrode surface and the electron transfer of the soluble cytochrome c3 on the surface which was not covered by the C form of cytochrome c3. Because of limited number of sites available for the diffusion-controlled electron transfer, and because these sites would be blocked by the B form of cytochrome c3 during the reduction process, the peak separation in Figure 8b more closely resembles a surface electron transfer process rather than a diffusion-controlled electron transfer process. The experimental results suggested that there was no mediated heterogeneous electron transfer observed in the time scale of the cyclic voltammetry (seconds). Thus, it is concluded that the heterogeneous electron transfer of the soluble cytochrome c3 is inhibited by both the B and the C forms of the adsorbed cytochrome c3 film on the mercury surface. In the case of the A form, we have proved that the adsorbed film does not inhibit the heterogeneous electron transfer of soluble cytochrome c3. If the adsorption is essential to the heterogeneous electron transfer, a smoothyl increasing curve should be obtained when the increasing phase of the curve of Figure 3 is extrapolated to the origin. When this is done, a positive intercept is obtained, suggesting that adsorbed cytochrome c3 in the A form does not promote heterogeneous electron transfer. 4. Radiolabeling Experiment. In order to measure the number of electrons involved in the electrode reaction of the three forms of cytochrome c3 on the mercury surface, the surface coverage of cytochrome c3 on the electrode was measured by using nonelectrochemical methods. For this reason, cytochrome c3 was labeled by attaching lZ5I primarily
Table 1. Surface Coverage of Cytochrome
on Hg at Dmerent
Potentials
incubation potential (vs AgCl/Ag)
conformation A
B C
open circuit (0.05 V)
-0.7 v -1.2 v
surface (mol/cm ) X 10"
1.10 f 0.05 0.94 f 0.2 0.67 f 0.1
charge (nC/cm2)
n
855 f 180 1.2 f 0.3
2144
* 160
3.3 f 0.5
I 0.00
I
I -0.20
I
I
I
-0.40
I -0.60
I
I
-0.80
E(V) vs AgCllAg
0.00
-0.20
-0.40
-0.60
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E(V) vs AgCVAg Figure 9. Muitisweep cycllc voltammogram of cytochrome q (69 pM) on a glassy carbon electrode. Scan rate, 0.1 VIS. The first and the 20th scans.
to the tyrosine residue. It was proved by experiments that the labeling did not change the electrochemical behavior of cytochrome c3. The surface coverage of cytochrome c3 on the mercury electrode was established by mweasuring the radioactivity of a mercury drop after treatment under various conditions. The experimental procedures were as follows. The mercury drop was soaked in the solution of radiolabeled cytochrome c3 for 5 min and then transferred into phosphate buffer solution. After being transferred, the drop was incubated in buffer solution either at open circuit, -0.7, or -1.2 V, respectively, for 5 min. The radioactivity of each drop was measured after the cyclic voltammograms of the drops had been taken. The charge was calculated from the area under the peak of the cyclic voltammogram. From the total charge and the surface coverage of the protein on the electrode, the number of electrons transferred in the electrode reaction was calculated. The results are summarized in Table 1. The number of electrons transferred in the reduction of the A form of cytochrome c3 was 1.2, which was consistent with previous results (0.9). The results indicated that cytochrome c3 molecules are spontaneously adsorbed on the mercury surface in form A. In this conformation, only one heme is accessible for the electrode reaction, and apparently no intramolecular electron transfer occurred in the adsorbed protein molecules on the time scale of seconds. The results also suggested that there should beat least three hemes involved in the electrode reaction of cytochrome c3 in conformation C. Which hemes participate in the electron transfer reaction is still a question to be answered. 11. Electrochemistry of Cytochrome c3 on a Glassy Carbon Electrode. Figure 9 shows a multisweep cyclic voltammogram of cytochrome c3 on a glassy carbon electrode. There was no change in the voltammograms after 20 scans. Evidently, no potential-triggered conformational transformation occurred on this surface. The surface coverage of cytochrome c3 on glassy carbon, measured by the radiolabeling method, was
Figure 10. Square wave voltammogramsof cytochrome q. Working electrode, glassy carbon. Amplitude, 10 mV. Frequency, 60 Hz. (a) Cytochrome q In the solution (69 pM). (b) Cytochrome q on the surface of the electrode.
1.1 X 10-l mol/cm2 (the geometric area of the electrode was used in the calculation). Since the real surface area of glassy carbon is always higher than the geometric area, the real surface coverage of adsorbed cytochrome c3 on glassy carbon surface could be somewhat lower. Only very small peaks were observed in cyclic voltammograms from the film transfer experiment. In square wave voltammetry experiments, two peaks appeared in the square wave voltammogram of a cytochrome c3 solution (Figure 1Oa). Film transfer experiments established that peak I corresponds to the adsorbed cytochrome c3, whereas peak I1 results from electrolysis of bulk cytochrome c3. The dependence of the peak potential of peak I indicated that the redox reaction of adsorbed cytochrome c3 on the glassy carbon electrode was reversible at frequencies up to 280 Hz. It was suggested that, due to the adsorption, the heme group can emerge from the protein backbone. The peak near -0.3 V can be attributed to the electrode reaction of a heme group in close contact with the electrode surface.32 However, the halfwidth of the square wave voltammogram in Figure 10b is too big for a single electron transfer process.33 The results suggest that the redox reaction of the adsorbed cytochrome c3 on a glassy carbon electrode is a consecutive multielectron transfer process with very small potential intervals. 111. Catalysis of Reduction of 0 2 by Cytochrome c3 Film. The cyclic voltammograms of oxygen in phosphate buffer on bare and cytochrome c3-modified glassy carbon electrodes are shown in Figure 1 1. The current for reduction of oxygen on cytochrome c3-modified electrodes was higher and the peak appeared at more positive potentials than that on a bare glassy carbon electrode. These results suggested that the reduction of oxygen is catalyzed by adsorbed cytochrome c3. However, as illustrated in Figure 12, the film continuously loses its catalytic activity with successive scans in oxygen-containing solutions. After being used to catalytically reduce oxygen in cytochrome c3solution, the electrode was put into a cytochrome (32) Manjaoui, A.; Haladjian, J.; Bianco, P.Electrochim. Acta 1990, 35, 177. (33) Lovric, M.; Komorsky-Lovric, S. J. Electroanal. Chem. 1988, 248, 239.
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I
I
0.5pA
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I
I
I
0.00
I -0.20
I
I
I
-0.40 E(V) vs AgCllAg
I -0.60
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Flgure 11. Cyclic voltammograms of oxygen. (a) CV of oxygen-free phosphate buffer on bare glassy carbon electrode. (b) CV of airsaturated phosphate buffer on bare glassy carbon electrode. (c) CV of air-saturated phosphate buffer on cytochrome q-modified electrode. Scan rate, 0.2 VIS.
-0.40
I
I
I
-0.20
-0.80
Figure 12. Cycllcvoltammogams of oxygen on cytochrome m o d i f i e d glassy carbon electrode. Scan rate, 0.1 V/s. The solution was purged with oxygen before each of the cyclic voltammograms to prevent depletion of oxygen near the electrode surface. The cyclic voltammograms were taken at (0) the first scan; (+) after five scans; and (0)after 25 scans.
I
I
I
I
-0.60
-0.80
E(V) v s AgCllAg
I E(V) vs AgCllAg
I -0.40
Flgure 13. Cyclic voltammogram of cytochrome q (69 MM)on glassy carbon electrode. Scan rate, 0.1 VIS. Working electrode: (a) freshly polished glassy carbon electrode; (b) glassy carbon electrode covered wlth cytochrome q which was degraded by the reduction product of oxygen.
0.00
0.00
I
-0.20
0.00
I
I
-0.40
I
I -0.60
I -0.80
E(V) vs AgCllAg Figure 14. Muitisweep cyclic voltammogram of cytochrome g (69 $4) on gold electrode. Scan rate, 0.1 VIS. (-)The first scan. (): The 12th scan.
+
c3 solution free of oxygen. The electrode reaction of cytochrome c3 on this electrode (Figure 13b) was no longer the same as that on a newly polished glassy carbon electrode (Figure 13a). This observation suggested that the adsorbed cytochrome c3 molecules on the glassy carbon surface were degraded by the oxygen reduction process. IV. Electrochemistry of Cytochrome q on a Gold Electrode. The cyclic voltammogram of cytochrome c3 on a gold electrode is presented in Figure 14. There was virtually no change after a number of scans. No detectable potential-dependent conformational transformation occurred on the gold surface. The surface coverage of cytochrome c3 on the gold electrode measured by radiolabeling method was 4.0 X 10-l2 mol/cm2 (the calculation was again based on the geometric surface area). Since the surface coverage was so low, no peak was found in the cyclic voltammogram of a cytochrome c3-modified electrode in buffer solution. A broad peak was observed in 3880
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-0.20
-0.30
-0.40
-0.50
-0.60
-0.70
E(V) vs AgCllAg Figure 15. Square wave voltammogram of cytochrome q. (-)Gold electrode in cytochrome q solution (69 pM). (-): Cytochrome q-adsorbed electrode in buffer solution.
+
the square wave voltammogram of the adsorbed cytochrome c3 on the gold surface (Figure 15). The peak appeared at the same potential as that of cytochrome c3 in solution. These results suggested that cytochrome c3 still maintains the same conformation as in aqueous solution after adsorption on a
gold surface. This conclusion is consistent with the previously reported electroreflectance results. l8
CONCLUSIONS Cytochrome c3 is strongly adsorbed on a mercury surface in three conformations. The change of conformation is irreversible and can be triggered by the applied potential. Conformation A. Cytochrome c3 molecules are spontaneously adsorbed on the surface of mercury in this conformation at a potential which is more positive than the potential of zero charge. The electrode reaction in this conformation is a oneelectron process. Unlike the cytochrome c3 in bulk solution, no intra- or intermolecular electron transfer occurs. The adsorbed cytochrome c3 in the A form has no effect on the heterogeneous electron transfer of the soluble cytochrome c3. Conformation B. When a potential between -0.4 and -0.8 V is applied to a mercury electrode, the conformation of cytochrome c3 changes to form B. The cytochrome c3 in this form is electroinactive and nonconductive. The heterogeneous electron transfer of soluble cytochrome c3 is blocked by the protein film. Conformation C. This conformation starts to form when a potential lower than -0.9 V is applied to a mercury electrode. The cytochrome c3 in this conformation is reduced at more negative potential than the soluble species. The number of electrons involved in the electrode reaction is 3.3, measured by the experiment combining radiolabeling and cyclic voltammetry. Even though the adsorbed cytochrome c3 in this conformation is electroactive, this adsorbed film does not mediate the heterogeneous electron transfer of the soluble species. The electron transfer reaction of cytochrome c3 in
bulk solution can take place only through the pores of the adsorbed film. The adsorption of cytochrome c3 on glassy carbon and gold surface is weaker than that on mercury. No potentialdependent conformational transformation appears on either electrode. On the gold surface, the redox potential of adsorbed cytochrome c3 is the same as that of soluble cytochrome c3. This implies that the adsorbed molecules maintain a conformation similar to that of thecytochrome c3 moleculein solution. On a glassy carbon surface, the conformation of adsorbed cytochrome c3 changes, such that the redox potential of adsorbed species is 200 mV higher than that of the soluble species. The behavior of cytochrome c3 on the electrode surface depends on the electrode material. The electron transfer of cytochrome c3 in solution is essentially decided by the nature of the adsorbed layer.
ACKNOWLEDGMENT This research was supported in part by the Office of Naval Research Grant NOOO14-90-5-1226 and by a U.S.-Japan Cooperative Research Grant from the National Science Foundation, I N T 91 16747. The generous donation of cytochrome c3 by Michael A. Cusanowich is gratefully acknowledged. Scientific Parentage of the Author. Dongbo Zhang, Ph.D. under G. S.Wilson, Ph.D. under A. M. Hartley, Ph.D. under J. J. Lingane, Ph.D. under I. M. Kolthoff. Received for review March 29, 1994. 1994." 0
Accepted August 18,
Abstract published in Aduance ACS Abstracts. October 1, 1994.
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