Langmuir 1991, 7, 3190-3196
3190
Spectroelectrochemical Study of the Redox Reaction Mechanism of Cytochrome c at a Gold Electrode in a Neutral Solution in the Presence of 4,4’-Bipyridyl as a Surface Modifier Takamasa Sagara, Hikaru Murakami, Satoshi Igarashi, Hisakuni Sato, and Katsumi Niki* Electrochemistry Laboratory, Department of Physical Chemistry, Yokohama National University, Tokiwadai, Hodogaya-ku, Yokohama 240, Japan Received August 29, 1990. I n Final Form: February 18, 1991 The redox reaction of cytochrome c at a gold electrode in the presence of 4,4’-bipyridyl (4-bpy) as a surface modifier was investigated spectroelectrochemically in a neutral aqueous solution. Two types of the redox processes of cytochrome c were studied in detail. One (process I) is the diffusion-controlled reaction of cytochrome c from the solution phase to the electrode surface covered by the adsorbed layer of 4-bpy, at the same formal potential as the native cytochrome c. The other (process 11) involves both the reaction of cytochrome c coadsorbed with 4-bpy on the gold electrode surface and the mediated reduction of cytochrome c in the solution phase. It was found that the formal potential of cytochrome c coadsorbed with 4-bpy became more positive as the concentration of 4-bpy increased. This is probably due to the fact that the coadsorbed 4-bpy acts to reduce the degree of unfolding of adsorbed cytochrome c. Electroreflectance spectra of each of the two processes were very different. It was found that the electronic structure of the cytochrome c coadsorbed with 4-bpy was different from that of the native cytochrome c.
Introduction It is well-known that proteins in general adsorb strongly on electrode surfaces from aqueous solution and that the adsorbed film plays an important role in the electrode reactions of these proteins from the bulk of the so1ution.l Usually, electron transfer proteins adsorbed directly on bare electrode surfaces do not exhibit a reversible electron transfer reaction and, in many cases, inhibit the redox reaction of the proteins in the solution phase.2 To establish reversible electron transfer reactions at the electrode interface, surface modifiers are often used. A variety of surface modifiers have been proposed for cytochrome c,3 and these modifiers are often referred to as electrontransfer promoters. The redox reaction mechanism of electron transfer proteins in the presence of surface modifiers, however, is still in question. Little is known about the structure of cytochrome c when it is interacting with a surface modifier. The reversible redox reaction of cytochrome c at the formal potential of the native protein is observed in the presence of surface modifiers, typically 4-pyridyl derivative^.^-^ In previous papers, we proposed a model of the interface based on the interaction between cytochrome c and bipyridyl derivatives constructed from ac impedance and electroreflectance (ER) measurementsa7p8
* To whom correspondence should
be addressed.
(1) Scheller, F. Bioelectrochem. Eioenerg. 1977, 4, 490. (2) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. In Comprehensiue Treatise ofElectrochemistry 10; Srinivasan, S., Chizmadzhev,Y. A., Bock-
ris, J. O’M., Conway, E. B., Yeager, E., Eds.; Plenum Press: New York,
1985; p 297. (3) Allen, P. M.; Hill, H. A. 0.; Walton, N. J. J. Electroanal. Chem. Interfacial Electrochem. 1984, 178, 69. (4) Eddowes, M. J.; Hill, H. A. 0. J . Chem. Soc., Chem. Commun. 1977, I l l . (5) Eddowes, M. J.; Hill, H. A. 0. J . Am. Chem. SOC.1979, 101,4461. (6) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Chem. Soc., Chem. Commun. 1982, 1032. (7) Sagara,T.; Niwa, K.; Sone, A.; Hinnen, C.;Niki, K. Langmuir 1990, 6. 254.
0743-7463/91/2407-3190$02.50/0
In the case of 4,4’-bipyridyl(I-bpy), 4-bpy coadsorbs with cytochrome c on a gold electrode surface, and cytochrome c from the solution phase reacts at the adsorption layer of 4-bpy. Cytochrome c coadsorbed with 4-bpy on a gold electrode exhibits a formal potential of -165 mV versus a silver/silver chloride electrode (Ag/AgCl in a saturated potassium chloride solution). This formal potential is 200 mV more negative than that of native cytochrome c but is 220 mV more positive than that of cytochrome c adsorbed on a bare gold electrode surface without surface modifiers. These observations suggest that the presence of 4-bpy reduces the extent that cytochrome c adsorbed on the electrode surface unfolds. The above mentioned reaction mechanism model has been also supported by the following results: (i) SERS (surface enhanced Raman scattering) signals of both cytochrome c and 4-bpy are observed at a silver electrode, and the coadsorption of cytochrome c and 4-bpy on the metal electrode surface is e ~ i d e n c e d(ii) ; ~ 4-bpy preadsorbed on a gold electrode is displaced by cytochrome c in a 4-bpy-free cytochrome c solution.10 I t has been suggested that the strength of interaction of cytochrome c with the 4-bpy-modified electrode should be different from that with the bis(4-pyridyl) disulfide (4-PyS) modified electrode. Cytochrome c in solution is immobilized on the adsorption layer of 4-PyS, and the redox reaction is observed at the formal potential of the native cytochrome c . 7 ~As ~ described in previous papers,’Jo the interaction between 4-PyS and cytochromec is stronger than that between 4-bpy and cytochrome c. It has been verified that the binding of 4-PyS to cytochrome c is much stronger than that of 4-bpy to cytochrome c.ll (8) Hinnen, C.; Niki, K. J. Electroanal. Chem. Interfacial Electrochem. 1989,264, 157. (9) Fan, K.-J.; Satake, I.; Ueda, K.; Akutsu, H.; Niki, K. In Redox Chemistry and Interfacial Eehauior of Biological Molecules; Dryhurst, G., Niki, K., Eds.; Plenum Press: New York, 1987; p 125. (10)Niwa, K.; Furukawa, M.: Niki, K. J . Electroanal. Chem. Interfacial Electrochem. 1988, 275, 245.
0 1991 American Chemical Society
Langmuir, Vol. 7, No. 12,1991 3191
Cytochrome c at a Gold Electrode
Hill and his colleagues proposed a standardized model of the interface where a surface modifier facilitates the electrode reaction of cytochrome c.3J2 In this model, they emphasize that one of the most important roles of the surface modifier is to provide an electrode surface that allowsthe protein to adsorb in a way so that the prosthetic group is relatively close to the electrode surface. On application of this model to the case of 4-bpy, there should be strong attractive interaction between the lysineenriched domain of cytochrome c and the nitrogen atom of 4-bpy adsorbed on the electrode surface to establish a favorable orientation of cytochrome c on the electrode surface.12 The aim of the present study is to further investigate the role of modifiers from the viewpoint of structure at a molecular level. An in situ spectroelectrochemical technique is very useful in elucidating the relationship between the structure and the electron transfer function of cytochrome c simultaneously. SERS is a very powerful tool to investigate the surface phenomena at the electrode surface, and its usefulness has been demonstrated by Cotton et al.13J4 Hildebrandt and Stockburger have demonstrated the spin-state change of cytochrome c on a silver electrode surface by using SERRS (surface enhanced resonance Raman scattering spectroscopy) measurem e n t ~ .It~ is ~ expected that in situ UV-vis reflectance spectroscopy (in other words, electroreflectance spectroscopy, ER)16will provide structural information in the form of an electronic spectrum. This method was first applied by Hinnen and Niki to investigate the redox reaction of cytochrome c in the presence of modifiers, although they did not discuss the structure of the cytochrome c molecule responsible for the electron transfer processes in detai1.8J7 In the present paper, the influence of 4-bpy on the electron transfer reaction of cytochromec is further studied by using measurements of dc and ac voltammetric responses, ER spectroscopy, and ER voltammetry.
Experimental Section Horse heart cytochrome c (type VI from Sigma ChemicalCo.) was purified chromatographically as described earlier.’* 4,4’Bipyridyl (Cbpy,reagent grade from Tokyo KaseiChemicalCo.) was used without further purification. Gold disk and flag (polycrystalline, >99.99 %) purchased from Tanaka Noble Metal Co., were used, respectively, for the ER and conventional voltammetric measurements. Water was purified to 16 MQcm through Milli Q (Millipore Co.). All other chemicals were reagent grade and used without further purification. For voltammetric measurements, a two-compartment water-jacketed cell was used, where the reference electrode compartment was separated by a Vycor glass rod. For the ER measurements, a specially designed spectroelectrochemical cell with a quartz optical window was used. The counter electrode was platinum wire. The electrode potentials were measuredagainst a silver/silver chlorideelectrode in a saturated potassium chloride solution. In the present paper, all potentials were referred to this electrode (+197 mVvs NHE). Prior to each measurement, the gold electrode was pretreated by using the same procedure described previ~usly.~ The instrumentation for the ER measurements was the same as described (11)Sagara, T.;Satake, I.; Murakami, H.; Akutsu, H.; Niki, K. J.Electroanal. Chem. Interfacial Electrochem. 1991, 331, 285. (12) Armstrong, F. A.; Hill, H. A. 0.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407. (13)Cotton, T. M. In Spectroscopy of Surfaces, Vol. 15; Clark, R. J. H.,Hester, R. H., Eds.; Wiley: New York, 1988; p 91. (14) Cotton, T. M.; Schultz, S. G.; Van Duyne, R. P. J.Am. Chem. SOC. 1980, 102, 7960. (15) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710. (16) Sagara, T.; Igarashi, S.; Sato, H.; Niki, K. Langmuir 1991,7,1005. (17) Hinnen,C.; Pars0ns.R.; Niki, K.J.ElectroanaL Chem.Znterfacial Electrochem. 1983, 147, 329. (18) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. Methods Enzymol. 1978,330, 128.
a 1 4 0 p ~cyt c l O m M 4-blpy
- 0.5
0.0
E vs
E w ~ aI
0.4 V
Figure 1. Cyclic voltammograms of 140 pM cytochrome c at a gold electrode in a 30 mM phosphate buffer solution containing 4-bpy at pH 7.0, with a sweep rate of 50 mV s-l. The concentrations of 4-bpy are (a) 10mM and (b) 30 pM. The voltammograms were measured 10 min after the immersion of the electrode in the electrolyte solutions. The notations of the peak potentials are given in Figure l a (see text).
elsewhere.16 The electrolyte was a 30 mM phosphate buffer solution at pH 7.0. The electrolyte, which contains neither cytochromec nor 4-bpy, is denoted as “basesolution”in the present paper. All of the measurements were carried out at 25 2 O C in anaerobic conditions. In ac voltammetry, pseudocapacitance and resistance were measured under an assumption that the capacitance and resistance are in series.
*
Results and Discussion Voltammetric Measurements. Figure 1 shows the dc voltammograms of the gold electrode in 140 pM cytochrome c solution at pH 7.0 containing 10 mM (Figure la) and 30 gM (Figure lb) 4-bpy. Two seta of anodic and cathodic peaks are observed in the dc voltammograms and two pseudocapacitance peaks in ac voltammograms. Both peaks represent the redox reaction of cytochrome c as confirmed by ER measurement^.^ The same phenomenon was observed at a gold electrode in the solution containing various concentrations of cytochrome c and 10 mM 4-bpy, as reported previ~usly.~ We define the redox reaction on the positive side as process I and that on the negative side as process 11. For each of the two processesat various concentrations of 4-bpy with a constant concentration of cytochrome c (140 pM), the following potentials were measured: EJI or 11) and E,(I or 11), the anodic and cathodic peak potentials in a dcvo1ta”ogram at a sweep rate of 50 mV s-l, respectively, and &(I or 11) and &(I or II), the peak potentials of pseudocapacitance and resistance in an ac voltammogram, respectively. The potentials characterizing process I remain constant, independent of the time passed after the immersion of a freshly prepared gold electrode into the electrolyte solution. On the other hand, the potentials characterizing process I1 vary slowly, and it takes about 2 h to reach steady-state values. The changes in the potentials were less than 60 mV. Hereafter, the potentials characterizing both processes I and I1 measured at 10 min after the immersion of a gold electrode in the solution are discussed. While the electrode was soaking for 10 min before the measurements, the electrode was maintained at an opencircuit potential and Ar gas was bubbled gently through the solution to prevent contamination by oxygen.
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3192 Langmuir, Vol. 7, No. 12, 1991
-0.4
-a3
I -0.2
1 -01
I
OD
E vs EAg/Aga 1
”
I 0.1
I
02
Figure 2. Plots of the potentials, Eo’(Z),E,,JII),Epa(II), Ec(II), and ER(II),of cytochrome c at a gold electrode in the presence of various concentrations of 4-bpy obtained by dc and ac volta”etries. The supporting electrolyte was 30 mM phosphate buffer at pH 7.0. The concentration of cytochrome c was 140
(4-bipy)
I
mM
Figure 3. Plot of pseudocapacitance at Ec(I1) as a function of the concentration of 4-bpy: frequency,30 Hz; ac amplitude, 7.1 mV. The concentration of cytochrome c was 140 rM,and the supporting electrolyte was 30 mM phosphate buffer solution. The capacitance is represented with an arbitrary scale.
fiM.
For process I, the relationship among the characteristic potentials can be written as (1) [Epc(I)+ Epa(I)]/2 = Ec(I) = ER(I) regardless of the concentration of 4-bpy, the sweep rate in dc voltammetry, and the frequency in ac voltammetry. Meanwhile, the peak current is proportional to the square root of the sweep rate, and the cathodic and anodic peak heights are equal. These facts reveal that process I is the quasi-reversible electrode reaction of cytochrome c in the solution phase. The value of eq 1can be equated with the formal potential of cytochrome c responsible for process I, EO’(1). The value of EO’(1) is independent of the concentration of 4-bpy and is very near to the formal potential of native cytochrome c. For process 11, at higher concentrations of 4-bpy ( 1 2 mM), the characteristics for the quasi-reversible reaction of adsorbed species are observed, as the peak current is nearly proportional to the sweep rate. Cytochrome c adsorbed on the electrode surface is responsible for process 11. At low concentrations of 4-bpy (I1mM), however, the asymmetry between anodic and cathodic processes of process I1 appears clearly as exemplified by the dc voltammogram of Figure lb. The height of the cathodic peak of process I1 is larger than that of the anodic peak. Since process I1 takes place always at more negative potential than Eo’(I),electron transfer to ferricytochrome c in the solution phase is thermodynamically downhill and thus possible from the electrode through the adsorbed layer of ferrocytochrome c, which is reduced in process 11. That is, the cathodic process in processI1 involves both reduction of cytochrome c adsorbed on the electrode surface and a mediated reduction of cytochrome c in the solution phase. On the other hand, electron transfer from ferrocytochrome c in the solution phase to ferricytochrome c adsorbed on the electrode surface, which is oxidized in process 11, is thermodynamically uphill. That is, the anodic process of process I1 is the reoxidation of cytochrome c adsorbed on the electrode surface. This would account for the difference between the anodic and cathodic peak heights in process 11. Figure 2 shows the dependence of both EO’(1) and the potentials characterizing process I1 on the concentration of 4-bpy where the concentration of cytochrome c is maintained constant at 140 p M . Ec(I1) and E~(11)were measured at 30 Hz. The value of EO’(1)is constant in the
concentration range between 10 mM and 50 pM of 4-bpy. At the concentrations of 4-bpy less than 10 pM, however, the peak at Eo’(I)is not observable by using either dc or ac voltammetric techniques. All the potentials characterizing process I1 shift to negative potentials as the concentration of 4-bpy decreases. The extent of the shift of Epc(II)is greater than that of Epa(11). The peak separation, Epa(II)- Epc(II),is greater than 150 mV at the sweep rate of 50 mV s-l at low concentrations of 4-bpy. Although the quantitative analysis of the lineshape of the voltammogram of process I1 is difficult a t present, the present results support the above mentioned interpretation of the response to process 11, i.e. the cathodic process in process I1 involves both the reduction of adsorbed cytochrome c and the reduction of cytochrome c in the solution phase mediated by cytochrome c adsorbed on the electrode surface. Thus, (Bpa(11) Epc(11))/2is not equal to the formal potential of cytochrome c adsorbed on the electrode surface. Frequency dependence of ac voltammogram was also measured at various concentrations of 4-bpy. When ac frequency was larger than 30 Hz, no frequency dependence of both Ec(I1) and ER(II)was observed. Because the contribution of cytochrome c the species in the solution phase to ac response is usually small at higher frequencies, Ec(I1)andER(I1) at 30 Hz might correspond to the formal potential of cytochrome c adsorbed on the electrode surface, Eo’(II). The plot of EO’(I1) and Epc(II)against 4-bpy concentrations are convergent to -0.38 V at lower concentrations of 4-bpy. The potential -0.38 V is the formal potential of cytochrome c adsorbed directly on the bare gold electrode ~ u r f a c e .At ~ lower concentrations of 4-bpy, the redox property of cytochrome c adsorbed on the electrode surface is more different from that of the native protein. This difference is probably due to an unfolding of the cytochrome c molecule, since it is known that the formal potentials of heme proteins become more negative as the exposure of the heme to the solution increases.lg The presence of 4-bpy coadsorbed with cytochrome c on the electrode surface serves to reduce the degree of unfolding of coadsorbed cytochrome c. Figure 3 shows the plot of pseudocapacitance at 30 Hz, from which the baseline values were subtracted, at Ec(I1) as a function of the concentration of 4-bpy. The pseudoca-
+
(19)Stellwagen, E. Nature 1978, 275, 73.
Cytochrome c at a Gold Electrode
0.01
0.01
0.1
1 10 (4-bipy] I mM
Figure 4. Plot of peak current of the redox process of cytochrome c at Ea’(I)as a function of the concentration of 4-bpy at a sweep rate of 50 mV s-1. The current is represented with an arbitrary
scale.
pacitance decreases sharply with the decrease of 4-bpy concentration from 10to 0.5 mM. The pseudocapacitance values are the lowest when the concentration of 4-bpy is 0.5 mM. The pseudocapacitance increases gradually with the decrease in 4-bpy concentration from 0.5 mM to 3 pM. Since the adsorption of cytochrome c and 4-bpy is competitive, the fraction of surface coverage with cytochrome c increases with a decrease in the concentration of 4-bpy. The degree of unfolding in cytochrome c increases with a decrease in 4-bpy concentration as discussed above. Although the amount of adsorbed cytochrome c becomes larger as the concentration of 4-bpy decreases, the pseudocapacitance exhibits a minimum. This is prabably due to a reduction of the electron transfer rate because of the greater extent of the unfolding of cytochrome c at lower concentrations of 4-bpy. The increase in the pseudocapacitance from 0.5 mM to 3 pM originates mainly with the increase of the amount of adsorbed cytochrome c. The result of Figure 3suggeststhat not onlythe structure but also the electron transfer kinetics are changed by the unfolding of cytochrome c. Therefore, it is likely that the change in electronic structure is related to the unfolding of cytochrome c coadsorbed with 4-bpy on the gold electrode surface. Figure 4 shows the peak height in dc voltammetry at EO’(1) as a function of 4-bpy concentration at the sweep rate of 50 mV s-l. The peak current decreases as the 4-bpy concentration decreases, and eventually, process I is hardly observed (see Figure 2). Since the electron transfer reaction of cytochrome c from the bulk of the solution through cytochrome c coadsorbed with 4-bpy is thermodynamically uphill, the electron transfer is only available a t the part of the electrode covered with 4-bpy, unless electron transfer via pinhole takes place without accompanying the unfolding of cytochrome c. Therefore, the results in Figure 4 are probably due to the decrement of effective area of the adsorbed layer of 4-bpy for the electrode reaction of cytochrome c in the solution phase with the decrease in the 4-bpy concentration. At lower concentrations of 4-bpy, the area on the electrode surface available for process I would be dispersed and fragmented. Amatore and his colleagues reported the way to analyze the dc voltammogram in such a case.*“ Armstrong and his colleagues suggested that the radial diffusion layer rather (20) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. Interfacial Electrochem. 1983, 147, 39.
Langmuir, Vol. 7,No.12,1991 3193
than a linear diffusion is formed and results in a sigmoidal waveshape if the distributed electrochemicallyactive sites behave as ultramicroelectrodes.z1~z2 However,the features of dc voltammograms, i.e. peak separation, peak current, and half-wave potential, are not in accordance with the criteria derived by Amatore and his colleagues.20 Even at low concentrations of 4-bpy, the lineshape is “peak-shaped” rather than “sigmoidal”. The voltammetric waveshapes at EO’(1) observed in the present experimental conditions are not similar to the curve simulated by Armstrong and his colleagues. Since EO’(1) is very near to the formal potential of the native cytochromec, the structure of cytochrome c reacting at the surface of the adsorbed layer of 4-bpy would be the same as the native protein. Structures of cytochrome c participating in both processes I and I1 are examined by ER spectroscopic measurements. ER Spectroscopic Measurements. Gold Electrode in a Mixture of Cytochrome c and 4-Bpy. The ER spectrum corresponds to the difference spectra between the reduced and oxidized species, which are responsible for the redox reaction at the electrode surface.16 By comparing the ER spectrum at the electrode surface with the difference spectrum in the homogeneous phase, it is possible to investigate the electronic structural change due to the adsorption of the electroactive species. Figure 5 shows the ER spectra of the gold electrode in (5 pM cytochrome c 5 mM 4-bpy) solution at EO’(1) = +80 mV (Figure 5a) and at EO’(I1) = -180 mV (Figure 5b). This value of EO’(I1) was determined from the peak potential in the ER voltammogram. The waveshapes of the spectra appear greatly different from each other, especially in the Soret band region. Figure 6 shows the difference spectrum of cytochrome c in the homogeneous phase. Since the reduced form of cytochrome c is obtained by a dithionite reduction, the spectra a t wavelengths shorter than 390 nm are altered upward due to the absorption of dithionite. In both parts a and b of Figure 5, a negative peak at 406-408 nm can be observed. The negative peak is less significant in Figure 6. The positive peak at 424 nm at EO’(1) is greater than the negative peak a t 406 nm for Eo’(I),while the positive peak at 424 nm for EO’(I1) is smaller than the negative peak at 408 nm for EO’(11). Note that the wavelengths of the positive peaks in both parts a and b of Figure 5 are about 6 nm longer than those in Figure 6. Both the negative peaks and the isosbestic pointa in Figure 5 in the Soret region also are shifted slightly toward red. The two positive peaks at the 01 and ,9 bands in both parts a and b of Figure 5 are similar to those in Figure 6. It is also notable that the spectral waveshape in the wavelength region between the Soret and ,9 bands in Figure 5b is bent significantly upward. Both parts a and b of Figure 5 are different from Figure 6, and it is obvious that the electronic structure of cytochrome c coadsorbed with 4-bpy is much different from that of the native cytochrome c responsible for the redox reduction at EO’(1). Figure 7 shows the ER spectrum of a gold electrode, on which cytochrome c is adsorbed, in the base solution. The important features of Figure 7 are (i) the negative peak in the Soret band region is much larger than the positive peak and (ii) the wavelength of the positive peak in the Soret band region is shifted 11 nm toward red.
+
(21)Armstrong, F. A.; Bond, A. M.; Hill, H. A. 0.; Psalti, I. S. M.; Zoski, C. G. J. Phys. Chem. 1989, 93,6485. (22) Armstrong, F. A.; Bond, A. M.; Hill, H. A. 0.;Oliver, B. N.; Psalti, I. S. M.J . Am. Chem. SOC.1989,111, 9185.
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3194 Langmuir, Vol. 7, No. 12, 1991
408
-180 m V
-1.5X10-‘
300
450
I
600
wavelength / n m
w a v e l e n g t h / nm
Figure 5. ER spectra of a gold electrode in (5 pM cytochrome c + 5 mM 4-bpy) in 30 mM phosphate buffer solution at pH 7.0, with a modulation of 100 mV and 21 Hz,and sweep rate of 12.5 nm min-l: (a, left) electrode potential +80 mV; (b, right) -180 mV. 1
0.5
P
2
9
-0.5
420
360
480
540
600
wavelength / nm
Figure 6. Difference absorption spectrum of cytochrome c (spectrumof ferricytochromec subtracted from ferrocytochrome c) in 30 mM phosphate buffer solution, pH 7.0.
300
400 wavelength I
500
600
nm
Figure 7. ER spectrum (solid line) of gold electrode at -324 mV, on which cytochromec was adsorbed by using a film transfer method, in the base solution: modulation of 100 mV and 14.24 Hz, and sweep rate of 25 nm mi+. The spectrum of Figure 6 is superimposed by using a broken line. By comparison of Figure 5b with Figure 7, it is revealed that the electronic structure of cytochrome c coadsorbed with 4-bpy is different from that of the native cytochrome c, but the structural change is not as significant as cytochrome c adsorbed directly on a bare gold electrode surface. Gold Electrode Modified by 4-Bpy in Cytochrome c Solution. The gold electrode modified by 4-bpy was prepared by a film transfer procedure from a 10 mM 4-bpy
solution. The modified electrode was then immersed in cytochrome c solution (10 pM), and the ER spectra were measured. In this case, EO’(1) = +80 mV and EO’(I1) = -240 mV were observed in the ER voltammogram. The appearance of process I1 suggests that the preadsorbed 4-bpy was partially desorbed from the electrode surface and that cytochrome c was coadsorbed with 4-bpy. Because the electrode is initially not in equilibrium with the electrolyte solution due to adsorption-dporption dynamics,the voltammetric response is dependent on time, as reported elsewhere.1° Thus, the electrode potential, where ER spectra were measured, was set exactly a t the peak potentials in the ER voltammograms, which were measured just before the measurements of ER spectra were taken. Figure 8 shows the ER spectra at the above mentioned gold electrode at both Eo’(I) and EO’(11). Figure 8a represents the real (solid line) and imaginary parts (broken line) of the ER signal at EO’(1). The spectrum of the real part is similar to the difference spectrum of Figure 6, while that of the imaginary part is inverted in terms of the sign of the signal. The response of the imaginary part is greater than that of the real part. These observations indicate that the contribution to the ER spectra at Eo’(I)from cytochrome c in the solution phase is rather significant. That is, both the concentration of ferricytochrome c and ferrocytochrome c as products of the electrode reaction near the electrode surface at Eo‘(I)are also modulated by the potential modulation. When the redox process of the surface species is not rapid enough to follow the potential modulation, the response of the imaginary part, which possesses the opposite sign to that of the real part, should be observed as calculated elsewhere.16 At Eo’(I), the concentration profile within the diffusion layer, is also altered by the potential modulation, which gives rise to the additional phase shift of -45O. Therefore, the imaginary part of the ER response becomes larger than the real part and has an opposite sign. That is, the diffusion process is dominant at EO’(1). Figure 8b represents the real (solid line) and imaginary (broken line) parts of the ER signal at EO’(I1). The ER signal arising from the redox reaction of cytochrome c is hardly detectable in the imaginary part.
Cytochrome c a t a Gold Electrode
Langmuir, Vol. 7, No. 12, 1991 3195
* I 5
300
400 500 wavelength I nm
600
b
1 t
300
.
I
I
I
400 500 wavelength I nm
600
Figure 8. ER spectra of gold electrode, which was premodified with 4-bpy, in 10 pM cytochrome c solution in 30 mM phosphate buffer at pH 7.0. The modification with 4-bpy was carried out by a film transfer method from a 10 mM 4-bpy solution: modulation of 100mV and 14.24 Hz;sweep rate of 25 nm min-l. Electrode potentials were as follows: (a) 80 mV @''(I)), solid, real part; broken, imaginary part; (b) -240 mV (Eo'(II)),solid, real part; broken, imaginary part.
These facts reveal that the ER response at E"'(1) corresponds to the reaction of cytochrome c in the solution phase and that at E"'(I1) corresponds mainly to the redox process of cytochrome c coadsorbed with 4-bpy. The real part of Figure 8b clearly resembles Figure 5b and Figure 7. I t is evident that the electronic structure of cytochrome c coadsorbed with 4-bpy is different from that of the native protein. Both the real and imaginary parts (the imaginary part should be examined after inverting the sign) in Figure 8a are very similar to the difference spectra represented in Figure 6. The contribution to the ER response at E"'(1) from cytochrome c adsorbed at the electrode surface is significantly low when compared to that from cytochrome c in the diffusion layer. The cytochrome c in the solution phase reacts on the adsorption layer of 4-bpy without unfolding. Role of 4-Bpy as a Surface Modifier. The binding of 4-bpy to cytochrome c is much weaker than that of 4-PyS." The results of binding measurements suggest that the interaction between the adsorption layer of 4-bpy with cytochrome c is not as strong as that of 4-PyS. In other words, the immobilized layer of cytochrome c is not formed on the adsorption layer of 4-bpy. The role of 4-bpy in the electrode reaction of cytochrome c in the solution phase is to provide a surface that prevents the unfolding of cytochrome c from the solution phase and facilitates the electron transfer without strong interaction. On the adsorption layer of 4-bpy, the diffusion-controlled electron transfer reaction of cytochrome c from the bulk of the solution, process I, takes place. In process I, both ferriand ferrocytochrome c maintain the native structure of
cytochrome c at thesurface of the adsorption layer of 4-bpy as revealed by the ER spectra. The role of 4-bpy as a surface modifier is revealed as different from that of 4-PyS. In the case of 4-PyS, on the other hand, cytochrome c is strongly immobilized on the adsorption layer. Through this immobilized layer, the electron transfer between the electrode and the protein in the solution takes place. The adsorption of 4-bpy to the electrode surface is also weaker than that of 4-PyS, and cytochromec coadsorbswith 4-bpy. The redox reaction of cytochrome c coadsorbed with 4-bpy in process I1 takes place at a formal potential at least 200 mV more negative than that of the native protein but more positive than the formal potentid of cytochrome c adsorbed on a bare gold electrode surface. The cytochrome c coadsorbed with 4-bpy is unfolded and has a different electronic structure from the native protein, but the degree of structural change is suppressed by the presence of coadsorbed 4-bpy as revealed by the ER spectra. Although 4-bpy cannot prevent the adsorption of cytochrome c in direct contact with the gold surface, 4-bpy plays a role in reducing the degree of unfolding of the coadsorbed cytochrome c. Structure of Cytochrome c. In the present study, ER spectroscopic technique provided us a detailed understanding of the electrode interface, especially from the viewpoint of the electronic structure of the protein. One remarkable feature highlighted by this spectroelectrochemical study is that the difference between the ER spectra and the spectrum of the native cytochrome c becomes larger as the formal potential of cytochrome c shifts toward negative. This poses an intriguing question: How is the change is the structure of cytochrome c related to the change in both the electronic spectrum and the value of EO'? Hildebrandt and Stockburger determined the spin-state change of cytochrome c on silver sol by using surface Cyenhanced Raman scattering (SERS) spe~troscopy:~3 tochrome c molecules, which are adsorbed on the surface of colloidal silver particles, are partially converted from a low spin state to a high spin state. Our SERS measurement revealed that ferrocytochrome c adsorbed directly on a silver electrode surface is mainly a high spin state.g In contrast, coadsorption of 4-bpy with cytochrome c gives rise to the partial transformation from the high spin state to the low spin stateagAfter our finding, Hildebrandt and Stockburger used SERRS to investigate in detail the states of cytochrome c adsorbed directly on a silver electrode surface as a function of electrode potential.15 Their conclusionwas that cytochrome c in the native state is in a six-coordinated low spin state (6cLS)and that another state exists on the silver electrode surface which involves two kinds of redox couples, one in a fivecoordinated high spin state (5cHS)with a formal potential of -0.31 V and the other in a 6cLS state with a formal potential of -0.41 V. The advantage of the ER technique is that the selective observation of the species participating in the electron transfer process at a given electrode potential is possible. Although the separate observations of the spectra of the oxidized and reduced species are not available now, what follows is a brief examination of the correspondence of the change in ER spectra to the spin state change by applying the two procedures. Procedure I. Shift toward red the spectral curve of the native ferrocytochrome c and, from the resulting curve, subtract the spectral curve of the native ferricytochrome C.
(23)Hildebrandt, P.; Stockburger, M. J.Phys. Chem. 1986,90,6017.
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Procedure 11. Multiply 0.5 to the spectral curve of the native ferrocytochrome c and, from the resulting curve, subtract the spectral curve of the native ferricytochrome C.
Procedure I results in a difference spectrum where both the negative and positive peaks in the Soret band region are enhanced and both the positive peak and the isosbestic point shift toward red. The spectrum produced by procedure I is similar to that in Figure 5b. It is known that among C-type cytochromes, the electronic spectra of cytochromes c’, which are in a 5cHS state, have a larger separation between the peaks of Soret bands of the ferri and ferro form.24 One possible explanation of the red shift in the ER spectrum of cytochrome c coadsorbed with 4-bpy is the partial change of the spin state from 6cLS to 5cHS, as suggested by our previous SERS measurements.9 (24) Meyer, T. E.; Kamen, M. D. Adu. Protein Chem. 1982, 35, 105.
Sagara et al.
Procedure I1 gives rise to spectra very similar to that in Figure 7 and the real part of Figure 8b. However, it is difficult to explain the meaning of the decrease in intensity of only the ferro form from the standpoint of the electronic structure of cytochrome c. To add detail to the discussion, separate measurements of the spectra of ferri- and ferrocytochrome c are required. For this purpose, experiments are now underway.
Acknowledgment. We are grateful to the Ministry of Education, Science and Culture, Japan, for the financial support of a Grant-in-Aid of the Scientific Research on Priority Area “Dynamic Interactions and Electric Properties of Macromolecular Complexes (No. 63612004 for K.N.)”. We wish to thank Dr. H. Nakagawa (Nakagawa Applied Research Co.) for his technical assistance in instrumentation. Registry No. 4-bpy, 553-26-4; cytochrome c, 9007-43-6.
