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Clay-Bridged Electron Transfer between Cytochrome P450cam and Electrode
Biochemical and Biophysical Research Communications 268, 740 –744 (2000) doi:10.1006/bbrc.2000.2200, available online at http://www.idealibrary.com on
Clay-Bridged Electron Transfer between Cytochrome P450 cam and Electrode Chenghong Lei,* ,† ,1 Ulla Wollenberger,* Christiane Jung,‡ ,2 and Frieder W. Scheller* ,2 *Analytical Biochemistry, University of Potsdam, c/o Im Biotechnologiepark, D-14943 Luckenwalde, Germany; †Fudan University, Shanghai, People’s Republic of China; and ‡Max Delbru¨ck Center for Molecular Medicine Berlin-Buch, Robert-Ro¨ssle-Strasse 10, D-13092 Berlin, Germany
Received January 4, 2000
We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450 cam on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s ⴚ1 with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450 cam. The formal potential of adsorbed P450 cam is ⴚ139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450 cam in solution. UV–VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450 cam in solution. However, P450 cam adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential. © 2000 Academic Press
Key Words: cytochrome P450 cam; electrochemistry; electron transfer.
Cytochromes P450 (P450) are heme enzymes which catalyze the monoxygenation of organic compounds involved in the metabolic processes of many animal and Abbreviations used: P450 cam, cytochrome P450 cam from Pseudomonas putida (CYP101); P420, denatured form of P450; Pdx, putidaredoxin; PdR, putidaredoxin reductase; FTIR, Fourier transform infrared spectroscopy; SMC, sodium montmorillonite colloid; GCE, glassy carbon electrode; Clay/GCE, clay-modified membrane on the electrode surface; PTFE, polytetrafluoroethylene; CV, cyclic voltammograms; P450/Clay/GCE, clay-modified electrode with adsorbed P450 cam. 1 Present address: Department of Chemical Engineering, Virginia Commonwealth University, Richmond, VA 23284-3028. 2 To whom correspondence may be addressed. (PD Dr. Jung) Fax: 0049-30-94063329, E-mail: [email protected]; or (Prof. Dr. Scheller) Fax: 0049-3371-681324, E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
human organs, plants, and bacteria (1). Among them, cytochrome P450 cam (P450 cam) is a camphor-inducible monoxygenase which was discovered in Pseudomonas putida (2). In catalyzing the hydroxylation of (1R)camphor to give exclusively 5-exohydroxy-camphor, electrons are transferred from NADH to P450 cam through the combined action of putidaredoxin (Pdx) and putidaredoxin reductase (PdR) (2). The negatively charged group of Asp38 in Pdx forms a salt bridge with Arg112 in the positively charged patch (Arg112 and Arg109, Arg79) in P450 cam to shuttle electrons between Pdx and P450 cam (3). For a potential application of cytochromes P450 in bioreactors or biosensors it might be suitable to substitute the biological electron delivery and transport system by artificial ones like electrochemical (4) or photochemical systems (5, 6). Both methods have been applied on cytochrome P450 since the early years of the P450 research. Several laboratories have used various methods to reduce electrochemically cytochromes P450 (7–10). The electron transfer rate constants, reported so far, are rather low and fall in the range of values lower than 0.01 s ⫺1. We applied a carbon electrode modified by the clay sodium montmorillonite, which has been reported to facilitate electron transfer processes (11), and observed a very fast heterogenous electron transfer to P450 cam with rate constants (5–150 s ⫺1) very similar to those reported for the biological P450 cam-Pdx system. This fast electron transfer might be caused by the optimal spacial orientation of P450 cam on the electrode surface induced by the negatively charged clay. MATERIALS AND METHODS Protein and clay. Cytochrome P450 cam from Pseudomonas putida was expressed in Escherichia coli strain TB1, isolated, purified and depleted from camphor as previously described (12). The protein stock solution used for the electrochemical studies was 40 M of substrate-free P450 cam in 50 mM Tris/HCl buffer, pH 7.4, 5% glycerol (v/v). For the infrared studies the stock solution was 1 mM of
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substrate-free P450 cam in 100 mM D 2O potassium phosphate buffer (corresponding to pH 7). Sodium montmorillonite colloid (SMC) was prepared from the purified montmorillonite powder (from Zhejiang province, China) in a similar way as previously described (12) to get a clay content of 1.25 g/liter. 120 l of SMC were mixed with 5 l of colloidal Pt and then diluted 4.4 times with water to obtain a pretreated sodium montmorillonite colloid (PSMC) (13). For the infrared studies the clay colloid (12.5 g/liter) was dialyzed against D 2O. Electrochemistry. Aliquots (5 l) of PSMC were deposited on a glassy carbon electrode (GCE, 3 mm in diameter) and allowed to dry at 20°C to form a clay-modified membrane on the electrode surface (Clay/GCE). 3 l of the 40 M substrate-free P450 cam stock solution in Tris-HCl buffer were added onto Clay/GCE and then put in a refrigerator overnight. In this period P450 cam was spontaneously adsorbed onto the clay-modified membrane. The modified electrode was rinsed with the working buffer (33 mM potassium phosphate buffer, pH 7) prior to use and thus P450/Clay/GCE was obtained. For comparative electrochemical studies hemin was dissolved in 1 M NaOH and then diluted with phosphate buffer to adjust a pH of 7.5 and a concentration of 0.5 mM. From this solution hemin was adsorbed onto the clay-modified electrode when the modified electrode was inserted into the hemin solution overnight. All electrochemical experiments were carried out with the Autolab PSTAT10 (Eco Chemie, Netherland). The electrochemical cell was equipped with a Ag/AgCl (1.0 M KCl) reference electrode and a platinum wire auxiliary electrode. Prior to measuring, 1 ml of the 33 mM potassium phosphate working buffer (pH 7) was carefully bubbled with argon for at least 20 min to remove oxygen. Electron transfer rates k were estimated by the formula k ⫽ m n F/RT published by Laviron for surface processes (14), where m ⫽ 1/3 for a voltammogram peak separation of 81 mV and n ⫽ 1 for the number of transfered electrons were used. , F, R and T are the scan rate, the Faraday constant, the gas constant and the temperature, respectively. FT-infrared studies. Infrared spectra were recorded using the Bruker IFS66 Fourier transform infrared spectrometer (Bruker, Germany). The IR sample solution was prepared with the D 2O clay reagent to get a final concentration of 350 M P450 cam and 8.12 g/liter clay. For studies in the absence of clay pure D 2O in a corresponding volume was used instead of the clay suspension. The P450 sample was placed in a demountable cell with CaF 2 windows separated by a PTFE spacer. For measurements of the amide I band of oxidized P450 cam a 23 m spacer and for the CO stretch signals of the carbon monoxide complex of P450 cam a 100 m spacer were taken. The preparation of the CO-P450 cam complex, the FTIR data treatment and baseline correction were the same as described previously (15, 16). UV/VIS absorption spectra were recorded on the UV-2101PC spectrometer (Shimadzu, Japan). For all the IR and UV/VIS experiments, the reference always comprised the same buffer in the absence and the presence of clay as the sample. All the experiments were carried out at 20 ⫾ 1°C. The P420 content has been determined from the Soret band difference spectrum between the carbon monoxide minus the reduced complex (18). The absorbance coefficients of 91 mM ⫺1 cm ⫺1 for the P450 peak at 446 nm, of 111 mM ⫺1 cm ⫺1 for the P420 peak at 420 nm and of 41 mM ⫺1 cm ⫺1 for the difference between the CO complex and the zero line at 420 nm have been used.
RESULTS Figure 1A shows cyclic voltammograms (CV) of P450/Clay/GCE at different scan rates. In the scan rate range from 2 to 12 V s ⫺1 , P450/Clay/GCE had almost symmetrical CV shapes, equal reduction and oxidation peak heights with a formal potential of ⫺361 mV (vs. Ag/AgCl); ⫺139 mV (vs NHE). The
FIG. 1. Cyclic voltammogram of cytochrome P450 cam adsorbed on the glassy carbon electrode modified with sodium montmorillonite at different scan rates (A) and plot of the peak current vs the scan rate (B); 33 mM potassium phosphate buffer, pH 7.
redox peak separation (⌬E P ) is 77 mV at 2 V s ⫺1 and 84 mV at 12 V s ⫺1 . The anodic as well as the cathodic peak current show a linear relationship with the scan rate (Fig. 1B). At a scan rate as low as 0.4 V s ⫺1 , only the reduction peak was found whilst almost no oxidation peak was visible (Fig. 1A, inset). From the integration of the anodic peak in the CV of P450/ Clay/GCE, the apparent surface coverage of the electroactive P450 cam was calculated to be in the order of 3.54 ⫻ 10 ⫺12 mol cm ⫺2 . Assuming a complete coverage of the electrode surface with a monolayer of the globular P450 cam , (triangle size 57 ⫻ 60 Å (16)) about 35% of the adsorbed P450 cam displayed the electroactivity.
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FIG. 2. Cyclic voltammogram of cytochrome P450 cam adsorbed on the glassy carbon electrode modified with sodium montmorillonite (A) in the presence of CO (dashed line) and replacement of CO by argon (dotted line) and (B) in the presence of metyrapone, 1.67 mM (dashed line); compared to the voltammogram in absence of an iron ligand (solid line); 33 mM potassium phosphate buffer, pH 7.
After the working buffer was bubbled with CO for 10 min, the formal potential of P450/Clay/GCE was positively shifted by 80 mV (Fig. 2A). When CO is replaced by argon the formal redox potential shifts back again to the original value. Addition of metyrapone induces a positive shift of the formal potential by 86 mV (Fig. 2B). Both CO and metyrapone bind specifically as a sixth ligand to the heme iron of P450 cam indicating that the observed electrochemical response does indeed originate from P450 cam . Addition of (1R)-camphor (500 M) to the working buffer did not shift the formal potential but increased the anodic peak area of the cyclic voltammogram by about 15% (voltammogram not shown). For an additional check we recorded the cyclic voltammogram of hemin adsorbed on the clay-modified electrode under the same experimental conditions as for the P450
study. Figure 3 shows that the cyclic voltammogram for hemin looks different from P450 cam . The formal potential of adsorbed hemin is ⫺330 mV (vs. Ag/ AgCl) and the redox peak separation (⌬E P ) is not constant in the scan rate range from 0.4 V s ⫺1 (89 mV) to 12 V s ⫺1 (151 mV). These differences to the behavior of the adsorbed P450 supports the aforementioned conclusion that the electrochemical response is really induced by the adsorbed P450. As noted above a formal redox potential of ⫺361 mV (vs Ag/AgCl) has been derived for P450 cam from the cyclic voltammogram in Fig. 1A. This value strongly deviates from the redox potential observed for substrate-free P450 cam in solution (⫺525 mV vs Ag/AgCl, ⫺303 mV (vs NHE) (19)). Therefore, the effect of clay on the P450 structure has been studied by spectroscopic methods. Figure 4A shows the UVVIS spectrum of oxidized P450 cam and its CO complex in the presence and in the absence of clay. There is no significant effect of clay on the spectrum of the oxidized protein neglecting the absorbance increase in the UV region due to light scattering. In the CO complex a slight increase of the P420 content by 9% is recognized compared to the sample without clay, which has a P420 content of 5%. The infrared spectra of the amide I band (Fig. 4B), which represents the secondary structure of P450 (15), do not show any difference whether clay is present or not. The second derivative of the amide I band is almost identical in both cases. Also the CO ligand stretch vibration at around 1940 and 1963 cm ⫺1 which is very sensitive to the structural changes at the enzyme active site (16) did not reveal any significant difference as shown in Fig. 4C.
FIG. 3. Cyclic voltammogram of hemin adsorbed on the glassy carbon electrode modified with sodium montmorillonite (dashed line) compared to the voltammogram of cytochrome P450 cam (solid line), scan rate of 10 V s ⫺1; 33 mM potassium phosphate buffer, pH 7.
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DISCUSSION
FIG. 4. Electronic absorption spectra of oxidized P450 cam in the absence (dashed line) and in the presence of 0.53 g/liter clay (dotted line) and that of reduced P450 cam complex with CO in the absence (solid line) and in the presence of 0.53 g/liter clay (dash/dot mixed line), 3.2 M P450 cam, 29 mM potassium phosphate buffer, pH 7.1 (A) and amide I⬘ infrared spectra and their second derivative, which are manually separated along the ordinate (B), and the CO ligand stretch vibration band, which has been area-normalized (C) in the absence (solid line) and in the presence of 8.12 g/liter clay (dotted line); 350 M P450 cam in 35 mM D 2O potassium phosphate buffer, corresponding to pH 7.
Although some electrochemical aspects of P450 were reported more than 20 years ago by Scheller et al. (20, 21), the direct, nonpromoted electrochemistry of P450 is rather difficult to obtain with nonmodified electrodes. The enzyme passivates the electrode and is denatured. The first direct electrochemistry by using freshly purified P450 cam in solution at the edge-plane graphite electrode was reported by Kazlauskaite et al. (7). Rusling’s group has found that P450 cam incorporated in lipid or polyelectrolyte film displayed the welldefined redox behavior from its heme Fe(II/III) (8). More recently, Lo et al. (10) demonstrated cyclic voltammograms on an edge-plane graphite electrode for various P450 cam mutants. In all the mentioned studies the electron transfer rate, so far reported, is rather slow with rate constants lower than 0.01 s ⫺1. Our studies with the clay modified carbon electrode gives clear indication for a reversible and fast electron transfer between the electrode and the adsorbed P450. The observed linearity of the peak current with the scan rate is characteristic for an surface process and allows to apply the theory by Laviron (14) for estimation of the rate constant. The rate constant is in a range of 5 to 152 s ⫺1 for the scan rates of 0.4 and 12 V s ⫺1, respectively. In the natural system the rate for the transfer of the first electron from putidaredoxin to P450 cam has been reported to lie in the range between 27 and 84 s ⫺1 (19, 22). This similarity suggests that the negatively charged clay (11) obviously mimics the electrostatics of the natural redox partner putidaredoxin and may hold the P450 in a productive orientation. In this orientation the active site of the adsorbed P450 cam is still accessible for iron ligands like CO and dioxygen. The positive shift of the formal potential by 80 mV due to CO binding resembles the expected value of 96 mV which we have estimated from the CO binding constant for substrate-free P450 cam of 5.7 ⫻ 10 4 mol ⫺1 (23) using the Nernst equation. Binding of residual dioxygen seems also to be taken place indicated by the missing oxidation peak in the voltammogram at slow scan rates as it has been similarly observed by Lo et al. (10). Not only the small ligands can entry the heme pocket of the adsorbed P450. Even the large metyrapone molecule has access to the active site of the adsorbed P450 indicated by the expected positive shift of the redox potential. The still unresolved problem is the shift of the formal potential of the adsorbed P450 cam by approximately 164 mV to more positive values compared to the substratefree P450 cam in solution. Positive shifts of the redox potential are generally observed when water is excluded from the heme pocket as in the case of camphor binding (24). Because the clay itself has no effect on the P450 structure in solution, we suggest that the adsorp-
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tion process leads to a partial dehydration of the P450 cam structure. This conclusion would also explain that the addition of camphor has only a minor effect on the electrochemical response. ACKNOWLEDGMENTS Dr. Chenghong Lei deeply appreciates the support by the Alexander von Humboldt Foundation (Bonn, Germany) which has awarded him a research fellowship. He also thanks Professor Fugen Zhou at Tongji University in Shanghai for offering the purified montmorillonite powder. We thank Dr. Jian Chen (University of Potsdam) for performing the hemin experiments. We gratefully acknowledge the financial support of this work by the Deutsche Forschungsgemeinschaft (INK 16/A1-1 and SK 35/3-1) and the Fonds der Chemischen Industrie. We thank Dr. W.-H. Schunck for critical reading of the manuscript.
REFERENCES 1. Lewis, D. F. V. (1996) Cytochromes P450 —Structure, Function and Mechanism, Taylor & Francis Ltd, London. 2. Gunsalus, I. C., Tyson, C. A., Tsai, R., and Lipscomb, J. D. (1971) Chem.-Biol. Interact. 4, 75–78. 3. Roitberg, A. E., Holden, M. J., Mayhew, M. P., Kurnikov, I. V., Beratan, D. N., and Vilker, V. L. (1998) J. Am. Chem. Soc. 120, 8927– 8932. 4. Estabrook, R. W., Faulkner, K. M., Shet, M. S., and Fisher, C. W. (1996) Adv. Enzymol. 272, 44 –51. 5. Hintz, M. J., and Peterson, J. A. (1980) J. Biol. Chem. 255, 7317–7325. 6. Contzen, J., and Jung, C. (1999) Biochemistry 49, 16253–16260. 7. Kazlauskaite, J., Westlake, A. C. G., Wong, L. L., and Hill, H. A. O. (1996) J. Chem. Soc. Chem. Commun. 2189 –2190.
8. Zhang, Z., Nassar, A. F., Lu, Z., Schenkman, J. B., and Rusling, J. F. (1997) J. Chem. Soc. Faraday Trans. 93, 1769 –1714. 9. Lvov, Y. M., Lu, Z., Schenkman, J. B., Zu, X., and Rusling, J. F. (1998) J. Am. Chem. Soc. 120, 4073– 4080. 10. Lo, K. K.-W. , Wong, L.-L., and Hill, H. A. O. (1999) FEBS Lett. 451, 342–346. 11. Ege, D., Ghosh, P. K., White, J. R., Equey, J.-F., and Bard, A. J. (1985) J. Am. Chem. Soc. 107, 5644 –5652. 12. Jung, C., Hui Bon Hoa, G., Schro¨der, K-L., Simon, M., and Doucet, J. P. (1992) Biochemistry 31, 12855–12862. 13. Lei, C., Lisdat, F., Wollenberger, U., and Scheller, F. W. (1999) Electroanalysis 11, 274 –276. 14. Laviron, E. (1979) J. Electroanal. Chem. 101, 19 –28. 15. Mouro, C., Jung, C., Bondon, A., and Simonneaux, G. (1997) Biochemistry 36, 8125– 8134. 16. Jung, C., Ristau, O., Schulze, H., and Sligar, S. G. (1996) Eur. J. Biochem. 235, 660 – 669. 17. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122–16130. 18. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370 –2378. 19. Fisher, M. T., and Sligar, S. G. (1985) J. Am. Chem. Soc. 107, 5018 –5019. 20. Scheller, F. W., Renneberg, R., Strnad, G., Pommerening, K., and Mohr, P. (1977) Bioelectrochem. Bioenerg. 4, 500 –507. 21. Dryhurst, G., Kadish, K. M., Scheller, F. W., and Renneberg, R. (1982) Biological Electrochemistry, Vol. 1, pp. 398 –521. Academic Press, New York. 22. Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J. D., Spencer, R. G. S., and Simonneaux, G. (1999) FEBS Lett. 455, 302–306. 23. Kato, M., Makino, R., and Iizuka, T. (1995) Biochim. Biophys. Acta 1246, 178 –184. 24. Poulos, T. L., Finzel, B. C., and Howard, A. J. (1986) Biochemistry 25, 5314 –5322.
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Clay-Bridged Electron Transfer between Cytochrome P450 cam and Electrode Chenghong Lei,* ,† ,1 Ulla Wollenberger,* Christiane Jung,‡ ,2 and Frieder W. Scheller* ,2 *Analytical Biochemistry, University of Potsdam, c/o Im Biotechnologiepark, D-14943 Luckenwalde, Germany; †Fudan University, Shanghai, People’s Republic of China; and ‡Max Delbru¨ck Center for Molecular Medicine Berlin-Buch, Robert-Ro¨ssle-Strasse 10, D-13092 Berlin, Germany
Received January 4, 2000
We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450 cam on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s ⴚ1 with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450 cam. The formal potential of adsorbed P450 cam is ⴚ139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450 cam in solution. UV–VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450 cam in solution. However, P450 cam adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential. © 2000 Academic Press
Key Words: cytochrome P450 cam; electrochemistry; electron transfer.
Cytochromes P450 (P450) are heme enzymes which catalyze the monoxygenation of organic compounds involved in the metabolic processes of many animal and Abbreviations used: P450 cam, cytochrome P450 cam from Pseudomonas putida (CYP101); P420, denatured form of P450; Pdx, putidaredoxin; PdR, putidaredoxin reductase; FTIR, Fourier transform infrared spectroscopy; SMC, sodium montmorillonite colloid; GCE, glassy carbon electrode; Clay/GCE, clay-modified membrane on the electrode surface; PTFE, polytetrafluoroethylene; CV, cyclic voltammograms; P450/Clay/GCE, clay-modified electrode with adsorbed P450 cam. 1 Present address: Department of Chemical Engineering, Virginia Commonwealth University, Richmond, VA 23284-3028. 2 To whom correspondence may be addressed. (PD Dr. Jung) Fax: 0049-30-94063329, E-mail: [email protected]; or (Prof. Dr. Scheller) Fax: 0049-3371-681324, E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
human organs, plants, and bacteria (1). Among them, cytochrome P450 cam (P450 cam) is a camphor-inducible monoxygenase which was discovered in Pseudomonas putida (2). In catalyzing the hydroxylation of (1R)camphor to give exclusively 5-exohydroxy-camphor, electrons are transferred from NADH to P450 cam through the combined action of putidaredoxin (Pdx) and putidaredoxin reductase (PdR) (2). The negatively charged group of Asp38 in Pdx forms a salt bridge with Arg112 in the positively charged patch (Arg112 and Arg109, Arg79) in P450 cam to shuttle electrons between Pdx and P450 cam (3). For a potential application of cytochromes P450 in bioreactors or biosensors it might be suitable to substitute the biological electron delivery and transport system by artificial ones like electrochemical (4) or photochemical systems (5, 6). Both methods have been applied on cytochrome P450 since the early years of the P450 research. Several laboratories have used various methods to reduce electrochemically cytochromes P450 (7–10). The electron transfer rate constants, reported so far, are rather low and fall in the range of values lower than 0.01 s ⫺1. We applied a carbon electrode modified by the clay sodium montmorillonite, which has been reported to facilitate electron transfer processes (11), and observed a very fast heterogenous electron transfer to P450 cam with rate constants (5–150 s ⫺1) very similar to those reported for the biological P450 cam-Pdx system. This fast electron transfer might be caused by the optimal spacial orientation of P450 cam on the electrode surface induced by the negatively charged clay. MATERIALS AND METHODS Protein and clay. Cytochrome P450 cam from Pseudomonas putida was expressed in Escherichia coli strain TB1, isolated, purified and depleted from camphor as previously described (12). The protein stock solution used for the electrochemical studies was 40 M of substrate-free P450 cam in 50 mM Tris/HCl buffer, pH 7.4, 5% glycerol (v/v). For the infrared studies the stock solution was 1 mM of
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substrate-free P450 cam in 100 mM D 2O potassium phosphate buffer (corresponding to pH 7). Sodium montmorillonite colloid (SMC) was prepared from the purified montmorillonite powder (from Zhejiang province, China) in a similar way as previously described (12) to get a clay content of 1.25 g/liter. 120 l of SMC were mixed with 5 l of colloidal Pt and then diluted 4.4 times with water to obtain a pretreated sodium montmorillonite colloid (PSMC) (13). For the infrared studies the clay colloid (12.5 g/liter) was dialyzed against D 2O. Electrochemistry. Aliquots (5 l) of PSMC were deposited on a glassy carbon electrode (GCE, 3 mm in diameter) and allowed to dry at 20°C to form a clay-modified membrane on the electrode surface (Clay/GCE). 3 l of the 40 M substrate-free P450 cam stock solution in Tris-HCl buffer were added onto Clay/GCE and then put in a refrigerator overnight. In this period P450 cam was spontaneously adsorbed onto the clay-modified membrane. The modified electrode was rinsed with the working buffer (33 mM potassium phosphate buffer, pH 7) prior to use and thus P450/Clay/GCE was obtained. For comparative electrochemical studies hemin was dissolved in 1 M NaOH and then diluted with phosphate buffer to adjust a pH of 7.5 and a concentration of 0.5 mM. From this solution hemin was adsorbed onto the clay-modified electrode when the modified electrode was inserted into the hemin solution overnight. All electrochemical experiments were carried out with the Autolab PSTAT10 (Eco Chemie, Netherland). The electrochemical cell was equipped with a Ag/AgCl (1.0 M KCl) reference electrode and a platinum wire auxiliary electrode. Prior to measuring, 1 ml of the 33 mM potassium phosphate working buffer (pH 7) was carefully bubbled with argon for at least 20 min to remove oxygen. Electron transfer rates k were estimated by the formula k ⫽ m n F/RT published by Laviron for surface processes (14), where m ⫽ 1/3 for a voltammogram peak separation of 81 mV and n ⫽ 1 for the number of transfered electrons were used. , F, R and T are the scan rate, the Faraday constant, the gas constant and the temperature, respectively. FT-infrared studies. Infrared spectra were recorded using the Bruker IFS66 Fourier transform infrared spectrometer (Bruker, Germany). The IR sample solution was prepared with the D 2O clay reagent to get a final concentration of 350 M P450 cam and 8.12 g/liter clay. For studies in the absence of clay pure D 2O in a corresponding volume was used instead of the clay suspension. The P450 sample was placed in a demountable cell with CaF 2 windows separated by a PTFE spacer. For measurements of the amide I band of oxidized P450 cam a 23 m spacer and for the CO stretch signals of the carbon monoxide complex of P450 cam a 100 m spacer were taken. The preparation of the CO-P450 cam complex, the FTIR data treatment and baseline correction were the same as described previously (15, 16). UV/VIS absorption spectra were recorded on the UV-2101PC spectrometer (Shimadzu, Japan). For all the IR and UV/VIS experiments, the reference always comprised the same buffer in the absence and the presence of clay as the sample. All the experiments were carried out at 20 ⫾ 1°C. The P420 content has been determined from the Soret band difference spectrum between the carbon monoxide minus the reduced complex (18). The absorbance coefficients of 91 mM ⫺1 cm ⫺1 for the P450 peak at 446 nm, of 111 mM ⫺1 cm ⫺1 for the P420 peak at 420 nm and of 41 mM ⫺1 cm ⫺1 for the difference between the CO complex and the zero line at 420 nm have been used.
RESULTS Figure 1A shows cyclic voltammograms (CV) of P450/Clay/GCE at different scan rates. In the scan rate range from 2 to 12 V s ⫺1 , P450/Clay/GCE had almost symmetrical CV shapes, equal reduction and oxidation peak heights with a formal potential of ⫺361 mV (vs. Ag/AgCl); ⫺139 mV (vs NHE). The
FIG. 1. Cyclic voltammogram of cytochrome P450 cam adsorbed on the glassy carbon electrode modified with sodium montmorillonite at different scan rates (A) and plot of the peak current vs the scan rate (B); 33 mM potassium phosphate buffer, pH 7.
redox peak separation (⌬E P ) is 77 mV at 2 V s ⫺1 and 84 mV at 12 V s ⫺1 . The anodic as well as the cathodic peak current show a linear relationship with the scan rate (Fig. 1B). At a scan rate as low as 0.4 V s ⫺1 , only the reduction peak was found whilst almost no oxidation peak was visible (Fig. 1A, inset). From the integration of the anodic peak in the CV of P450/ Clay/GCE, the apparent surface coverage of the electroactive P450 cam was calculated to be in the order of 3.54 ⫻ 10 ⫺12 mol cm ⫺2 . Assuming a complete coverage of the electrode surface with a monolayer of the globular P450 cam , (triangle size 57 ⫻ 60 Å (16)) about 35% of the adsorbed P450 cam displayed the electroactivity.
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FIG. 2. Cyclic voltammogram of cytochrome P450 cam adsorbed on the glassy carbon electrode modified with sodium montmorillonite (A) in the presence of CO (dashed line) and replacement of CO by argon (dotted line) and (B) in the presence of metyrapone, 1.67 mM (dashed line); compared to the voltammogram in absence of an iron ligand (solid line); 33 mM potassium phosphate buffer, pH 7.
After the working buffer was bubbled with CO for 10 min, the formal potential of P450/Clay/GCE was positively shifted by 80 mV (Fig. 2A). When CO is replaced by argon the formal redox potential shifts back again to the original value. Addition of metyrapone induces a positive shift of the formal potential by 86 mV (Fig. 2B). Both CO and metyrapone bind specifically as a sixth ligand to the heme iron of P450 cam indicating that the observed electrochemical response does indeed originate from P450 cam . Addition of (1R)-camphor (500 M) to the working buffer did not shift the formal potential but increased the anodic peak area of the cyclic voltammogram by about 15% (voltammogram not shown). For an additional check we recorded the cyclic voltammogram of hemin adsorbed on the clay-modified electrode under the same experimental conditions as for the P450
study. Figure 3 shows that the cyclic voltammogram for hemin looks different from P450 cam . The formal potential of adsorbed hemin is ⫺330 mV (vs. Ag/ AgCl) and the redox peak separation (⌬E P ) is not constant in the scan rate range from 0.4 V s ⫺1 (89 mV) to 12 V s ⫺1 (151 mV). These differences to the behavior of the adsorbed P450 supports the aforementioned conclusion that the electrochemical response is really induced by the adsorbed P450. As noted above a formal redox potential of ⫺361 mV (vs Ag/AgCl) has been derived for P450 cam from the cyclic voltammogram in Fig. 1A. This value strongly deviates from the redox potential observed for substrate-free P450 cam in solution (⫺525 mV vs Ag/AgCl, ⫺303 mV (vs NHE) (19)). Therefore, the effect of clay on the P450 structure has been studied by spectroscopic methods. Figure 4A shows the UVVIS spectrum of oxidized P450 cam and its CO complex in the presence and in the absence of clay. There is no significant effect of clay on the spectrum of the oxidized protein neglecting the absorbance increase in the UV region due to light scattering. In the CO complex a slight increase of the P420 content by 9% is recognized compared to the sample without clay, which has a P420 content of 5%. The infrared spectra of the amide I band (Fig. 4B), which represents the secondary structure of P450 (15), do not show any difference whether clay is present or not. The second derivative of the amide I band is almost identical in both cases. Also the CO ligand stretch vibration at around 1940 and 1963 cm ⫺1 which is very sensitive to the structural changes at the enzyme active site (16) did not reveal any significant difference as shown in Fig. 4C.
FIG. 3. Cyclic voltammogram of hemin adsorbed on the glassy carbon electrode modified with sodium montmorillonite (dashed line) compared to the voltammogram of cytochrome P450 cam (solid line), scan rate of 10 V s ⫺1; 33 mM potassium phosphate buffer, pH 7.
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DISCUSSION
FIG. 4. Electronic absorption spectra of oxidized P450 cam in the absence (dashed line) and in the presence of 0.53 g/liter clay (dotted line) and that of reduced P450 cam complex with CO in the absence (solid line) and in the presence of 0.53 g/liter clay (dash/dot mixed line), 3.2 M P450 cam, 29 mM potassium phosphate buffer, pH 7.1 (A) and amide I⬘ infrared spectra and their second derivative, which are manually separated along the ordinate (B), and the CO ligand stretch vibration band, which has been area-normalized (C) in the absence (solid line) and in the presence of 8.12 g/liter clay (dotted line); 350 M P450 cam in 35 mM D 2O potassium phosphate buffer, corresponding to pH 7.
Although some electrochemical aspects of P450 were reported more than 20 years ago by Scheller et al. (20, 21), the direct, nonpromoted electrochemistry of P450 is rather difficult to obtain with nonmodified electrodes. The enzyme passivates the electrode and is denatured. The first direct electrochemistry by using freshly purified P450 cam in solution at the edge-plane graphite electrode was reported by Kazlauskaite et al. (7). Rusling’s group has found that P450 cam incorporated in lipid or polyelectrolyte film displayed the welldefined redox behavior from its heme Fe(II/III) (8). More recently, Lo et al. (10) demonstrated cyclic voltammograms on an edge-plane graphite electrode for various P450 cam mutants. In all the mentioned studies the electron transfer rate, so far reported, is rather slow with rate constants lower than 0.01 s ⫺1. Our studies with the clay modified carbon electrode gives clear indication for a reversible and fast electron transfer between the electrode and the adsorbed P450. The observed linearity of the peak current with the scan rate is characteristic for an surface process and allows to apply the theory by Laviron (14) for estimation of the rate constant. The rate constant is in a range of 5 to 152 s ⫺1 for the scan rates of 0.4 and 12 V s ⫺1, respectively. In the natural system the rate for the transfer of the first electron from putidaredoxin to P450 cam has been reported to lie in the range between 27 and 84 s ⫺1 (19, 22). This similarity suggests that the negatively charged clay (11) obviously mimics the electrostatics of the natural redox partner putidaredoxin and may hold the P450 in a productive orientation. In this orientation the active site of the adsorbed P450 cam is still accessible for iron ligands like CO and dioxygen. The positive shift of the formal potential by 80 mV due to CO binding resembles the expected value of 96 mV which we have estimated from the CO binding constant for substrate-free P450 cam of 5.7 ⫻ 10 4 mol ⫺1 (23) using the Nernst equation. Binding of residual dioxygen seems also to be taken place indicated by the missing oxidation peak in the voltammogram at slow scan rates as it has been similarly observed by Lo et al. (10). Not only the small ligands can entry the heme pocket of the adsorbed P450. Even the large metyrapone molecule has access to the active site of the adsorbed P450 indicated by the expected positive shift of the redox potential. The still unresolved problem is the shift of the formal potential of the adsorbed P450 cam by approximately 164 mV to more positive values compared to the substratefree P450 cam in solution. Positive shifts of the redox potential are generally observed when water is excluded from the heme pocket as in the case of camphor binding (24). Because the clay itself has no effect on the P450 structure in solution, we suggest that the adsorp-
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tion process leads to a partial dehydration of the P450 cam structure. This conclusion would also explain that the addition of camphor has only a minor effect on the electrochemical response. ACKNOWLEDGMENTS Dr. Chenghong Lei deeply appreciates the support by the Alexander von Humboldt Foundation (Bonn, Germany) which has awarded him a research fellowship. He also thanks Professor Fugen Zhou at Tongji University in Shanghai for offering the purified montmorillonite powder. We thank Dr. Jian Chen (University of Potsdam) for performing the hemin experiments. We gratefully acknowledge the financial support of this work by the Deutsche Forschungsgemeinschaft (INK 16/A1-1 and SK 35/3-1) and the Fonds der Chemischen Industrie. We thank Dr. W.-H. Schunck for critical reading of the manuscript.
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