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Electroenzyme-Catalyzed Oxidation of Styrene and cis-β-Methylstyrene Using Thin Films of Cytochrome P450cam and Myoglobin Xiaolin Zu,† Zhongqing Lu,† Zhe Zhang,† John B. Schenkman,‡ and James F. Rusling*,† Department of Chemistry, U-60, University of Connecticut, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received June 2, 1999. In Final Form: June 2, 1999 Protein-polyion films grown layer-by-layer and cast protein-surfactant films were employed on electrodes for catalytic oxidation of styrene derivatives to epoxides. Cytochrome P450cam and myoglobin in these films mediated the electrochemical reduction of oxygen to hydrogen peroxide, which activates these heme proteins to catalyze olefin oxidation. Compared to bare electrodes with the proteins dissolved in solution, ultrathin protein-polyion films on Au electrodes coated with mercaptopropane sulfonate gave the best catalytic activities for the oxidations. Improved performance of protein-polyion films is related to efficient, reversible heme FeIII/FeII electron transfer and better mechanical stability than the surfactant films. Furthermore, dependence of product stereochemistry on oxygen availability in the reaction medium for the oxidation of cis-β-methylstyrene suggested two pathways for olefin oxidation, which had not been reported previously for cyt P450 enzymes. The stereoselective pathway depends on an active, high-valent iron-oxygen intermediate as in the natural enzyme system, while the nonstereoselective pathway may involve a peroxyl radical near the protein surface.
We previously incorporated the iron heme proteins myoglobin (Mb) and cytochrome P450cam (cyt P450cam) into films of insoluble surfactants and lipids on electrodes,1 and showed that they could be used for anaerobic electrochemical catalysis of organohalide reductions.2,3 More recently, we constructed alternately layered films of these proteins and polyions on electrodes and demonstrated aerobic electrode-driven enzyme-like conversion of styrene to styrene oxide.4 These reactions are “doubly catalytic”, featuring mediated electrochemical reduction of protein-bound dioxygen to hydrogen peroxide, followed by peroxide-initiated, enzyme-catalyzed oxidation of the olefinic bond of styrene. Facilitation of reversible electrochemical heme FeIII/FeII conversion is a key to the success of both types of films. Cytochrome P450 enzymes in human liver metabolize lipophilic pollutants and drugs, often to toxic products.5-9 The natural catalytic cycle includes binding of substrate to cyt P450FeIII, followed by reduction to cyt P450FeII, † ‡
University of Connecticut. University of Connecticut Health Center.
(1) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (2) Nassar, A.-E. F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 10986-10993. (3) Zhang, Z.; Nassar, A.-E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769-1774. (4) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (5) (a) Ortiz de Montellano, P. R., Ed. Cytochrome P450, 2nd ed.; Plenum: New York, 1995. (b) Schenkman, J. B., Greim, H., Eds. Cytochrome P450; Springer-Verlag: Berlin, 1993. (c) Omura, T., Ishimura, Y., Fujii-Kuriyama, Y., Eds. Cytochrome P450, 2nd ed.; Kodansha/ VCH: Tokyo, 1993. (6) (a) Guengerich, F. P.; MacDonald, T. L. Acc. Chem. Res. 1984, 17, 9-16. (b) Guengerich, F. P.; MacDonald, T. L. FASEB J. 1990, 4, 24532459. (7) Gonzalez, F. J. Trends Pharm. Sci. 1992, 13, 346-352. (8) (a) Jacoby, W. B., Ed. Enzymatic Basis of Detoxification; Academic: New York, 1980; Vols. I and II. (b) Singer, B.; Grunberger, D. Molecular Biology of Mutagens and Carcinogens; Plenum: New York, 1983.
which binds dioxygen. The resulting cyt P450FeII-O2 is reduced by a second electron and ultimately forms an active iron-oxo complex which transfers oxygen to the substrate. Electrons for the natural catalytic cycle of cytosolic cytochrome P450cam in Pseudomonas putida are supplied by NADH via the enzymes putidaredoxin reductase and putidaredoxin. While camphor is the natural substrate of cytochrome P450cam, it also oxidizes camphor analogues10-13 and other substrates such as styrenes,14,15 alkylbenzenes,16 tetralone,17 and tetralin.18 Styrene is epoxidized by cytochrome P450cam to styrene oxide,19,20 which can damage DNA.19-21 When activated by hydrogen peroxide, Mb catalyzes oxidations via a ferrylmyoglobin (•MbFeIVdO) radical.22 (9) (a) Yang, C. S.; Lu, A. Y. H. In Mammalian Cytochromes P450; Guengerich, F. P., Ed.; CRC Press: Boca Raton, FL, 1987; Vol. 2, pp 1-17. (b) Nelson, S. D.; Harvison, P. J. In Mammalian Cytochromes P450; Guengerich, F. P., Ed.; CRC Press: Boca Raton, FL, 1987; Vol. 2, pp 19-79. (10) Atkins, W. M.; Sligar, S. G. J. Biol. Chem. 1988, 263, 1884218849. (11) Atkins, W. M.; Sligar, S. G. J. Am. Chem. Soc. 1989, 111, 27152717. (12) White, R. E.; McCarthy, M.-B.; Egeberg, K. D.; Sligar, S. G. Arch. Biochem. Biophys. 1984, 228, 493-502. (13) Eble, K. S.; Dawson, J. H. J. Biol. Chem. 1984, 259, 1438914393. (14) Ortiz de Montellano, P. R.; Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H. J. Am. Chem. Soc. 1991, 113, 3195-3196. (15) Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1992, 114, 6987-6993. (16) (a) Loida, P. J.; Sligar, A. G. Protein Eng. 1993, 6, 207-212. (b) Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1998, 353, 285-296. (17) Watanabe, Y.; Ishimura, Y. J. Am. Chem. Soc. 1989, 111, 410411. (18) Grayson, D. A.; Tewari, Y. B.; Mahew, M. P.; Vilker, V. L.; Goldberg, R. N. Arch. Biochem. Biophys. 1996, 332, 239-247. (19) Bond, J. A. CRC Crit. Rev. Toxicol. 1989, 17, 227-249. (20) Pauwels, W.; Vodiceka, P.; Servi, M.; Plna, K.; Veulemans, H.; Hemminki, K. Carcinogenesis 1996, 17, 2673-2680. (21) Latham, G. J.; Zhou, L.; Harris, C. M.; Lioyd, R. S. J. Biol. Chem. 1993, 268, 23527-23530.
10.1021/la990685k CCC: $18.00 © 1999 American Chemical Society Published on Web 08/05/1999
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Chart 1
Careful mechanistic studies23 have revealed two different pathways for Mb-H2O2 oxidation of styrenes. One pathway features transfer of the ferryl oxygen to the olefin, preserves the olefin stereochemistry, and incorporates an oxygen from H2O2 into the epoxide. cis-β-Methylstyrene is converted to cis-β-methylstyrene oxide by this pathway. The second pathway involves oxidation of styrene by a peroxyl radical on the protein surface derived from reaction of dioxygen with •MbFeIVdO and results in a mixture of trans- and cis-β-methylstyrene oxides. Thus, cis-β-methylstyrene oxidation can be used as a stereochemical probe to reveal the relative importance of each pathway. Like Mb, cyt P450s can also be activated by organic peroxides and hydrogen peroxide.5,24,25 It has been suggested that the natural enzyme process and hydrogen peroxide assisted oxidations catalyzed by cyt P450s share a common active intermediate. In this paper, we evaluate Mb and cyt P450cam in polyion and surfactant films (Chart 1) on electrodes for oxidation of styrene and cis-β-methylstyrene and show that cyt P450cam in polyion films provides the best catalytic activity. More importantly, results suggest two competing pathways for olefin oxidation, with stereoselectivity controlled by the oxygen content of the medium.
Figure 1. Cyclic voltammograms at 0.2 V s-1 in pH 7.4 buffer in a sealed cell on Au (0.16 cm2) for Au-MPS-(PDDA/P450)2 films: (a) solid line, under Ar; (b) solid line, after passing 50 mL of oxygen through the solutions; (c) dashed line, solution saturated in styrene after passing 50 mL of oxygen; and (d) dashed line, bare Au electrode after passing 50 mL of oxygen. used were sodium poly(styrenesulfonate) (PSS) and poly(dimethyldiallylammonium chloride) (PDDA). Carbon cloth electrodes (1.5 × 6 cm) were coated by soaking in 0.25 mM Mb in 5 mM aqueous vesicle dispersions of surfactant didodecyldimethylammonium bromide (DDAB) or 30 µM cyt P450cam in 2 mM DDAB and drying in air overnight. Electrolyses were done in 4 mL of pH 7.4 buffer saturated with styrene or cis-β-methylstyrene (ca. 10 mM) in a sealed, divided three-electrode cell at -0.60 V vs SCE and 4 °C. Chemical oxidations were done by adding hydrogen peroxide to solutions the same as those for electrolyses. Product mixtures were extracted with CH2Cl2 and analyzed by gas chromatography.4,26
Experimental Section
Results
Chemicals. Horse heart myoglobin was from Sigma. Purification of Pseudomonas putida cyt P450cam expressed in E. coli DH5R containing P450cam cDNA was described previously.3 Buffer was pH 7.4 TRIS + 100 mM KCl. Chemical sources were reported previously.3,4 Apparatus and Procedures. Electrochemical methods were described previously.3,4 Polyion-protein films were prepared by layer-by-layer growth on gold electrodes coated with mercaptopropane sulfonate (MPS), as described previously.4 Polyions
Voltammetry. Cyclic voltammetry in anaerobic pH 7.4 buffers gave reversible peaks for the heme FeIII/FeII couples of cyt P450cam and Mb in DDAB and polyion films as reported previously.1,3,4 With oxygen in solution, a large reduction wave was observed at the potential of heme FeIII reduction (Figure 1), accompanied by disappearance of the peak for oxidation of the heme FeII. The new reduction peak was 200 mV positive of the direct reduction of oxygen on gold. This peak increased slightly in height when the solution was saturated with styrene. Buffer solutions containing only styrene gave no significant peaks on gold or pyrolytic graphite (PG) electrodes between 0.5 and -1.0 V vs SCE. Cyclic voltammetry of Mb and cyt P450 dissolved in anaerobic pH 7.4 buffer on bare Au or PG electrodes gave no peaks. In aerobic solutions, a 20-40% larger peak current for reduction of oxygen was found with 7-80 µM of either protein in solution, compared to solutions with oxygen but no protein.27 Oxidation of Styrene. Electrolyses were done under oxygen at applied potentials on the plateau of the catalytic CV oxygen reduction waves. Reactions with Mb and cyt P450cam in protein/polyion films on Au electrodes, protein-DDAB films on carbon cloth electrodes, and protein in solution on Au and carbon electrodes were compared. The major products were styrene oxide and
(22) (a) George, P.; Irvine, D. H. Biochem. J. 1952, 52, 511-517. (b) King, N. K.; Winfield, M. E. J. Biol. Chem. 1963, 238, 1520-1528. (c) King, N. K.; Winfield, M. E. Aust. J. Biol. Sci. 1966, 19, 211-217. (d) Yonetoni, T.; Schleyer, H. J. Biol. Chem. 1967, 242, 1974-1979. (e) Galaris, D.; Cadenas, E.; Hochstein, P. Free Radical Biol. Med. 1989, 6, 473-478. (f) Galaris, D.; Cadenas, E.; Hochstein P. Arch. Biochem. Biophys. 1989, 273, 497-504. (g) Galaris, D.; Eddy, L.; Arduini, A.; Cadenas, E.; Hochstein, P. Biochem. Biophys. Res. Commun. 1989, 160, 1162-1168. (h) Giuvlivi, C.; Cadenas, E. FEBS Lett. 1993, 332, 287290. (i) Turner, J. J. O.; Rice-Evans, C. A.; Davies, M. J.; Newman, E. S. R. Biochem. J. 1991, 277, 833-837. (j) Davies, M. J. Biochim. Biophys. Acta 1991, 1077, 86-90. (k) Dee, G.; Rice-Evans, C. A.; Obeyesekera, S.; Meraji, S.; Jacobs, M.; Bruckdorfer, K. R. FEBS Lett. 1991, 294, 38-42. (23) (a) Ortiz de Montellano, P. R.; Catalano, C. E. J. Biol. Chem. 1985, 260, 9265-9271. (b) Rao, S. I.; Wilks, A.; Ortiz de Montellano, P. R. J. Biol. Chem. 1993, 268, 803-809. (c) Ortiz de Montellano, P. R.; Rao, S. I.; Wilks, A. Life Chem. Rep. 1994, 12, 29-32. (d) Choe, Y. S.; Rao, S. I.; Wilks, A.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1994, 314, 126-131. (24) (a) Nordblom, G.; White, R. E.; Coon, M. J. Arch. Biochem. Biophys. 1976, 175, 524-533. (b) White, R. E.; Sligar, S. G.; Coon, M. J. J. Biol. Chem. 1980, 255, 11108-11111. (25) (a) Pratt, J. M.; Ridd, T. I.; King, L. J. J. Chem. Soc., Chem. Commun. 1995, 2297-2298. (b) Anari, M. R.; Josephy, P. D.; Henry, T.; O’Brein, P. J. Chem. Res. Toxicol. 1997, 10, 582-588 and references therein.
(26) Extraction and chromatographic analysis was done immediately at the end of a reaction, since styrene oxide is hydrolyzed to give benzaldehyde at the rate of about 0.5%/min at room temperature.27 (27) Zu, X. Ph.D. Thesis, University of Connecticut, Storrs, CT, 1998.
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Table 1. Yields for Styrene Oxidation Using Myoglobin and cyt P450cam at 4 °C by Electrolysis at -0.6 V vs SCE under Oxygen or with H2O2 Open to Air reaction systema
amt of protein (nmol)
Au-MPS-(Mb/PSS)2 Au-MPS-(Mb/PSS)2 + catalased Mb-DDAB (CC)c Mb in solution (CC)c Mb in solution (Au) Mb + catalased (CC) Au-MPS-(PDDA/P450)2 Au-MPS-(PDDA/P450)2 + catalased P450-DDAB (CC)c P450 in solution (Au) P450 in solution (CC) P450 + catalased (CC)
0.77 0.77 110 325 325 325 0.43 0.43 14 28 28 28
Electrolyses 6.4 2.3 25 45 50 4.6 6 2.2 120 12 210 17 Controls (Electrolyses) 2 2 12
no protein (Au-MPS) no protein (Au) no protein (CC) Mb + 20 mM H2O2 P450 + 20 mM H2O2 no protein + 20 mM H2O2
styrene oxide (nmol)
325 28
Chemical Reaction (Air) 172 166 2
benzaldehyde (nmol)
turnoverb no. (h-1)
[H2O2] found (mM)
18 6.8 32 60 56 11 7 8 21 8 40 14
3.8 0.4 0.1 0.1 0.1 0.01 9.3 0.5 7.2 0.35 7.0 0.2
20 0 10 5 5 0 20 0 10 10 10 0
6.4 7.4 53 118 26 39
2 2 3 0.5 5.8
20 20 20
a All reactions for 1 h in 4 mL pH 7.4 buffer (50 mM TRIS + 0.1 M KCl) with O bubbled through the solutions (first 20 min) saturated 2 in styrene (ca. 10 mM). Film symbols: protein/PDDA indicates individual layers of protein and polyion; (protein/PDDA)2 indicates two successive bilayers of these components. Film thicknesses estimated from quartz crystal microbalance (QCM) measurements in ref 4 are as follows: MPS-(Mb/PSS)2, 11 nm; MPS-(P450/PDDA)2, 12 nm. Amounts of protein in films were estimated from QCM estimates of ng of protein per cm2 electrode area for each protein layer (ref 4). Protein-DDAB indicates cast film of the two mixed components; amounts of protein estimated from weight cast on electrode. Results are averages of two or more runs, analyses by gas chromatography with reproducibility (10%. Au electrode was 1 × 5 cm (A ) 10 cm2) with film on both sides. b nmol of styrene oxide (less control)/nmol of myoglobin or cytochrome P450cam/h. c CC ) 1.5 × 6 cm carbon cloth, active electrochemical area 230 cm2. d 3000 units catalase added.
benzaldehyde (Table 1). Reproducibility of product yields was within (10% for replicate reactions. Styrene oxide is the main enzyme-catalyzed product, but considerable benzaldehyde results from reaction of styrene with hydrogen peroxide14,15,23 formed during electrolysis. Thus, we focused mainly on styrene oxide. Turnover numbers for styrene oxide formation showed that cyt P450cam had better catalytic activity for styrene oxidation in all systems studied (Table 1).28 Also, proteins in layered polyion films had larger turnover numbers than any other catalytic environment. We do not expect that the nature of the polyion has significant influence on turnover, as it had little influence on voltammetric properties in previous studies.4 For Mb, DDAB films and solutions gave equally poor catalytic activities. While cyt P450cam showed the best activity in polyion films, DDAB films and cyt P450 solutions on bare carbon cloth cathodes had turnover numbers approaching those in polyion films (Table 1). The carbon cloth electrode, with a 20-fold greater active surface area than the gold electrode, gave a much better turnover number than Au for cyt P450cam in solution, but not for Mb. In all electrolyses with protein present, hydrogen peroxide was produced in amounts significantly larger than those obtained in control experiments without protein (Table 1). Addition of catalase to the electrolysis solutions to destroy H2O2 caused greatly decreased turnover numbers for styrene oxide production. Cyt P450cam was much more active than Mb for styrene oxide produced by addition of H2O2. (28) Cyt P450cam was stored at -78 °C in the camphor-bound form. Similar turnover numbers were obtained whether camphor was removed before the experiment or not, so nearly all experiments were done without removing camphor. During film preparation or in solution, since no camphor was present, the enzyme is likely to end up in the camphorfree form.
Oxidation of cis-β-Methylstyrene. The main products were trans- and cis-β-methylstyrene oxides.29 Mb and cyt P450cam gave similar turnover numbers in polyion films for this substrate (Table 2). In electrolyses with DDAB films or in solution, cyt P450cam was more active than Mb. The same order of activity was found for reactions driven by addition of H2O2. All electrolyses under O2 except on Au with cyt P450cam in solution gave more trans- than cis-β-methylstyrene oxide (Table 2). For the H2O2 addition oxidations which were done open to air, this order was reversed and both Mb and cyt P450 gave more cis- than trans-oxide. Oxidations of cis-β-methylstyrene were then examined with different availabilities of oxygen in solution using protein-polyion films and dissolved proteins. With Mb, electrolyses with the reaction cell open to air always gave a larger cis/trans ratio but less total oxide products than electrolyses under oxygen (Figure 2). No organic electrolysis products were found in the absence of oxygen, i.e., when nitrogen was bubbled through the solution. In oxidations by H2O2 addition, the cis/trans ratio increased and the total oxide yields decreased with decreasing oxygen availability. When nitrogen was bubbled through H2O2 solutions, cis-β-methylstyrene oxide was the only oxide product. For cyt P450cam, the cis/trans product ratio showed the same increasing trend with decreasing oxygen availability, except that actual numerical ratios were larger (Figure 3). Figures 2 and 3 show the generally better enzyme activity of cyt P450cam compared to Mb. In solution, the increase in the fraction of cis-oxide is probably not significant. For cyt P450cam/polyion films, saturation with oxygen during electrolysis resulted in trans > cis, (29) Minor oxidation products as reported in refs 14, 15, and 23b were also found at trans when the reaction cell was open to air. As with Mb, the cis/trans ratio increased and the total oxide yields decreased with decreasing oxygen availability. In oxidations by H2O2 addition, removal of all oxygen resulted in formation of cis-β-methylstyrene oxide only. The stability of the protein films was monitored by reversible cyclic voltammetry (see Supporting Information) in anaerobic buffer before and after electrolyses. The polyion films containing Mb or cyt P450cam showed reversible FeIII reduction peaks which were about 20% decreased after an hour of electrolysis in a system open to air and 30% decreased after an hour of electrolysis under oxygen. In DDAB films 30-40% decreases in the FeIII reduction peaks for Mb and cyt P450cam were found after an hour of electrolysis open to air, but no peaks remained after an hour of electrolysis with oxygen saturation. Thus,
Figure 3. Yield of total β-methylstyrene oxides and % cis-βmethylstyrene from Cyt P450cam catalysis for eight experiments under different conditions and different availabilities of oxygen. All reactions were for 1 h at 4 °C in 4 mL pH 7.4 buffer saturated in cis-β-methylstyrene. Where indicated, O2 (first 20 min) or N2 were bubbled through the reaction solutions, or the solution was open to ambient air. Amounts of protein and other conditions used were the same as in Table 2 for equivalent systems. Data are averages of two or more runs, reproducibility (10%: (1) electrolysis on Au with Cyt P450cam in solution and O2 bubbling; (2) electrolysis on Au with Cyt P450cam in solution open to air; (3) electrolysis using Au-MPS-(PDDA/P450)2 and O2 bubbling (4) electrolysis using Au-MPS-(PDDA/P450)2 open to air; (5) electrolysis using Au-MPS-(PDDA/P450)2 and N2 bubbling; (6) Cyt P450cam in solution and O2 bubbling + 0.5 mM H2O2; (7) Cyt P450cam in solution open to air + 20 mM H2O2; (8) Cyt P450cam in solution and N2 bubbling + 0.5 mM H2O2.
decomposition of both proteins during electrolysis is faster when more oxygen is present. Discussion Spectroelectrochemical studies of Mb during electrolysis in the presence of oxygen in buffer solutions, microemulsions,30 and thicker cast polyanion films31 revealed visible spectra characteristic of ferrylmyoglobin and ferrylmyoglobin radical. The fact that styrenes are oxidized during (30) Onuoha, A. C.; Zu, Z.; Rusling, J. F. J. Am. Chem. Soc. 1997, 119, 3979-3986. (31) (a) Kong, J.; Mbindyo, J. K. N.; Rusling, J. F. Biophys. Chem., in press. (b) The films refered to here were 1-4 µm thick to obtain sufficient optical absorbance. Films in the present study were 11-12 nm (see Table 1, footnote a).
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electrolysis using our ultrathin polyion films (Tables 1 and 2) is consistent with the formation of ferrylmyoglobin radical (•MbFeIVdO), since this species is capable of styrene oxidation but nonradical ferrylmyoglobin is not.23d The active oxidant in cyt P450cam oxidations is too unstable5,6 to be observed by spectroelectrochemistry. •MbFeIVdO is most likely formed by reaction of MbFeIII with H2O2 generated by electrolysis.30 The high valent active6 cyt P450(FeO)3+ is likely to be formed in an analogous way. H2O2 dependence of the oxidation of styrene during electrolysis is shown by the large decrease in turnover number when the peroxide-destroying enzyme catalase is included (Table 1). This decrease is probably caused by limited formation of •MbFeIVdO when H2O2 is destroyed. The data obtained herein are consistent with the “doubly catalytic” pathway for electrochemically driven styrene oxidation with Mb, suggested earlier based on studies in buffer solutions and microemulsions30 (Scheme 1). The
Scheme 1 MbFeIII + e- ) MbFeII (at electrode)
(1)
MbFeII + O2 f MbFeII-O2
(2)
MbFeII-O2 + 2e- + 2H+ f MbFeII + H2O2 (at electrode) (3) •
MbFeIVdO + H2O2 f •MbFeIVdO + H2O •
MbFeIVdO + H2O2 f MbFeIVdO + O2
(4) (5)
•
MbFeIVdO + styrene f styrene oxide + MbFeIII (6)
electrode provides electrons to reduce metmyoglobin (MbFeIII, eq 1) to MbFeII, which reacts rapidly with oxygen to give MbFeII-O2 (eq 2). Hydrogen peroxide is produced by catalytic electrochemical reduction of MbFeII-O2 (eq 3), apparently regenerating MbFeIII. Hydrogen peroxide reacts with MbFeIII in solution to give radical •MbFeIVdO, which oxidizes styrene in the well-known catalytic peroxide driven process (eq 6). •MbFeIVdO can oxidize styrene, but nonradical MbFeIVdO cannot.23d Our data are consistent with cyt P450cam in films on electrodes catalyzing styrene oxidation in a similar fashion. Turnover numbers for conversion of styrene and cisβ-methylstyrene to their respective oxides were consistently larger in the polyion films than in other protein environments (Tables 1 and 2). This can be correlated with the superior protein heme FeIII/FeII electron-transfer properties of film-coated electrodes compared to the proteins in solution on bare electrodes.1-4 Also, cyclic voltammogram before and after electrolyses showed that the proteins are more stable under reaction conditions in polyion films than in DDAB films, which may undergo mechanical damage during electrolyses with stirring.2 Protein damage is also likely to be caused by reaction of amino acid residues with H2O2.32,33 It is unlikely that substrate mass transport is an issue in the observed turnover differences, since it is not expected to be a limiting factor in catalysis with either type of film.1,2,34 It is instructive to compare the turnover numbers in polyion films with those of cyt P450cam using the full (32) Yao, K.; Falick, A. M.; Patel, N.; Correia, M. A. J. Biol. Chem. 1993, 268, 59-65 and references therein. (33) Tschirret-Guth, R. A.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1996, 335, 93-101 and references therein. (34) Lvov, Y. M.; Rusling, J. F. Unpublished results.
Scheme 2
complement of natural electron donor, putidaredoxin, and putidaredoxin reductase. Such experiments in solution at 25 °C gave turnovers of 60 h-1 for styrene and 32 h-1 for cis-β-methylstyrene.15 Considering the higher temperature, our values of 9 h-1 for styrene and 7 h-1 for cis-β-methylstyrene at 4 °C are consistent with similar total activity for the electrochemical method with MPS(PDDA/P450)2 films compared to the native enzyme system. However, the native enzyme system is completely stereospecific, while introduction of oxygen in electrochemical or hydrogen peroxide methods degrades the stereoselectivity (see below). Cis/trans ratios of β-methylstyrene oxides (Table 2, Figures 2 and 3) can be explained by competing iron-oxo and protein radical pathways. These pathways have been elucidated in detail for Mb activated by H2O2,23 but not to our knowledge for cyt P450s. For horse heart MbH2O2 oxidation of styrene in solution, 18O labeling showed that 78% of the oxygen in styrene oxide was derived from molecular oxygen and 35% from H2O2,23a although quantitative assignment to each pathway is difficult because Mb produces O2 from H2O2 during the reaction. Oxidation of cis-β-methylstyrene by Mb-H2O2 coupled with 18O labeling suggested23b that ferryl oxygen from •MbFeIVdO derived from hydrogen peroxide ended up in cis-βmethylstyrene oxide in a stereospecific oxygen transfer (Scheme 2, top). The second oxidation pathway features reaction of molecular oxygen with a radical on an amino acid residue of •MbFeIVdO to give a peroxyl radical [•O-O-MbFeIVdO] which then oxidizes the olefin in a nonstereoselective radical process, yielding trans and cis oxide isomers (Scheme 2, bottom). Recent site-directed mutagenesis studies implicated a surface tryptophan residue on Mb as the site of peroxidation.35 Results (Table 2, Figures 2 and 3) are consistent with the existence of the two pathways in cis-β-methylstyrene oxidations catalyzed by Mb and cyt P450cam. For MbH2O2 in reactions open to air, the 66:34 cis/trans-βmethylstyrene oxide ratio (Table 2) was similar to those reported previously for native Mb in solution.23b An increase in the amount of oxygen changed the ratio to 31:69, and total elimination of oxygen gave exclusively cis-β-methylstyrene oxide. This is consistent with a shift toward the ferryloxy pathway as the oxygen availability decreases, because of the decreasing rate of forming protein peroxyl radicals. Similar trends were found here for H2O2cyt P450 oxidations without electrolysis. A similar dependence of cis-β-methylstyrene product stereochemistry on oxygen availability was found in the electrode-driven oxidations catalyzed by Mb and cyt P450cam. Cis/trans oxide ratios were larger when the (35) DeGray, J. A.; Gunther, M. R.; Tschirret-Guth, R. A.; Ortiz de Montellano, P. R.; Mason, R. P. J. Biol. Chem. 1997, 272, 2359-2362.
Catalyzed Oxidation of Styrene
reaction system was open to air than under oxygen (Figures 2 and 3), but the difference in solution electrolyses may be insignificant. The decrease in cis/trans oxide ratio under oxygen is more dramatic for the protein-polyion films. For electrolyses on Au with cyt P450cam in solution, the cis/trans ratio remains relatively large even under oxygen, suggesting a strong preference for the iron-oxo pathway when cyt P450cam is dissolved in solution and perhaps an influence of the electrode surface. On the other hand, saturation with oxygen when the amount of hydrogen peroxide was small gave a low cis/trans product ratio with cyt P450cam. While the two oxidation pathways in Scheme 2 are well documented for Mb-H2O2,23 we are unaware of such reports concerning cyt P450-H2O2. However, our results clearly indicate one pathway, presumably iron-oxygen based, which strictly preserves the stereochemistry of the reactant as in the native enzyme system.14,15 The second, competing dioxygen-dependent pathway is nonstereoselective. It is possible, but yet to be confirmed, that the second pathway involves a protein peroxyl radical as in Mb, in which the peroxyl radical is presumed to form from reaction of •X-MbFe(IV)dO with O2.23,36 It is of interest that high oxygen availability during electrolysis also (36) Also, participation of superoxide radical, a possible decomposition product of a ferric-peroxo intermediate,5 cannot be ruled out at present.
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promotes protein decomposition in the films. Decomposition of Mb in the Mb-H2O2 system via dimerization is also thought to be mediated by protein radicals.33 Conclusions Compared to solutions and surfactant films, ultrathin polyion films grown layer-by-layer on Au-MPS electrodes gave the best catalytic activities for electrochemicalenzyme oxidations of styrenes catalyzed by cyt P450cam and Mb. These films enabled studies of enzyme-like catalysis employing only tiny amounts of protein, less than 1 nmol/electrode. Dependence of product stereochemistry on the amount of oxygen available revealed two pathways for olefin oxidation by cyt P450-H2O2 systems driven by electrolysis or by addition of H2O2. Acknowledgment. E. coli DH 5R-containing cyt P450cam cDNA was the generous gift of Dr. J. A. Peterson, Dallas. This work was supported by Grant No. ES03154 from the National Institute of Environmental Health Science (NIEHS), NIH. Contents are solely the responsibility of the authors and do not necessarily represent official views of NIEHS, NIH. Supporting Information Available: One figure and detailed experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org. LA990685K
