Electron-Transfer Reactions and Functionalization of Cytochrome

The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, ...
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Electron-Transfer Reactions and Functionalization of Cytochrome P450cam Monooxygenase System in Reverse Micelles Hirofumi Ichinose,† Junji Michizoe,‡ Tatsuo Maruyama,‡ Noriho Kamiya,‡ and Masahiro Goto*,†,‡ Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, 4-1-8, Honmachi, Kawaguchi, Saitama 332-0012, Japan and Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-8581 Japan Received January 27, 2004. In Final Form: March 30, 2004 Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a “monooxygenation” mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media.

Introduction Enzymes catalyze highly selective chemical transformations under mild conditions. Because of their great advantages as catalysts, many researchers have made an effort to utilize enzymatic reactions for a series of practical applications.1 Since a number of organic substrates exhibit poor solubility in aqueous solution due to their hydrophobicity, enzyme-based chemical transformations in organic solvents are an attractive research field in bioengineering.2,3 However, native enzymes do not always exhibit nativelike structure and function in nonaqueous media. Reverse micelles, which are thermodynamically stable self-assemblies formed by surfactant molecules, provide unique nanosized aqueous environments in organic media.4 The water pools in the micellar cores are surrounded by surfactant molecules and sheltered from exposure to bulk organic solvents. Because of their unique aqueous environment, reverse micelles possess a superior potential to entrap proteins in organic media without denaturation.5,6 Therefore, research has focused on the utilization of reverse micelles to enhance enzymatic activity in organic media. Using the reverse micellar system, a series of * To whom correspondence should be addressed. Phone: +81-92-642-3575. Fax: +81-92-642-3575. E-mail: mgototcm@ mbox.nc.kyushu-u.ac.jp. † Japan Science and Technology Agency. ‡ Kyushu University. (1) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409, 258-268. (2) Go¨lken, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 69-74. (3) Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A. Ind. Eng. Chem. Fundam. 1986, 25, 603-612. (4) Oldfield, C. Biotechnol. Genet. Eng. Rev. 1994, 12, 255-327. (5) Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A. Ind. Eng. Chem. Fundam. 1986, 25, 603-612. (6) Michizoe, J.; Okazaki, S.; Goto, M.; Furusaki, S. Biochem. Eng. J. 2001, 8, 129-134.

enzymes have been found to exhibit their catalytic activities in organic solvents; however, most of these studies were directed to single-enzyme-based chemical transformations. Since a number of biocatalysts consist of multiprotein components and, in particular, proteinmediated electron-transfer reactions are one of the most essential events involved in a series of bioprocesses, we explored the functionalization of a multicomponent enzyme system in reverse micelles. We focused on cytochrome P450 monooxygenase (P450), a remarkably diverse oxygenation catalyst found in nature,7 because this enzyme is thought to be potentially useful in various oxygenation reactions. Among a series of P450s, the camphor-hydroxylating P450 from soil bacterium Pseudomonas putida, cytochrome CYP101 (P450cam; EC1.14.15.1), has been intensively studied as a model enzyme for the heme-thiolate monooxygenases.8-10 Since these investigations reveal that P450cam is capable of oxidizing a series of unnatural hydrophobic substrates,11-14 realizing a P450cam system in reverse micelles can significantly expand the utility in nonaqueous biocatalytic processes. (7) Nelson, D. R.; Kamataki, T.; Waxman, D. J.; Guengerich, F. P.; Estabrook, R. W.; Feyereisen, R.; Gonzalez, F. J.; Coon, M. J.; Gunsalus, I. C.; Gotoh, O.; Okuda, K. DNA Cell Biol. 1993, 12, 1-51. (8) Gunsalus, I. C.; Wagner, G. C. Methods Enzymol. 1978, 52, 166188. (9) Mueller, E. J.; Loida, P. J.; Sligar, S. G. In Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; pp 83-124. (10) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195, 687-699. (11) England, P. A.; Harford-Cross, C. F.; Stevenson, J.-A.; Rouch, D. A.; Wong, L.-L. FEBS Lett.1997, 424, 271-274. (12) Stevenson, J.-A.; Westlake, A. C. G.; Whittock, C.; Wong, L.-L. J. Am. Chem. Soc. 1996, 118, 12846-12847. (13) Jones, J. P.; O’Hare, E. J.; Wong, L.-L. Eur. J. Biochem. 2001, 268, 1460-1467. (14) Grayson, D. A.; Vilker, V. L. J. Mol. Catal. B: Enzymatic 1999, 6, 533-547.

10.1021/la049752n CCC: $27.50 © 2004 American Chemical Society Published on Web 05/18/2004

Cytochrome P450cam Monooxygenase System

In the present study, we first investigated a proteinmediated electron-transfer reaction between truncated cytochrome b5 (tCyt-b5; water-soluble heme-containing domain of cytochrome b5) and a bacterial FAD-containing PdR in a reverse micellar system. Furthermore, we demonstrated the monooxygenation reaction catalyzed by cytochrome P450cam in reverse micelles. This is the first report on a protein-mediated electron-transfer reaction and the functionalization of a multicomponent P450 system in organic media. Experimental Section Chemicals. Bis(2-ethylhexyl)sulfosuccinate sodium salt (aerosol-OT; AOT) and isooctane were purchased from Aldrich and used without further purification. Tween 85 was purchased from Kishida Chemical Co. Ltd. (1R)-(+)-Camphor, ampicillin sodium salt, and NADH were purchased from Wako Pure Chem. Co. δ-Aminolevulinic acid was purchased from COSMO BIO. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was purchased from TaKaRa. Deionized water was obtained using a Milli-Q System (Millipore). Construction of Protein-Expression Plasmids. The nucleotide sequences of PCR primers used are summarized in Table S1 (Supporting Information). The gene encoding cytochrome b5 was synthesized as previously described.15 The gene fragment encoding tCyt-b5 was amplified by PCR, performed with the primer combination of Cyt-1 and Cyt-2 (Table S1). The amplified DNA fragment was digested with NdeI/XhoI and inserted into the same restriction sites in the pET22 vector. The genes encoding putidaredoxin reductase (PdR), putidaredoxin (Pdx), and cytochrome P450cam (P450cam) were amplified from whole cell DNA of P. putida strain PpG 786 (ATCC 29607) by PCR using Pyrobest DNA polymerase (TaKaRa).16 Amplification of each cDNA was performed using the primer combinations of camA-1 and cam-2 for PdR, camB-1 and camB-2 for Pdx, and camC-1 and camC-2 for P450cam (Table S1). The rare start codon (GTG) for the PdR gene was changed to an ATG initiation codon. The amplified cDNAs were sequenced with an automated DNA Sequencer (CEQ 2000XL DNA Analysis System; BECKMAN COULTER) using the CEQ DTCS-Quick Start kit (BECKMAN COULTER). The gene fragments encoding Pdx and P450cam were digested with NdeI/XhoI and inserted into the same restriction sites in the pET22 vector. The gene fragment encoding PdR was digested with NdeI/EcoRI and inserted into the same restriction sites in the pET22 protein expression vector. The constructed protein expression plasmids, pET-b5 for tCyt-b5, pET-camA for PdR, pET-camB for Pdx, and pET-camC for P450cam, are illustrated in Figure S1 (Supporting Information). Expression and Purification of Recombinant Proteins. The protein expression plasmids were transformed into Escherichia coli strain BL21(DE3)pLysS. Transformants were grown in 50 mL of LB medium supplemented with ampicillin (100 mg/L) at 37 °C. The overnight cultures were used to inoculate 1 L of TB medium supplemented with ampicillin (100 mg/L), and cells were grown at 37 °C to an optical density (OD600) of 0.8. The bacterial cultures for the protein expression of recombinant P450cam and tCyt-b5 were also supplemented with δ-aminolevulinic acid (500 µM). When the OD600 reached a value of 0.8, the temperature was lowered to 27 °C, and IPTG was then added to the culture to give a final concentration of 0.5 mM. The culture was grown for 24 h. The cells were harvested by centrifugation and disrupted by sonication in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 10 mM β-mercaptoethanol. The lysates were centrifuged (30 000g, 30 min) at 4 °C and then purified by DEAE anion exchange chromatography. The isolated recombinant proteins were further purified with a Ni-NTA column (HiTrap Chelating HP; Amersham Biosciences) and/or a gel filtration column (HiPrep 16/60 Sephacryl S-200 HR; Amersham Biosciences). All the recombinant proteins were purified to homogeneity and (15) Beck-von Bodman, S. B.; Schuler, M. A.; Jollie, D. R.; Sligar, S. G. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9443-9447. (16) Chakrabarty, A. M. Annu. Rev. Genet. 1976, 10, 7-30.

Langmuir, Vol. 20, No. 13, 2004 5565 analyzed by SDS-PAGE. The recombinant proteins were successfully overexpressed using a pET/E. coli system as soluble and catalytically active form. A typical yield for 1 L of culture media was around 10 mg of homogeneous protein. The concentrations of purified tCyt-b5, P450cam, PdR, and Pdx were determined by measuring the optical densities at 410 ( ) 130.0 mM-1 cm-1), 417 ( ) 115.0 mM-1 cm-1), 454 ( ) 10.0 mM-1 cm-1), and 415 nm ( ) 11.1 mM-1 cm-1) .8,15 Spectroscopic Measurement of Ferric and Ferrous tCyt-b5. A reversed micellar solution harboring tCyt-b5 and PdR was prepared by direct injection of 58 µL of aliquot solution (50 mM Tris-HCl buffer, pH 7.4, containing 173 µM tCyt-b5 and 86 nM PdR) into 940 µL of an isooctane solution containing AOT (100 mM) and Tween 85 (100 mM). Absorption spectra were recorded after the addition of 2 mL of NADH (25 mM) solution or H2O into the reverse micellar solution harboring tCyt-b5 and PdR. The reverse micelles were prepared at Wo ([H2O]/[surfactant]) ) 16.7. The reaction mixture (1 mL) consisted of tCyt-b5 (10 µM), PdR (5 nM), NADH (0.05 or 0 mM) in an isooctane solution containing AOT (100 mM), and Tween 85 (100 mM). All the reverse micellar solutions in the present study were stable and homogeneous during the experimental processes. The absorption spectra were recorded on a UV-vis spectrophotometer (JASCO Ubest-570; JASCO) with a spectral bandwidth of 1.0 nm and a 1-cm light path. Reactions were carried out at room temperature. Kinetic Measurement of tCyt-b5 Reduction by NADH/ PdR. A reversed micellar solution harboring tCyt-b5 was prepared by direct injection of 50 µL of aliquot solution (50 mM Tris-HCl buffer, pH 7.4, containing 173 µM tCyt-b5) into 850 µL of isooctane solution containing AOT (100 mM) and Tween 85 (100 mM). Reversed micellar solutions harboring PdR or NADH were prepared by direct injection of 50 µL of aliquot solution (50 mM Tris-HCl buffer, pH 7.4, containing 20 µM PdR or 100 mM NADH) into 950 µL of isooctane solution containing 100 mM each of AOT and Tween 85. To initiate tCyt-b5 reduction, 50 µL of PdR reverse micellar solution was exogenously added into premixed tCyt-b5 (900 µL) and NADH (50 µL) reverse micellar solution. As a comparison, tCyt-b5 reduction was initiated by the exogenous addition of 50 µL of NADH reverse micellar solution into premixed tCyt-b5 (900 µL) and PdR (50 µL) reverse micellar solution. After gently inverting the mixture, the reaction system immediately turned into a homogeneous solution. The final reaction mixture (1 mL) consisted of tCyt-b5 (8.7 µM), PdR (50 nM), and NADH (0.25 mM) in an isooctane solution of AOT (100 mM) and Tween 85 (100 mM). The time course of tCyt-b5 reduction was monitored at 424 nm. Reactions were conducted at room temperature. Camphor Hydroxylation in the Reverse Micellar System. The reverse micellar solution harboring P450cam, Pdx, and P450 was prepared by direct injection of aliquot enzyme cocktail (50 mM Tris-HCl buffer, pH 7.4, containing 25 mM KCl, 12.5 µM PdR, 125 µM Pdx, and 25 µM P450cam) into an isooctane solution of camphor, AOT, and Tween 85. The reaction was initiated by the exogenous addition of an aqueous NADH solution. The reverse micelles were prepared at Wo ([H2O]/[surfactant]) ) 27.8. The reaction mixture (1 mL) consisted of PdR (1 µM), Pdx (10 µM), P450cam (2 µM), camphor (2 mM), KCl (2 mM), NADH (2 mM), AOT (100 mM), and Tween 85 (100 mM) in isooctane. Product Analysis. GC/MS analysis was performed at 70 eV on a Shimadzu GCMS-QP5050A equipped with a 30 m fused silica column (HP-5, Agilent Technologies). The oven temperature was programmed to ramp from 80 to 300 °C at a rate of 8 °C /min.

Results and Discussion Protein-Protein Interaction and Electron-Transfer Reaction in Reverse Micelles. Most redox proteins catalyze electron-transfer reactions by directly interacting with their redox partners. In a reverse micellar system, however, it has commonly been accepted that the macromolecular protein is segregated in the nanospaced micellar core. Therefore, it has been suggested that the exchange rate of macromolecules such as proteins might be much

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slower than that of small molecules.17 This feature might be disadvantageous for efficient protein-protein interaction. In fact, not much is known about how a multicomponent protein system could work in reverse micelles. We investigated a protein-mediated electron-transfer reaction using bacterial flavoprotein PdR. PdR shuttles electrons from NADH to a wide variety of electron-carrier proteins; however, Pdx is the natural redox partner of PdR in the bacterial camphor hydroxylating system. Due to the difficulty of performing a spectroscopic characterization of electronic states of Pdx in reverse micelles, the hemecontaining electron-transfer protein tCyt-b5 was chosen as an electron acceptor for PdR to better understand the possible involvement of the protein-mediated electrontransfer reaction in the reverse micellar system. Enzymatic reactions in reverse micelles have been extensively investigated using ionic surfactants, for example, AOT. However, a strong electrostatic interaction between surfactant headgroups and enzymes is known to often cause protein denaturation. When PdR was solubilized in the AOT reverse micellar solution, the absorption spectrum of PdR was significantly changed from that in an aqueous buffer system (data not shown), suggesting that a strong electrostatic interaction caused significant conformational change around the flavin prosthetic group. On the other hand, it has been shown that nonionic surfactants relieve protein denaturation caused by such electrostatic interactions.18,19 To avoid protein denaturation, a hybrid reverse micellar system formed with anionic AOT and nonionic Tween 85 was chosen in the present study. Figure 1 shows the absorption spectra of tCyt-b5 in the reverse micellar solution. Ferric tCyt-b5 appeared to be effectively converted to the ferrous form by the addition of NADH and PdR. The absorption spectra of the ferric and ferrous tCyt-b5 in the reverse micellar solution were similar to those in the aqueous buffer solution. Thus, one can conclude that PdR and tCyt-b5 were successfully encapsulated in AOT/Tween 85 reverse micelles. It also implies that tCyt-b5 can serve as a probe molecule to evaluate a protein-mediated electron-transfer reaction. These results indicate that reverse micelles allow proteinmediated electron-transfer reactions to take place. Although it has commonly been accepted that reverse micellar systems involve rapid molecular exchange events for low-molecular-weight compounds, protein-protein interactions have been poorly characterized in reverse micelles. If reverse micelles do not allow interactions between macromolecular proteins separately packed in different micellar cores, PdR would be capable of transporting electrons to only tCyt-b5 coexisting in the same micellar cores. On the other hand, if reverse micelles allow interactions between proteins separately packed in different micellar cores, PdR would be capable of catalyzing the electron-transfer reaction from NADH to tCyt-b5, which was segregated in different micellar cores. To clarify the mechanism of protein-mediated electron-transfer reaction in reverse micelles, tCyt-b5 reduction was attempted by mixing the separately prepared reverse micellar solutions. Figure 2 shows the time course of tCytb5 reduction by NADH via PdR in AOT/Tween 85 reverse micelles. As shown in the figure, the tCyt-b5 reduction was found to require both NADH and PdR. In addition, (17) Nicot, C.; Waks, M. Biotechnol. Genet. Eng. Rev. 1996, 13, 267314. (18) Naoe, K.; Nishino, M.; Ohsa, T.; Kawagoe, M.; Imai, M. J. Chem. Technol. Biotechnol. 1999, 74, 221-226. (19) Shioi, A.; Kishimoto, T.; Adachi, M.; Harada, M. J. Chem. Eng. Jpn. 1997, 30, 1130-1133.

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Figure 1. Cytochrome b5 reduction catalyzed by putidaredoxin reductase. The reaction mixture (1 mL) consisted of tCyt-b5 (10 µM), PdR (5 nM), and NADH (0.05 mM) in an isooctane solution of AOT (100 mM) and Tween 85 (100 mM). The reaction was initiated by adding NADH. The reverse micelles were prepared at Wo ([H2O]/[surfactant]) ) 16.7. The absorption spectrum of ferrous tCyt-b5 was scanned 2 min after the addition of NADH.

Figure 2. Time-course analysis of tCyt-b5 reduction by PdR in reverse micellar system. The reaction mixture (1 mL) consisted of tCyt-b5 (8.7 µM), PdR (A, B, C, 50 nM; D, 0 nM), and NADH (A, B, D, 0.25 mM; C, 0 mM) in an isooctane solution of AOT (100 mM) and Tween 85 (100 mM). The reaction was initiated by the exogenous addition of reverse micellar solution harboring NADH (A) or PdR (B). The time course of tCyt-b5 reduction was monitored at 424 nm.

it was clearly shown that the reaction profiles were virtually identical for the two reaction systems (the reaction initiated by sequential addition of PdR reverse micellar solution into premixed tCyt-b5/NADH reverse micelles and the reaction involving sequential addition of NADH reverse micellar solution into premixed tCyt-b5/ PdR reverse micelles) (Figure 2). These results imply that

Cytochrome P450cam Monooxygenase System

rapid protein-protein interaction takes place in the reverse micellar system, which facilitates the proteinmediated electron-transfer reaction. Thus, it can be concluded that protein-protein interaction in the reverse micellar system allows “micelle-to-micelle” electrontransfer reactions to take place. On the other hand, if reverse micelles allow a rapid protein exchange event as well as small molecules, one mechanism involves the electron-transfer reaction in transiently combined reverse micelles when all the components are contained, which results in “one-micelle” electron-transfer reactions. Although further studies are required to better understand the reaction mechanism, protein-mediated electrontransfer reactions in the reverse micellar system would enable us to utilize multicomponent enzyme systems in organic media. Functionalization of Bacterial Cytochrome P450 in Organic Media. A reverse micellar system was clearly shown to be useful for a protein-mediated electron-transfer reaction in organic media. Thus, we attempted to activate the P450 monooxygenase system in reverse micelles. The bacterial P450cam enzyme system consists of three protein components, a heme-containing P450cam, an iron-sulfur protein putidaredoxin (Pdx), and an FAD-containing putidaredoxin reductase (PdR), as the set of monooxygenation catalysts.8 In this monooxygenase system, electrons are transferred from NADH via the flavin group of PdR to the 2Fe-2S center of Pdx and then to the heme iron of P450cam. In addition to the role of Pdx as an electron carrier, Pdx also plays an “effector” role by directly interacting with P450cam.20 Therefore, the mechanism of camphor hydroxylation catalyzed by the P450cam system involves site-specific direct interaction of protein components. Figure 3 shows absorption spectra for P450cam in AOT/ Tween 85 reverse micelles. The Soret band for the ferric low-spin state of P450cam was observed at 417 nm (Figure 3). When P450cam was incubated with d-camphor in reverse micelles, P450cam was effectively converted to the ferric high-spin state (Figure 3). The absorption maximum of the Soret band was shifted to 447 nm when P450cam was incubated with PdR, Pdx, d-camphor, and NADH under CO-saturated conditions. These results indicate that the heme environments of P450cam were virtually identical to those in the aqueous buffer solution. Furthermore, the CO-complex formation strongly suggests the electron transfer from NADH to P450cam by way of Pdx and PdR in AOT/Tween 85 reverse micelles. Finally, we tried to reconstruct the P450cam reaction system in AOT/Tween 85 reverse micelles by directly injecting a premixed enzyme cocktail containing PdR, Pdx, and P450cam. The reaction was initiated by the additional injection of NADH into the reverse micellar solution harboring all the protein components. Figure 4 shows the gas chromatography-mass spectrometry (GC/MS) chromatogram of products generated in the reverse micellar system. The GC retention times and the MS fragmentation patterns of the product were identical to those of 5-exohydroxycamphor. The P450cam-mediated reaction did not proceed at all without NADH. Omitting either PdR or Pdx from the protein components did not result in any product formation either. It has been well documented that P450 enzymes are capable of oxidizing substrates via the peroxide shunt pathway. In the peroxygenation reaction, H2O2 reacts with a P450 enzyme to form its high-valent ferryl-oxy complex, (20) Lipscomb, J. D.; Sligar, S. G.; Namtvedt, M. J.; Gunsalus, I. C. J. Biol. Chem. 1976, 251, 1116-1124.

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Figure 3. Absorption spectra of P450cam in reverse micelles. Absorption spectra were recorded for the ferric low-spin state (s), the camphor-bounded ferric high-spin state (‚ ‚ ‚), and the ferrous-CO state with camphor (- - -) of P450cam (3.3 µM) in AOT (100 mM)/Tween 85 (100 mM) reverse micelles in isooctane. The reverse micelles were prepared at Wo ([H2O]/[surfactant]) ) 16.7. The CO-bounded ferrous state of P450cam was prepared by mixing PdR (5 nM), Pdx (10 nM), NADH (0.04 mM), and CO in the reverse micellar solution.

which is a key intermediate in the oxygen-transfer reaction.21 To clarify whether a peroxidative reaction is involved or not, the same reaction was attempted using hydrogen peroxide or m-chloroperbenzoic acid. No product formation was observed from those reactions, implying that the oxygen-transfer reactions must have proceeded via a “monooxygenation” mechanism. However, it also appeared that only 20% of starting substrate was converted to hydroxylated products in the reverse micellar system. Since the monooxygenation reaction catalyzed by P450 is well known to undergo a branched uncoupling reaction,22,23 the uncoupled NADH consumption from product formation might explain the low yield observed in this reaction system. In the present study, we demonstrated the capability of reverse micelles to facilitate protein-mediated electrontransfer reactions, which led us to activate a multicomponent enzyme system in organic media. Although further investigation is required to achieve efficient monooxygenation catalysis, the present study may help us to utilize a P450 monooxygenase system in the enzymatic transformation of hydrophobic substrates. Abbreviations used. AOT, bis(2-ethylhexyl)sulfosuccinate sodium salt; IPTG, isopropyl-1-thio-β-D-galactopyranoside; P450, cytochrome P450; PdR, putidaredoxin reductase; Pdx, putidaredoxin; tCyt-b5, truncated soluble cytochrome b5. Acknowledgment. This research was supported by the Precursory Research for Embryonic Science and (21) Imai, M.; Shimada, H.; Watanabe, Y.; Matushima-Hibiya, Y.; Makino, R.; Koga, H.; Horiuchi, T.; Ishimura, Y. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7823-7827. (22) Jacob, J.; Grimmer, G.; Raab, G.; Schmoldt, A. Xenobiotica 1982, 12, 45-53. (23) Shou, M.; Grogan, J.; Mancewicz, J. A.; Krausz, K. W.; Gonzalez, F. J.; Gelboin, H. V.; Korzekwa, K. R. Biochemistry 1994, 33, 64506455.

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Figure 4. GC/MS analysis of oxidized products from the P450cam reaction in the reversed micellar system. The reaction was initiated by the addition of NADH (A, 2 mM; B, 0 mM) into the reversed micellar solution harboring 2 µM P450cam, 10 µM Pdx, 1 µM PdR, and 2 mM KCl. The reaction mixture was incubated for 1 h at 37 °C. Reaction products were analyzed directly by GC/MS using 2 mM n-dodecane as the internal standard. (Inset) Mass spectrum of 5-exo-hydroxycamphor.

Technology (PRESTO) of Japan Science and Technology Agency (to H.I. and M.G.), a Grant-in-Aid for the 21st Century COE Program, “Functional Innovation of Molecular Informatics” from the Ministry of Education, Culture, Science, Sports and Technology of Japan (to M.G.), and partly by Showa-Shell Sekiyu Foundation for Promotion of Environmental Research (to N.K.).

Supporting Information Available: Table containing nucleotide sequences of the primers for PCR (Table S1) and figure showing constructed protein expression plasmids, pET-b5 for tCyt-b5, pET-camA for PdR, pET-camB for Pdx, and pET-camC for P450cam (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org. LA049752N