Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.
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.
Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, 4-1-8, Honmachi, Kawaguchi, Saitama 332-0012, Japan.
Hirofumi
H
Ichinose
Junji
J
Michizoe
Tatsuo
T
Maruyama
Noriho
N
Kamiya
Masahiro
M
Goto
D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004579:Electron Transport; D008823:Micelles; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology; D012997:Solvents; D013057:Spectrum Analysis
D008823:Micelles; D012997:Solvents; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase