| Project/Area Number |
61480469
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | School of Medicine,Keio University |
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Principal Investigator |
ISHIMURA Yuzuru School of Medicine, Keio University, Professor, 医学部, 教授 (40025599)
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| Co-Investigator(Kenkyū-buntansha) |
IIZUKA Tetsutaro School of Medicine, Keio University, Associate Professor (present affiliation; T, 医学部(現理化学研究所無機化学部門主任研究員), 助教授 (30029475)
WATANABE Yoshihito School of Medicine, Keio University, Assistant Professor, 医学部, 助手 (10201245)
MITANI Fumiko School of Medicine, Keio University, Assistant Professor, 医学部, 助手 (60041852)
MAKINO Ryu School of Medicine, Keio University, Assistant Professor, 医学部, 講師 (40101026)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1986: ¥5,400,000 (Direct Cost: ¥5,400,000)
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| Keywords | oxygen activation / infrared spectroscopy / resonance Raman spectroscopy / cytochrome P450cam O-O stretching frequency / プチダレドキシン / ペルオキシダーゼ / Compound II / 振動スペクトル法 / 共鳴ラマン散乱 / 赤外吸収スペクトル法 / 酸素錯体 / ヘム置換法 / 西洋ワサビペルオキシダーゼ / シトクロームP450 |
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| Research Abstract |
The electronic structure of oxygensted and carbonyl complexes of hemecontaining enzymes has been examined by infrared and resonance Raman spectroscopic methods. By comparing the results on cytochrome P450_<cam> with those on other enzymes, the oxygen activation mechanism of P450_<cam> has been beduced as follows.
1) Effector action of substrate; by analyzing the effects of a substrate on the Fe-CO stretch of P450_<cam>-CO complex, the substrate, camphor was found to interact with the Fe-CO portion, thereby weakening the strength of C-O bond.
2) O-O stretch of P450_<cam>-O_2 complex; the O-O stretching frequency of P450_<cam>-O_2 complex detected at 1141 dm ^<-1> was almost identical with that for the oxygenated form of myoglobin, suggesting that the electronic strucxture of both proteins was Fe^<3+>-O_2^-.
3) Effector action of putidaredoxin; by analyses of the C-O stretch in P450_<cam>-CO complex, putidaredoxin was found to bind stoichiometrically to P450_<cam>- CO complex with a weakening in the strength of the C-O bond. Weakening of the C-N bond in C^<15>N^-- ferric P450_<cam> complex was also observed by ^<15> N NMR analyses. From these findings, we speculate that the binding of putidaredoxin facilitates the O-O bond cleavage by weakening the O-O bond strength, being a most important step in the oxygen activation by cytochrome P450_<cam>.
4) The electronic structure of catalytically active intermediate, whose formation has been hypothesized during the one-electron reduction of oxygenated P450_<cam>, has been considered to be isoelectronic to peroxidase intermediate, compound I of II. Even for the peroxidase intermediate, however, its structure remained unclear. From this point of view, the coordination structure of compound II of native, and heme- and metalsubstituted peroxidase was analyzed by a resonance Raman spectroscopy. The results indicate definitively that the coordination structure of compound II is Fe(IV)=O,but not Fe(IV)-OH,
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