| Project/Area Number |
62480460
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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 | Keio University, School of Medicine |
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Principal Investigator |
MAKINO Ryu Associate Professor, School of Medicine, Keio University, 医学部, 講師 (40101026)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Yoshifumi Assistant Professor, Pharmaceutical Science, Tokyo Universit, 薬学部, 助手 (70107390)
WATANABE Yoshihito Assistant Professor, School of Medicine, Keio University, 医学部, 助手 (10201245)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Keywords | Cytochrome P450 / Horseradish peroxidase / Chloroperoxidase / Oxygenated form / Resonance Raman spectrum / 酸素化型反応中間体 / 赤外吸収スペクトル / チトクロムP450cam / 酸素化型 / アロマターゼ反応 / O_2伸縮振動 |
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| Research Abstract |
Heme-containing enzymes yield nearly identical reaction intermediates such as Fe(III)-O2^- and Fe(IV)=O species. Nevertheless, the reactive oxygen in the intermediates was incorporated into the substrate in the reaction by cytochrome P450, while that is meraly reduced to water in the peroxidase reaction. To elucidate the reactivity difference, effects of the fifth axial and distal side amino acids of the heme-iron on the structure of the heme-bound ligand were analyzed by resonance Raman and infrared spectroscopic methods. The findings are summarized as follows; 1) From the comparison of the stretching frequencies of nitric oxide (NO) bound to cytochrome P450cam, chloroperoxidase (CPO) and horseradish peroxidase (HRP), it was suggested that the cystein thiolate as the fifth axial ligand in P450cam and CPO donated the electron density to the heme-bound ligand, thereby decreasing the N-O bond strength. 2) The presence of a dissociable amino acid residue was found to locate in the distal
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side of the heme in peroxidases (HRP and CPO), while that was absent in P450cam. Further, in a reaction intermediate, compound II, of HRP the distal amino acid residue was responsible for hydrogen-bond fromation with the oxygen of Fe(IV)=O, thereby regulating the reactivity of compound II. In all the enzymes examined, the addition of substrate affected the C-O stretching frequency of the ligand carbon monoxide, indicating that the bound-ligand interacted with the suvstrate. 3) The stretching vibration of the bound oxygen in the oxygenated form of CPO located at around 1130 cm^<-1> which essentially agreed with those of P450cam and myoglobin.
These results suggest that the reactivity of the heme-bound oxygen, i.e. the function of the heme-enzymes is determined by the interaction with the amino acid residue in the distal side of the heme-iron, rather than the effect of the electron conating capacity of the fifth axial ligand. Interestingly, the O-O stretching frequency of oxygenated forms was found to be independent of an electronic character of the fifth axial amino acid residue. Less
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