| Project/Area Number |
12640549
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Inorganic chemistry
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| Research Institution | Cente for Integrative Biosience |
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Principal Investigator |
FUJII Hiroshi Okazaki National Research Institutes, Center for Integrative Bioscience, Associate Professor, 統合バイオサイエンスセンター, 助教授 (80228957)
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| Co-Investigator(Kenkyū-buntansha) |
FUNAHASHI Yasuhiro Okazaki National Research Institutes,Institutes for Molecular Science,Research, 分子科学研究所, 助手 (00321604)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | nitrite reductase / heme / nitric oxide / reaction mechanism / artificial enzvme / protein mutation / ミューテ-ション |
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| Research Abstract |
In this project, we tried to alter the function of myoglobin, which serves as an oxygen storage in a muscle, to nitrite reductase, which reduces nitrite anion to nitric oxide in denitrification process. To prepare the artificial nitrite reductase from myoglobin, we first synthesized the active site model heme of the nitrite reductase. Since heme-d_1, which has been known as an active site of the nitrite reductase, has a dioxoisobacteriochlorin core, we prepared dioxoisobacterichlorin from dimethyldeutrioporphyrin with the oxidation by osmium tetraoxide and the pinocol rearrangement. The absorption spectrum of the synthesized heme-d_1 model was similar to that of nitrite reductase, suggesting the heme-d_1 model works as an active site of the artificial nitrite reductase. Therefore, we further tried to reconstitute the heme-d_1 model into apomyoglobin. The heme-d1 model formed a stable 1 : 1 complex with the apomyoglobin. The spectroscopic data of the complex were close to those of nitrite reductase. On the basis of these results, we further tried to add the protein mutations into the complex. We prepared L29H/F45H/H64Y myoglobin mutant because the structure of the mutant seems to be close to that of nitrite reductase. The mutant myoglobin was successfully expressed by e-coli and isolated. The triple mutant was also formed stable complex with heme-d1 model complex. The artificial nitrite reductase mimics not only the spectroscopic properties but also the reactivity of nitrite reducatse.
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