| Project/Area Number |
09308026
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
YOSHIKAWA Shinya Himeji Institute of Technology, Professor, 理学部, 教授 (40068119)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAI Hiroaki Osaka University, Research Associate, 蛋白質研究所, 助手 (00272162)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥34,400,000 (Direct Cost: ¥34,400,000)
Fiscal Year 1999: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1998: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1997: ¥24,900,000 (Direct Cost: ¥24,900,000)
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| Keywords | Cytochrome c Oxidase / membrane protein / proton pump / active transport / heme protein / mitochondria / electron transfer complex / 生体エネルギー変換 / 電子伝達 / 結晶化 / 酸化還元滴定 |
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| Research Abstract |
A. Cytochrome c Oxidase.
Fully oxidized form : X-ray diffraction spots for the fully oxidized form were obtained up to 1.65 A resolution. The diffraction patterns have provided x-ray structures at 2.30 A resolution at room temperature and at 2.0 A resolution at liquid nitrogen temperature. A peroxide bridging between FeィイD2a3ィエD2 and CuィイD2BィエD2 in the OィイD22ィエD2 reduction site and a covalent linkage between His242 and Tyr244 are seen at 2.30 A resolution. Two independent hydrogen bond networks connect the OィイD22ィエD2 reduction site with matrix space.
Fully reduced form : The OィイD22ィエD2 reduction site in the fully reduced form does not have any peroxide, giving a trigonal planer coordination to CuィイD2BィエD2ィイD11+ィエD1, the stability of which results in the unusual stability of the FeィイD2a3ィエD2ィイD12+ィエD1-OィイD22ィエD2. One of aspartate residue (Asp51) migrates to be exposed to the bulk water phase in the intermembrane space. Asp51 in the fully oxidized state is connected to the matrix space by a hydrogen bond network so that Asp51 can take up protons from the matrix space. Thus, that Asp51 is the site for proton pumping.
Other studies : Redox titration experiments showed that 2 extra electron acceptor sites are present besides the four redox active metal sites in the fully oxidized state as prepared. FTIR results showed that on reduction, one carboxyl group is dissociated, which is most likely to be that of Asp51. These results strongly support the above x-ray structural analysis.
B. NADH-ubiquinone oxidoreductase
Reproducibility of the purification method has been improved significantly. As the first step for elucidation of the reaction mechanism of this enzyme, the initial steady state of the enzyme reaction has been analyzed to reveal an ordered sequential mechanism with ubiquione (QィイD21ィエD2)-NADH-NADィイD1+ィエD1-QィイD21ィエD2HィイD22ィエD2, as the order of substrate bindings and product releasings.
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