Activation mechanism of the Active Site of [NiFe] hydrogenas
| Project/Area Number |
16074214
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | University of Hyogo |
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Principal Investigator |
HIGUCHI Yoshiki University of Hyogo, Graduate School of Life Science, Professor (90183574)
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| Co-Investigator(Kenkyū-buntansha) |
小森 博文 兵庫県立大学, 大学院生命理学研究科, 助手 (30382261)
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| Project Period (FY) |
2004 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 2007: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2006: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | [NiFe] hydrogenase / ultr-high resolution x-ray structure analysis / sulfet-reducing baclerium / neutron structure analysis / fuel cell / bio-hydrogen / metello-protein / deuterated protein / 超高分解能X線構造解析 / CO結合型ヒドロゲナーゼ / 準動的X線結晶解析法 / クリーンエネルギー |
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| Research Abstract |
[NiFe] hydrogenase is composed of two subunits and has a Ni-Fe active site in the large subunit. Ni is coordinated by four cysteine sulfurs (two of them form a bridge between Fe and Ni). Fe has additional non-protein diatomic ligands, and a third bridge in the oxidized form. The as-purified (inactive-oxidized), Ni-C (active-reduced) and CO-inhibited forms of the [NiFe] hydrogenase from D. v. Miyazaki F were already reported. Recently, it was found that the oxidized form is a mixture of Ni-A and Ni-B, and the enzyme is activated from Ni-A to Ni-C through Ni-B.
1. In this project, we have discovered the protocol to prepare pure Ni-A from Ni-B by using 50 mM Na_2S and exposure to O_2, and elucidated the crystal structures of Ni-A and Ni-B. We found that Ni-B has a monatomic non-protein bridging ligand (X_<B1>), whereas the Ni-A has a diatomic species (X_<A1>-X_<A2>). In addition, the sulfurs of cysteines are found to have a modified atomic species (X_<546>) in both Ni-A and Ni-B.
2. In orde
… More
r to clarify the activation mechanism of H_2 at the active site, we have succeeded in preparing the large single crystal (1.0 mm^3) of the enzyme in D_2O solution. Neutron diffraction experiments showed that- the crystal diffract about 10 A. The crystallization condition for the larger crystals is being improved.
3. The Ni-Fe active site is matured by a series of the proteins coded in the hyp operon. HypE is involved in the biosynthesis of CN which is coordinated to Fe. We determined the crystal structures of HypE in the absence and presence of ATP at 2.0 and 2.6 A resolution, respectively. Comparison of the structures reveals that the binding of ATP does not entail an overall structural change. The residue Cys341 at the C-terminus, whose thiol group is supposed to be carbamoylated prior to the nitrile group synthesis, is completely buried within the protein, and is located in the vicinity of the ・-phosphate group of the bound ATP. The obtained structure suggests that the catalytic reaction occurs in this configuration but that a conformational change is required for the carbamoylation of Cys341.
4. CooA is a transcription factor, and is responsible for the expression of CO-tolerant hydrogenases in some bacteria. We have determined the crystal structure of an imidazole (Im)-bound CooA from C. hydrogenoformans (Ch-CooA) at 2.2 A. The structure of Ch-CooA reveals that Im binds to the heme Fe, and replaces the N-terminus, as does CO. Even though the ligand exchange, Im-bound Ch-CooA remains in the inactive form. These results indicate that the release of the N-terminus resulting from Im-binding is not sufficient to activate CooA. The structure provides new insights into the structural changes required to achieve activation. Less
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Report
(5 results)
Research Products
(58 results)
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[Journal Article] Metalation of Expanded Porphyrins: A Chemical Trigger Used To Produce Molecular Twisting and Mobius Aromaticity2008
Author(s)
Y, Tanaka, S.Saito, S.Mori, N.Aratani, H.Shinokubo, N.Shibata, Y.Higuchi, Z.S.Yoon, K.S.Kim, S.B.Noh, J.K.Park, D.Kim, A.Osuka
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Journal Title
Angew.Chem.Int.Ed. 47
Pages: 681-684
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Peer Reviewed
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[Journal Article] A Dinuclear Ni(mu-H)Ru Complex Derived from H_22007
Author(s)
S. Ogo, R. Kabe, K. Uehara, B. Kure, T. Nishimura, S. C. Menon, R. Harada, S. Fukuzumi, Y. Higuchi, T. Ohhara, T. Tamada, R. Kuroki
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Journal Title
Science 316
Pages: 585-587
Description
「研究成果報告書概要(和文)」より
Related Report
Peer Reviewed
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[Journal Article] A Dinuclear Nl(mu-H)Ru Complex Derived from H_22007
Author(s)
S. Ogo, R. Kabe, K. Uehara, B. Kure, T. Nishimura, S. C. Menon, R. Harada, S. Fokozumi, Y. Higuchi, T. Ohhara, T. Tamada, R. Kuroki
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Journal Title
Science 316
Pages: 585-587
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] A Dinuclear Ni(mu-H)Ru Complex Derived From H_22007
Author(s)
S.0go, R.Kabe, K.Uehara B.Kure, T.Nishimura, S.C.Menon, R.Harada, S.Fukuzumi, Y.Higuchi, T.Ohhara, T.Tamada, R.Kuroki
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Journal Title
Science 316
Pages: 585-587
Related Report
Peer Reviewed
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[Journal Article] High Fidelity Self-Sorting Assembling of meso-Cinchomeronimide Appende dmeso-meso Linked Zn(II) Diporphyrins2006
Author(s)
T.Kamada, N.Aratani, T.Ikeda, N.Shibata, Y.Higuchi, A.Wakamiya, S.Yamaguchi, K.S.Kim, Z.S.Yoon, D.Kim, A.Osuka
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Journal Title
J. Am. Chem. Soc. 128
Pages: 7670-7678
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[Journal Article] Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F2004
Author(s)
N.Shibata, K.Suto, E.Ichimura, K.Yoshimura, K.Muneo, S.Tomigami, Y.Morimoto, M.Ogata, T.Yagi, Y.Higuchi, N.Yasuoka
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Journal Title
Prot.Pep.Let 11-1
Pages: 93-96
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