2013 Fiscal Year Final Research Report
Reaction Mechansim of [NiFe]-hydrogenase by Neutron Structural Chmeistry
| Project/Area Number |
24657077
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | University of Hyogo |
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Principal Investigator |
HIGUCHI Yoshiki 兵庫県立大学, 生命理学研究科, 教授 (90183574)
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| Project Period (FY) |
2012-04-01 – 2014-03-31
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| Keywords | ヒドロゲナーゼ / X線結晶解析 / 反応機構 / Ni酵素 / 燃料電池 |
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| Research Abstract |
The goal of this study is to elucidate the reaction mechansim of at the active site of [NiFe]-hydrogenase by neutron diffraction study. For this purpose, we have tried to prepare large crystals of hydrogenase in high quality in D2O.
We have improved the crystallization condition by using 2-methyl-2,4-pentanediol(MPD) as a precipitant in D2O. We found that MPD is oxidized to be 4-hydroxy-4-methyl-2-pentanone(HMPO) which causes a serious damage to the quality of the crystals. We have successfully determined the more suitable crystallization condition under anaerobic condition. As a result, we have succeeded in prearation of a crystal with a volume of 11 mm3. In addition, we have found the condition to freeze the large crystals without damage. Unfortunately, the reactor (neutron source) of JAEA in Tokai is still in preparatory period due to a serious disaster in the area of East Japan in 2011, we will try to do neutron diffraction study in the near future.
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[Presentation] Purification and Crystallization of NAD+-reducing [NiFe]-hydrogenase2014
Author(s)
H. Nakagawa, M. Taketa, M. Habukawa, K. Kihira, N. Shibata, H. Nishihara, K.-S. Yoon, S. Ogo, Ma. Ishii, Y. Igarashi, Y. Shomura and Y. Higuchi
Organizer
第1回ピコバイロジー研究所国際シンポジウム
Place of Presentation
兵庫
Year and Date
20140107-08
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