Reaction Mechansim of [NiFe]-hydrogenase by Neutron Structural Chmeistry

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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.