Structural Study of the Mechanism of Hydrogen Activation on Ni-enzymes

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We have studied newly found NAD+-reducing four subunits [NiFe] hydrogenase and O_2-tolerant membrane-bound [NiFe] hydrogenase (MBH) in this project. In 2010-2012, we have developed the new protocols for inoculation of the bacterium and purification for NAD+-reducing four subunits [NiFe] hydrogenase. For MBH, the x-ray crystal structures of as-isolated, H2-reduced and chemically super-oxidized MBH at high resolution has been successfully determined. Overall structure of the molecule and the coordination geometry of the Dinuclear Ni-Fe active site of the MBH are almost identical to those of the standard O_2-sensitive [NiFe] hydrogenase. The proximal iron-sulfur (Fe-S) cluster, however, has a [4Fe-3S]-6Cys structure. When the enzyme is chemically oxidized, the cluster supplies additional electrons to the Ni-Fe active site, and a deprotonated amide nitrogen of the polypeptide backbone coordinates the Fe atom stabilizing the super-oxidized state of the cluster.