Activation mechanism of the Active Site of [NiFe] hydrogenas

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[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