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X-ray crystallography is the most powerful tool for obtaining structural information about protein molecules, affording accurate and precise positions for all of the atoms in the protein except for hydrogen. However, hydrogen species play crucial roles in the physiological functions of enzymes, including molecular recognition through hydrogen bonding and catalytic reactions involving proton transfer. Neutron crystallography enables direct identification of the positions of hydrogen spe-cies. [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an enzyme that catalyzes the reversible oxidation of molecular hydrogen. It contains a bimetallic Ni–Fe active site for the catalytic reaction and three Fe–S clusters for electron transfer. Previous X-ray structure analyses of the enzyme under various oxidation conditions have revealed that the active site changes its coordination structure depending on the redox state. In the inactive air-oxidized form, an oxygen species was identified between the Ni and Fe atoms, whereas in the active H2-reduced form, subatomic-resolution X-ray structure analysis and single-crystal EPR analyses indicated a hydride ligand between the two metal atoms. However, the assignment of the hydride moiety by X-ray crystallography remains controversial, and the proton transfer pathways in the molecule are still ambiguous. To allow neutron diffraction experiments, large crystals of [NiFe]-hydrogenase were prepared by the vapor diffusion method with the macroseeding technique according to the two-dimensional phase diagram (protein concentration vs. precipitant concentration). Neutron diffraction data were collected at approximately 2.0 Å resolution at cryogenic tem-perature using a gas-stream cooling system to trap short-lived intermediates in the catalytic reaction. |
