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A membrane-bound hydrogenase fromDesulfovibrio vulgarisMiyazaki F is a metalloenzyme that contains a binuclear Ni–Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni–Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni–Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Investigation has been made to evaluate the effect of REM addition on the solidification macrostructure of commercial ingots and continuously cast slabs. The results are as follows:(1) REM addition brings about diminution of macro-segregation, enlargement of equiaxed zone, and reduction of loose structure.(2) Also, the starting position of the formation of the inverse V-segregation moves toward the center of ingot and the segregated area expands. The length of segregation line and the segregation ratio are reduced.(3) Autoradiographic observation does not show large differences in macrosolidification rate. However, mushy zone at the bottom of an ingot is formed earlier.(4) REM-oxides, sulphides, and oxysulphides are mainly precipitated in dendrite arms. The constituents of inclusions are varied depending on the methods of REM addition.Non-metallic inclusions formed by REM addition seem to act as nucleation catalyst and thus improve not only the microstructure but also the macrostructure of the casts.