STRUCTURE DETERMINATION OF NiFe HYDROGENASE. Yoshiki Higuchi, Noritake Yasuoka*, Division of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan, ^*Department of Life Science, Himeji Institute of Technology, 1479-1 Kanaji, Kamigori, Hyogo 678-12, Japan

NiFe hydrogenase from sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F is a membrane protein composed of heterodimer of 62.5 and 28.8 kDa. It has two Fe₄S₄, one Fe₃S₄ clusters, and one nickel atom as active centers. The single crystals of hydrogenase was prepared from 40% of 2-methyl-2,4,pentanediol solution ( space group is P 2₁2₁2₁, a =101.0 b =126.5 c =66.5Å). The structure determination has been carried out by the multiple isomorphous replacement (MIR) method combined with the multiwavelength anomalous diffraction (MAD) effect from native crystal. Six derivatives were successfully prepared and showed effective changes in their diffraction intensity. Native anomalous diffraction data were collected at five wavelengths (1.040, 1.489, 1.730, 1.743 and 1.750Å) from one crystal. Native phases were obtained from MIR and MAD data set using the MLPHARE in CCP4 program package. The initial electron density map at 3.0Å was not in good quality but had many ghost peaks around heavy atom binding sites. The quality of electron density map was slightly improved by iterative solvent flattening and histogram mapping. The main chain tracing was carried out at 3.5 Å map, and new molecular envelope was recalculated from this main chain backbone model. The phases were further improved by solvent flattening using this envelope. The new electron density map calculated at 3.0Å was clear enough to locate the side chain atoms and even carbonyl oxygen atoms. The structure of hydrogenase is now in under refinement using the program XPLOR. The R-value was reduced to 0.30 from the initial model (0.510) by only positional refinement. The structure comparison with NiFe hydrogenase from the different strain, Desulfovibrio gigas will be discussed.