X-RAY CRYSTAL STUDIES OF HYDROXYLAMINE OXYDOREDUCTASE. Igarashi N, Moriyama H, Fujiwara T, Fukumori Y and Tanaka N, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226 Japan

The autotropic bacterium Nitrosomonas europaea obtains energy for growth by the oxidation of ammonia to nitrite. Hydoxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite. The enzyme contains eight hemes per subunit (63kDa) which participate in catalytic function and electron transport. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. Crystallization of HAO was performed by the vapor diffusion method using ammonium sulfate as precipitant. The space group was P63 with cell dimensions a = b = 96.2 Å c = 265.6 Å and with two subunits per asymmetric unit. It was found that the crystal specimen was hemihedrally twinned, which was firstly detected by the facts that the distribution of intensities reflected on a varied degree of twinning for each crystal and showed a pseudo 6/mmm Laue symmetry and lack of heavy-atom cross vectors between positions related bythe pseudo heigher symmetry operation in MIR. To deconvolute the intensities into the separated ones, the Britton's principle (Acta Cryst., A28, 296-297, 1972) was applied. The resultant indicated that the pseudo higher symmetry operation disappeared in the difference Patterson map. The observation of the crystallizing process shown that HAO crystal was composed of the two twin domains, which was caused by the crystal growth from two very fine crystals orientated exactly in the opposite direction from each other. To collect the untwinned data set, the uniform part at the edge of a crystal was aimed to input the X-ray using the fine focused SR beam (BL6A at PF). Two heavy atom derivatives were prepared by soaking in 1mM HgAc2 and 1.4mM K₂PtC_l4, respectively. The heavy-atom binding sites were confirmed by both isomorphous and Bijvoet difference Patterson map. The structure determination by MIRAS method is now in progress.