CRYSTALLIZATION OF NITRIC OXIDE REDUCTASE CYTOCHROME P450NOR FROM FUSARIUM OXYSPORUM. Park S.-Y., Shimizu H, Adachi S, Udai P. S., Iizuka T., Shiro Y., *Shoun H., #Nakagawa A., #Tanaka I., The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351 -01, Japan, *Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan, #Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan
Cytochrome P450 nitric oxide reductase purified from Fusarium oxysporum (P450nor, Mr = 46 kDa) is an unique heme enzyme that catalyzes the reduction of nitric oxide with electrons directly transfer from NADH. The cytochrome P450nor was crystallized with the vapour diffusion method using the sitting drop technique. The crystals were grown in 100 mM MES buffer at pH 5.5 using PEG 4000 as precipitant. The crystal belongs to the space group P212121 in an orthorhombic system. The cell dimensions are determined a = 55.02 Å, b = 82.42 Å and c = 87.06 Å The asymmetric unit contains one molecule of P450nor protein with a corresponding crystal volume per protein mass (Vm) of 2.17 Å3 Da-1. X-ray data collection were carried out at station BL6A of the Photon Factory, Japan. After data reduction (processing package DENZO and SCALEPACK), native data set consists of 80156 measurements of 23943 unique reflections with an R merge 5.2 %. The completeness of the data set is 87.1 % for the 100 - 2.0 Å range. Position of the Fe atom from anomalous dispersion; Data collection were performed with a Weissenberg camera for macromolecular crystallography and imaging plates as detector of the Photon Factory (BL6A, [[lambda]] = 1.70 Å). A total of 58480 observations corresponding to 13386 unique reflections were collected to 2.5 Å resolution with R merge 5.1 %. Completeness of the data set is 97.3 % for the 100 - 2.5 Å range. No serious radiation damage to the crystal was detected during the data collection. Bijvoet anomalous Patterson map shows clear Fe-Fe self vectors on the Harker sections. We intend to make use of the anomalous-dispersion effect of iron atom combined with multiple isomorphous replacement for structure determination. Preparation of heavy atom derivatives for phase determinations are in progress.