Crystallographic Studies of Flavoreductases in Electron Transport Systems of Liver Microsomes

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We have carried out X-ray crystal structure analyses of two flavoreductases (NADH-cytochrome b_5 reductase and HADPH-cytochrome P450 reductase) located in two electron transport pathways in liver microsomes where NADH and HADPH function as initial electron donors, respectively.NADH-cytochrome b_5 reductase has so far been crystallized by ourselves. We searched two good heavy-atom derivatives in order to obtain the electron density map based on the multiple isomorphous replacement procedure. In spite of many attempts, however, we could find only one derivative. Therefore, we calculated the electron density map at 5A resolution by the single isomorphous replacement. This electron density map was in good quality which enabled us to distinguish between the protein and solvent regions. Furthermore, we collected intensity data at the higher resolutions and used synchrotron radiation to measure precise intensities including anomalous dispersion effects. We also drawn the electron density map based on these data. We tried to interpret these maps, and then we estimated the outline of the molecular shape and roughly traced the folding of this enzyme. We also estimated the FAD binding position which is one of the most important key points in this projects. However, to make more precise discussion, we need better quality electron density maps. It is the most important problem in the near future to improve the quality of these density maps.We have tried to crystallize HADPH-cytochrome P450 reductase. Initially we searched precipitants effective to this enzyme and were successful in finding a few reagents. We tried many crystallization conditions using these precipitants. But, we could not yet obtained any good crystals suitable for X-ray diffraction works.