Neutron structural analysis of NADH-cytochrome reductase

Hirano, Yu; Yamada, Mitsugu*; Kurihara, Kazuo; Shoyama, Yoshinari*; Kuroki, Ryota; Kusaka, Katsuhiro*; Kimura, Shigenobu*; Takeda, Kazuki*; Miki, Kunio*; Tamada, Taro

NADH-cytochrome reductase (b5R), a flavoprotein consisting of NADH- and FAD- domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome . The reaction catalyzed by plays a role in fatty acid synthesis, cholesterol synthesis, and xenobiotic oxidation as a member of the electron transport chain on the endoplasmic reticulum. We have already determined the crystal structures of both the fully reduced and oxidized forms of porcine liver b5R by X-ray crystallography, but, its detail mechanism, especially hydride/proton transfers and exact states of semiquinone, still remains unknown. The hydrogen information obtained by neutron crystallography will be essential for the real understanding of catalytic cycle of the . A large crystal with the size of almost 2 mm was transferred to cryo-protectant solution by stepwise soaking method, and then were flash-frozen in a cold nitrogen gas stream. Using this crystal, we collected neutron data to 1.4 resolution at BL03 (iBIX), MLF, J-PARC, and then collected to 0.85 resolution at BL5A, PF, KEK. Crystallographic refinement using both neutron and X-ray data is in progress.