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Preliminary neutron diffraction studies of NADH-cytochrome ${it b$_{5}$}$ reductase at J-PARC

Hirano, Yu; Yamada, Mitsugu*; Kurihara, Kazuo; Kusaka, Katsuhiro*; Kimura, Shigenobu*; Miki, Kunio*; Tamada, Taro

NADH-cytochrome ${it b$_{5}$}$ 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 ${it b$_{5}$}$. The reaction catalyzed by b5R 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 b5R. For neutron diffraction experiments, we prepared large crystals with the size of almost 2 mm$$^{3}$$. Large crystals were transferred to cryo-protectant solution by stepwise soaking method, and then were flash-frozen in a cold nitrogen gas stream. In preliminary neutron experiment, we confirmed some diffraction spots above 1.4 ${AA}$, resolution from a crystal with the size of 1.8 mm$$^{3}$$ under 100 K at BL03 (iBIX), MLF, J-PARC after 14 hours exposure at 300 KW accelerator power.

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