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Report No.

Elucidations of the catalytic cycle of NADH-cytochrome ${it b$_{5}$}$ reductase by X-ray crystallography

Tamada, Taro; Yamada, Mitsugu*; Takeda, Kazuki*; Matsumoto, Fumiko*; Kimura, Shigenobu*; Kuroki, Ryota; Miki, Kunio*

NADH-cytochrome ${it b$_{5}$}$ reductase (b5R) is a flavoprotein consisting of NADH- and FAD- domains, and catalyzes the electron transfer from two electron carriers of NADH to one electron carrier of cytochrome ${it b$_{5}$}$ (Cb5). The structure of the fully reduced form of porcine liver b5R was determined using a crystal grown under anaerobic condition. In the reduced b5R structure, which was determined at 1.68 ${AA}$ resolution, the relative location of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent accessible surface area of FAD, and created a new hydrogen bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat, which is similar to that in the oxidized form, and is stacked with the nicotinamide ring of NAD$$^{+}$$. Both of reduced and oxidized b5R structures could explain how prevents the backflow and accelerates the transfer of electrons to one-electron acceptors, such as Cb5, in the catalytic cycle. Furthermore, the crystallographic analysis by the cryo-trapping method, which controls the exposure time of the fully reduced crystals against the air, suggested that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.



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