title: Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer.
abstract: NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration 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 as in the oxidized form and stacked together with the nicotinamide ring of NAD(+). Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.
authors.affiliation: Molecular Biology Research Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195, Japan.
authors.forename: Mitsugu
authors.initials: M
authors.lastname: Yamada
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.forename: Kazuki
authors.initials: K
authors.lastname: Takeda
authors.forename: Fumiko
authors.initials: F
authors.lastname: Matsumoto
authors.forename: Hiraku
authors.initials: H
authors.lastname: Ohno
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kosugi
authors.forename: Kiyofumi
authors.initials: K
authors.lastname: Takaba
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Shoyama
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kimura
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D042966:Cytochrome-B(5) Reductase / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D013552:Swine
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D042966:Cytochrome-B(5) Reductase
keywords: FAD; FNR; crystal structure; e.s.d.; electron transfer; estimated standard deviation; ferredoxin–NADP(+) oxidoreductase; flavin adenine dinucleotide; oxidoreductase; reaction intermediate