title: Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding.
abstract: The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Chuanwu
authors.initials: C
authors.lastname: Xia
authors.forename: Djemel
authors.initials: D
authors.lastname: Hamdane
authors.forename: Anna L
authors.initials: AL
authors.lastname: Shen
authors.forename: Vivian
authors.initials: V
authors.lastname: Choi
authors.forename: Charles B
authors.initials: CB
authors.lastname: Kasper
authors.forename: Naw May
authors.initials: NM
authors.lastname: Pearl
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D009249:NADP; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase