NDLI: Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

Content Provider	PubMed Central
Author	Xia, Chuanwu Hamdane, Djemel Shen, Anna L. Choi, Vivian Kasper, Charles B. Pearl, Naw May Zhang, Haoming Im, Sang-choul Lucy, Waskell Kim, Jung-ja P.
Copyright Year	2011
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 Asp147 and Arg514 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 Gly631–Asn635 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 Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 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.
Related Links	http://dx.doi.org/10.1074/jbc.M111.230532
Starting Page	16246
File Format	PDF
ISSN	1083351X
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	18
Volume Number	286
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2011-05-06
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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