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Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding
- Source :
- Journal of Biological Chemistry. 286:16246-16260
- Publication Year :
- 2011
- Publisher :
- Elsevier BV, 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 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.
- Subjects :
- Flavin Mononucleotide
Stereochemistry
Mutation, Missense
Flavin mononucleotide
Flavoprotein
Flavin group
Crystallography, X-Ray
Biochemistry
Protein Structure, Secondary
Electron Transport
chemistry.chemical_compound
Oxidoreductase
Animals
Molecular Biology
NADPH-Ferrihemoprotein Reductase
chemistry.chemical_classification
Cofactor binding
biology
Cytochrome P450 reductase
Cell Biology
Electron transport chain
Protein Structure, Tertiary
Rats
Amino Acid Substitution
chemistry
Enzymology
biology.protein
NADPH binding
Oxidation-Reduction
NADP
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 286
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....e5b3e88bbf2069f5402eca37690c2b7d