title: An appraisal of multiple NADPH binding-site models proposed for cytochrome P450 reductase, NO synthase, and related diflavin reductase systems.
abstract: The diflavin reductases exemplified by mammalian cytochrome P450 reductase catalyze NADPH dehydrogenation and electron transfer to an associated monooxygenase. It has recently been proposed that double occupancy of the NADPH dehydrogenation site inhibits the NADPH to FAD hydride transfer step in this series of enzymes. This has important implications for the mechanism of enzyme turnover. However, the conclusions are drawn from a series of pre-steady-state stopped-flow experiments in which the data analysis and interpretation are flawed. Recent data published for P450-BM3 reductase show a decrease in the rate constant for pre-steady-state flavin oxidation with increasing NADP(+) concentration. This is interpreted as evidence of inhibition by multiple substrate binding. A detailed reanalysis shows that the data are in fact consistent with a simple single-binding-site model in which reversible hydride transfer causes the observed effect. Data for the related systems are also discussed.
authors.affiliation: School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK. Simon.Daff@ed.ac.uk
authors.forename: Simon
authors.initials: S
authors.lastname: Daff
mesh_terms: D000409:Alanine / Q000235:genetics; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; D003545:Cysteine; D000409:Alanine