title: Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase.
abstract: Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp(634), Ala(635), Arg(636) and Asn(637); human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp(679)) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c(3+) turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp(677) and slower release of NADP(+). In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c(3+) reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg(636) influences catalysis is discussed.
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC, V1V 1V7, Canada.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Mothersole
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC, V1V 1V7, Canada.
authors.forename: Carla E
authors.initials: CE
authors.lastname: Meints
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC, V1V 1V7, Canada.
authors.forename: Alex
authors.initials: A
authors.lastname: Louder
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC, V1V 1V7, Canada. Electronic address: kirsten.wolthers@ubc.ca.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Wolthers
mesh_terms: D000409:Alanine / Q000737:chemistry; D017360:Arabidopsis; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001410:Bacillus megaterium; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005415:Flavins; D001224:Aspartic Acid; D009249:NADP; D014364:Tryptophan; D045304:Cytochromes c; D001120:Arginine; D009251:NADPH-Ferrihemoprotein Reductase; D000409:Alanine
keywords: Cytochrome P450 reductase; Flavin enzymology; Stopped-flow spectroscopy