title: Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes.
abstract: Herein, we compare the kinetic properties of CPR from Arabidopsis thaliana (ATR2), with CPR from Artemisia annua (aaCPR) and human CPR (hCPR). While all three CPR forms elicit comparable rates for cytochrome c(3+) turnover, NADPH reduction of the FAD cofactor is ∼50-fold faster in aaCPR and ATR2 compared to hCPR, with a kobs of ∼500 s(-1) (6 °C). Stopped-flow analysis of the isolated FAD-domains reveals that NADP(+)-FADH2 charge-transfer complex formation is also significantly faster in the plant enzymes, but the rate of its decay is comparable for all three proteins. In hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled to subsequent FAD to FMN electron transfer, indicating that the former catalytic event is slow relative to the latter. In contrast, interflavin electron transfer is slower than NADPH hydride transfer in aaCPR and ATR2, occurring with an observed rate constant of ∼50 s(-1). Finally, the transfer of electrons from FMN to cytochrome c(3+) is rapid (>10(3) s(-1)) in all three enzymes and does not limit catalytic turnover. In combination, the data reveal differences in rate-determining steps between plant CPR and their mammalian equivalent in mediating the flux of reducing equivalents from NADPH to external electron acceptors.
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna BC, V1V 1V7, Canada.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Whitelaw
authors.affiliation: Department of Chemistry, University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna BC, V1V 1V7, Canada.
authors.forename: Rochelle
authors.initials: R
authors.lastname: Tonkin
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. Electronic address: kirsten.wolthers@ubc.ca.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Wolthers
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D031172:Artemisia annua / Q000201:enzymology* / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D003574:Cytochrome c Group; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; C017165:cytochrome c(3); D009251:NADPH-Ferrihemoprotein Reductase
keywords: Charge-transfer complex; Cytochrome P450 reductase; Flavin; Stopped-flow spectroscopy