title: Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains.
abstract: The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].
authors.affiliation: Centre for Mechanisms of Human Toxicity, University of Leicester, Hodgkin Building, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: A
authors.initials: A
authors.lastname: Gutierrez
authors.forename: L Y
authors.initials: LY
authors.lastname: Lian
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
authors.forename: N S
authors.initials: NS
authors.lastname: Scrutton
authors.forename: G C
authors.initials: GC
authors.lastname: Roberts
mesh_terms: D000818:Animals; D004579:Electron Transport; D004735:Energy Transfer; D005347:Fibroblasts / Q000201:enzymology; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D051381:Rats; D013053:Spectrophotometry / Q000379:methods
chemical_list: D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D006859:Hydrogen; D009251:NADPH-Ferrihemoprotein Reductase