Relaxation kinetics of cytochrome P450 reductase: internal electron transfer is limited by conformational change and regulated by coenzyme binding.
The kinetics of internal electron transfer in human cytochrome P450 reductase have been studied using temperature-jump relaxation spectroscopy. Temperature perturbation of CPR reduced at the two-electron level with NADPH yields biphasic absorption transients at 450 and 600 nm. The observed rate, 1/tau, for the fast phase is 2200 +/- 300 s(-1). The absence of this phase in fluorescence transients and in absorption transients collected with dithionite-reduced enzyme indicates this phase does not report on electron/hydride transfer and is consistent with its origin in local conformational change in the vicinity of the FAD isoalloxazine ring. The slow phase (1/tau = 55 +/- 2 s(-1)) observed in the absorption transients obtained with CPR reduced at the two-electron level with NADPH reports on internal electron transfer: FAD(sq)-FMN(sq) --> FAD(ox)-FMN(hq). The observed rate of this transient is slower (1/tau = 11 +/- 0.5 s(-1)) in CPR reduced to the two-electron level by dithionite rather than NADPH, demonstrating that coenzyme binding has an important influence on the observed rate of internal electron transfer. Temperature perturbation experiments with CPR reduced with 10-fold molar excess of NADPH produce monophasic absorption transients (1/tau = 20 +/- 0.2 s(-1)) reporting on internal electron transfer: FAD(sq)-FMN(hq) --> FAD(hq)-FMN(sq). The observed rate constants for electron transfer are substantially less than those expected from analysis of CPR by electron-transfer theory (approximately 10(10) s(-1)). Potential gating mechanisms have been investigated using the temperature-jump method. Observed rates for electron transfer were unaffected in experiments performed in deuterated solvent, indicating that deprotonation does not gate the reaction. Introduction of glycerol into the sample significantly decreased the observed rate for internal electron transfer, suggesting conformational gating of the reaction. Replacement of Trp-676 with His-676 reduces approximately 2-fold the observed rate of internal electron transfer in two-electron-reduced enzyme, whereas the observed rate for FAD(sq)-FMN(hq) --> FAD(hq)-FMN(sq) transfer is increased approximately 13-fold in the W676H mutant reduced with a 10-fold molar excess of NADPH. The studies reveal altered redox properties of the FAD in W676H CPR. The data are discussed in the context of previous stopped-flow studies of human CPR and the X-ray crystallographic structure of rat CPR.
Gutierrez
Aldo
A
Biological NMR Centre, University of Leicester, Medical Sciences Building, P.O. Box 138, University Road, Leicester LE1 9HN, UK.
Paine
Mark
M
Wolf
C Roland
CR
Scrutton
Nigel S
NS
Roberts
Gordon C K
GC
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005347:Fibroblasts; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D003067:Coenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase