Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytoc hrome P450 oxidoreductase were determined by anaerobic redox titration of t he diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain El [oxidized/semiquinone] = -286 +/- 6 mV, E-2 [semiquinone/ reduced] = -371 +/- 7 mV; FMN domain E-1 = -43 +/- 7 mV, E-2 = -280 +/- 8 mV) and the soluble diflavin reductase (E-1 [FMN] = -66 +/- 8 mV, E-2 [FMN] = -269 +/- 10 mV; E1 [FAD] = -283 +/- 5 mV, E2 [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue sem iquinone state, with the FMN semiquinone being most intense due to larger s eparation (similar to 200 mV) of its two couples. Both the FMN domain and t he soluble reductase are purified in partially reduced, colored form from t he Escherichia coil expression system, either as a green reductase or a gra y-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reduc tase domain of neuronal nitric oxide synthase. However, they differ markedl y from those of yeast and bacterial CPRs, pointing to an important evolutio nary difference in electronic regulation of these enzymes.