Quantitation of FAD-Dependent Cytochrome P450 Reductase Activity by Photoreduction

NADPH cytochrome P450 reductase binds two flavin cofactors, FMN and FAD, per molecule of reductase. We have developed an assay to quantitate the reduction activity of FMN-bound flavoprotein. This Tris–light assay system takes advantage of the ability of photoactivated flavins to release electrons to acceptors. In turn, electrons derived from Tris buffer restore the flavin to the unexcited, ground state which can again undergo photoactivation to release another electron. FMN-bound reductase, supplied with reducing equivalents from a Tris–light electron generating system, reduces ferricyanide at a rate of 1.8 μmol/min/nmol reductase. Holoreductase in this system is able to catalyze ferricyanide reduction at a rate of 1.6 μmol/min/nmol reductase, while FAD-bound reductase has no activity. The 8-NH2-FAD and 8-OH-FAD analog-reconstituted FMN-bound reductase catalyzes the reduction of ferricyanide at rates of 0.43 and 0.28 μmol/min/nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide reduction at a rate of 1.1 μmol/min/nmol reductase. FAD or its analogs at the concentrations used to reconstitute enzymatic activity do not support the reduction of ferricyanide in the Tris–light system in the absence of reductase protein. The free flavins, i.e., FMN, 8-OH-FAD, 8-NH2-FAD, and riboflavin, are able to support ferricyanide reduction at a rate of 0.40, 0.52, 0.87, and 0.16 μmol/min/nmol flavin, respectively. This is the first report of an enzymatic assay specific for FMN-bound NADPH cytochrome P450 reductase activity in the absence of its FAD cofactor. Moreover, this report describes the use of an assay procedure based on the provision of reducing equivalents by a Tris–light system which may be useful for other flavin redox enzymes in the absence of reduced pyridine nucleotides or biopterin cofactors.