QUANTITATION OF FAD-DEPENDENT CYTOCHROME-P450 REDUCTASE-ACTIVITY BY PHOTOREDUCTION

NADPH cytochrome P450 reductase binds two flavin cofactors, FMN and FA D, per molecule of reductase. We have developed an assay to quantitate the reduction activity of FMN-bound flavoprotein. This Tris-light ass ay system takes advantage of the ability of photoactivated flavins to release electrons to accepters. In turn, electrons derived from Tris b uffer restore the flavin to the unexcited, ground state which can agai n undergo photoactivation to release another electron. FMN-bound reduc tase, supplied with reducing equivalents from a Tris-light electron ge nerating system, reduces ferricyanide at a rate of 1.8 mu mol/min/nmol reductase. Holoreductase in this system is able to catalyze ferricyan ide reduction at a rate of 1.6 mu mol/min/nmol reductase, while FAD-bo und reductase has no activity. The 8-NH2-FAD and 8-OH-FAD analog-recon stituted FMN-bound reductase catalyzes the reduction of ferricyanide a t rates of 0.43 and 0.28 mu mol/min/nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide re duction at a rate of 1.1 mu mol/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 a t a rate of 0.40, 0.52, 0.87, and 0.16 mu mol/min/nmol flavin, respect ively. 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 proc edure based on the provision of reducing equivalents by a Tris-light s ystem which may be useful for other flavin redox enzymes in the absenc e of reduced pyridine nucleotides or biopterin cofactors. (C) 1996 Aca demic Press, Inc.