Quantitation of FAD-dependent cytochrome P450 reductase activity by photoreduction

Analytical Biochemistry
A V Hodgson, H W Strobel

Abstract

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 Trislight 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 mumol/min/ nmol reductase. Holoreductase in this system is able to catalyze ferricyanide reduction at a rate of 1.6 mumol/ 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 mumol/min/ nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide reduction at a rate of 1.1 mumol/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 sys...Continue Reading

Citations

May 19, 2006·Journal of Bacteriology·Stuart WrightShahid Khan
Mar 14, 2007·Angewandte Chemie·Frank HollmannManfred T Reetz
Feb 13, 2002·Biosensors & Bioelectronics·Victoria V ShumyantsevaAlexander I Archakov

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