Mechanism of cytochrome P450 reductase from the house fly: evidence for anFMN semiquinone as electron donor

The interaction of recombinant house fly (Musca domestica) P450 reductase w ith NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one m olecule of NADPH, whereas the rate of oxidation of a second molecule of NAD PH is too slow to account for the observed rates of catalysis. This demonst rates that house fly P450 reductase does not require a priming reaction wit h NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectrosc opy revealed that the enzyme forms tao types of neutral FMN semiquinone. On e serves as the catalytic intermediate of cytochrome c reduction, and anoth er one is an 'air-stable' semiquinone, which reduces cytochrome c 3000 time s more slowly. The results show that the reduction state of the house fly P 450 reductase during catalysis cycles in a 0-2-1-0 sequence. (C) 1999 Feder ation of European Biochemical Societies.