The interaction of recombinant house fly (Musca domestica) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one molecule of NADPH, whereas the rate of oxidation of a second molecule of NADPH is too slow to account for the observed rates of catalysis. This demonstrates that house fly P450 reductase does not require a priming reaction with NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectroscopy revealed that the enzyme forms two types of neutral FMN semiquinone. One serves as the catalytic intermediate of cytochrome c reduction, and another one is an 'airstable' semiquinone, which reduces cytochrome c 3000 times more slowly. The results show that the reduction state of the house fly P450 reductase during catalysis cycles in a 0-2-1-0 sequence.
Murataliev, M. B., & Feyereisen, R. (1999). Mechanism of cytochrome P450 reductase from the house fly: Evidence for an FMN semiquinone as electron donor. FEBS Letters, 453(1–2), 201–204. https://doi.org/10.1016/S0014-5793(99)00723-1