Mechanism of cytochrome P450 reductase from the house fly: evidence for an FMN semiquinone as electron donor.
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.
Department of Entomology and Center for Toxicology, University of Arizona, Tucson 85721-0036, USA. marat@ag.arizona.edu
M B
MB
Murataliev
R
R
Feyereisen
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D017022:Flow Injection Analysis; D006793:Houseflies / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000378:metabolism
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C043609:flavin mononucleotide semiquinone; D009249:NADP; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase