Role of the electron transfer system in microsomal drug monooxygenase reaction catalyzed by cytochrome P-450.
Four protein components of the hepatic microsomal electron transfer system, NADPH-cytochrome P-450 reductase, cytochrome P-450, NADH-cytochrome b5 reductase, and cytochrome b5, all purified from liver microsomes of phenobarbital-pretreated rabbits, were co-reconstituted into liposomes of egg yolk phosphatidylcholine. The electron transfer rates between the four protein components were measured directly by the stopped-flow method with the reconstituted systems of different compositions, and the effect of the change of the composition on the monooxygenase activity was simultaneously determined. The results obtained led to the following conclusions: (i) The first of the two electrons required for the monooxygenase reaction is exclusively supplied via NADPH-cytochrome P-450 reductase, whereas the second one is preferentially supplied via cytochrome b5. (ii) The rate-limiting step of the overall monooxygenase reaction is the introduction of the second electron, or a step later than that, if the second electron is sufficiently supplied. (iii) All four proteins seem to distribute randomly on the plane of liposomal membranes, and the interaction between them is caused by the lateral diffusion of the proteins.
D000818:Animals; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D004579:Electron Transport; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D010713:Phosphatidylcholines; D011817:Rabbits
D003573:Cytochrome b Group; D008081:Liposomes; D010713:Phosphatidylcholines; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase