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CYP2C9 Protein Interactions with Cytochrome b₅: Effects on the Coupling of Catalysis

Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (C.W.L., T.S.T.); Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc, Seattle, Washington (L.C.W.); and Department of Chemistry and Program in Physical Biosciences, Washington State University, Pullman, Washington (J.P.J.)

The hemoprotein cytochrome b₅ (cyt b5) has been demonstrated to affect the kinetics of drug oxidation by the microsomal cytochromes P450 (P450s). However, the mechanisms through which cyt b5 exerts these effects are variable and P450 isoform-dependent. Whereas the effects of cyt b5 on the major drug-metabolizing enzymes CYP2D6, CYP2E1, and CYP3A4 are well studied, fewer studies conducted over limited ranges of cyt b5 concentrations have been performed on CYP2C9. In the present study with CYP2C9, cyt b5 exerted complex actions upon P450 oxidative reactions by affecting the rate of metabolite formation, the consumption of NADPH by cytochrome P450 reductase, and uncoupling of the reaction cycle to hydrogen peroxide and water. Cytochrome b₅ devoid of the heme moiety (apo-b5) exhibited effects similar to those of native cyt b5. All rates were highly dependent on the cyt b5 to CYP2C9 enzyme ratio, suggesting that the amount of cyt b5 present in an in vitro incubation is an important factor that can have an impact on the reliability of extrapolating in vitro generated data to predict the in vivo condition. The major effects of cyt b5 are hypothesized to result from a cyt b5-induced conformational change in CYP2C9 that results in an increased collision frequency between the iron-oxygen species (Cpd I) and the substrate, and a decrease in the oxidase activity. Together, these findings suggest that cyt b5 can alter multiple steps in the P450 catalytic cycle via complex interactions with P450 and P450 reductase.

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				M. Subramanian, M. Low, C. W. Locuson, and T. S. Tracy CYP2D6-CYP2C9 Protein-Protein Interactions and Isoform-Selective Effects on Substrate Binding and Catalysis Drug Metab. Dispos., August 1, 2009; 37(8): 1682 - 1689. [Abstract] [Full Text] [PDF]

				H. Zhang, D. Hamdane, S.-C. Im, and L. Waskell Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4 J. Biol. Chem., February 29, 2008; 283(9): 5217 - 5225. [Abstract] [Full Text] [PDF]