title: Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation.
abstract: Understanding the detailed metabolic mechanisms of membrane-associated cytochromes P450 is often hampered by heterogeneity, ill-defined oligomeric state of the enzyme, and variation in the stoichiometry of the functional P450.reductase complexes in various reconstituted systems. Here, we describe the detailed characterization of a functionally homogeneous 1:1 complex of cytochrome P450 3A4 (CYP3A4) and cytochrome P450 reductase solubilized via self-assembly in a nanoscale phospholipid bilayer. CYP3A4 in this complex showed a nearly complete conversion from the low- to high-spin state when saturated with testosterone (TS) and no noticeable modulation due to the presence of cytochrome P450 reductase. Global analysis of equilibrium substrate binding and steady-state NADPH consumption kinetics provided precise resolution of the fractional contributions to turnover of CYP3A4 intermediates with one, two, or three TS molecules bound. The first binding event accelerates NADPH consumption but does not result in significant product formation due to essentially complete uncoupling. Binding of the second substrate molecule is critically important for catalysis, as the product formation rate reaches a maximum value with two TS molecules bound, whereas the third binding event significantly improves the coupling efficiency of redox equivalent usage with no further increase in product formation rate. The resolution of the fractional contributions of binding intermediates of CYP3A4 into experimentally observed overall spin shift and the rates of steady-state NADPH oxidation and product formation provide new detailed insight into the mechanisms of cooperativity and allosteric regulation in this human cytochrome P450.
authors.affiliation: Department of Biochemistry, College of Medicine, University of Illinois, Urbana, Illinois 61801, USA.
authors.forename: Ilia G
authors.initials: IG
authors.lastname: Denisov
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baas
authors.forename: Yelena V
authors.initials: YV
authors.lastname: Grinkova
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
mesh_terms: D000494:Allosteric Regulation; D002384:Catalysis; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D010084:Oxidation-Reduction; D013739:Testosterone / Q000378:metabolism
chemical_list: D013739:Testosterone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase