title: Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries.
abstract: Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl]>50mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type>E48G/E49G>D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4.
authors.affiliation: Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, United States.
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.affiliation: Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, United States. Electronic address: rauchus@med.umich.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D001667:Binding, Competitive; D055162:Biocatalysis; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D009154:Mutation; D009543:Nifedipine / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000494:pharmacology; D011374:Progesterone / Q000378:metabolism*; D013499:Surface Properties; D013739:Testosterone / Q000378:metabolism*
chemical_list: D010743:Phospholipids; D013739:Testosterone; D011374:Progesterone; D015786:Cytochromes b5; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009543:Nifedipine
keywords: Allostery; CYP3A4; Cytochrome P450; Cytochrome b(5); Drug oxidation; Testosterone