title: Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis.
abstract: The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA. yoshitaka.yamaguchi@shionogi.co.jp
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Kishore K
authors.initials: KK
authors.lastname: Khan
authors.forename: You Ai
authors.initials: YA
authors.lastname: He
authors.forename: You Qun
authors.initials: YQ
authors.lastname: He
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007097:Imines; D066298:In Vitro Techniques; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011524:Protoporphyrins / Q000378:metabolism; D051381:Rats
chemical_list: D007097:Imines; D011524:Protoporphyrins; C032609:N-phenylprotoporphyrin IX; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C011981:phenyldiazene; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase