title: Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium.
abstract: We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55+/-6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD, 9500 Gilman Drive, La Jolla, CA 92093, USA. ddavydov@ucsd.edu
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Elena V
authors.initials: EV
authors.lastname: Sineva
authors.forename: Srinivas
authors.initials: S
authors.lastname: Sistla
authors.forename: Nadezhda Y
authors.initials: NY
authors.lastname: Davydova
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Frank
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001896:Boron Compounds; D002462:Cell Membrane / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004579:Electron Transport; D005415:Flavins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism*; D036103:Nanotechnology; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001426:Bacterial Proteins; D001896:Boron Compounds; D005415:Flavins; D005456:Fluorescent Dyes; D011510:Proteolipids; C020485:proteoliposomes; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases