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 |
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Authors: | Dmitri R Davydov Elena V Sineva Nadezhda Y Davydova Stephen G Sligar |
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Institution: | a Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD, 9500 Gilman Drive, La Jolla, CA 92093, USA b Department of Biochemistry and Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, USA |
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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. |
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Keywords: | Cytochrome P450 3A4 Flavin domain of cytochrome P450BM-3 Kinetics of electron transfer Spin equilibrium Nanodisc Liposome Oligomerization Lifetime FRET BODIPY Cysteine-depleted mutant |
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