title: Kinetics of dithionite-dependent reduction of cytochrome P450 3A4: heterogeneity of the enzyme caused by its oligomerization.
abstract: To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.
authors.affiliation: Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, 301 University Boulevard, Galveston, Texas 77555, USA. d.davydov@UTMB.edu
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Harshica
authors.initials: H
authors.lastname: Fernando
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baas
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism*; D004227:Dithionite / Q000494:pharmacology*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D011510:Proteolipids
chemical_list: D001704:Biopolymers; D011510:Proteolipids; C020485:proteoliposomes; D004227:Dithionite; D051544:Cytochrome P-450 CYP3A