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.

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…Scanning stop-flow kinetic experiments were performed using a SF-MiniMixer Stopped-Flow apparatus (KinTek Corporation Austin, TX) with a 5 mm path length quartz cell combined with a rapid scanning MC2000-2 CCD-spectrometer (Ocean Optics, Inc., Dunedin, FL) as described previously [19]. All experiments were performed in 100 mM Na-HEPES buffer, pH 7.4, containing 1 mM dithiothreitol and 1 mM EDTA.…”

“…We also used PCA in the analysis of the series of spectra obtained in rapid kinetic experiments [19]. This analysis gave us the curves representing time-dependent changes in the concentrations of the ferric low-spin, ferric high-spin, and the ferrous carbonyl complexes of P450 and P420 states of the heme protein.…”

“…Elucidating the mechanisms of CYP3A4 cooperativity is complicated by the persistent conformational heterogeneity observed, whereby the enzyme is thought to be represented by at least two conformers with different ligand-binding properties [19][20][21][22][23][24]. This functional heterogeneity appears to be linked closely to the oligomeric state of the enzyme [19,20].…”

“…This functional heterogeneity appears to be linked closely to the oligomeric state of the enzyme [19,20]. Microsomal cytochromes P450 are known to form oligomers both in solution [25][26][27] and in membranes [28][29][30][31][32][33], and direct association between P450s has been recognized as an important determinant of function [34][35][36][37].…”

“…Microsomal cytochromes P450 are known to form oligomers both in solution [25][26][27] and in membranes [28][29][30][31][32][33], and direct association between P450s has been recognized as an important determinant of function [34][35][36][37]. In particular, functional heterogeneity of a single P450 species caused by oligomerization has been revealed in interactions with substrates [19,38,39].…”