The association of cytochrome P-450 and NADPH-cytochrome P-450 reductase in phospholipid membranes

doi:10.1016/0003-9861(84)90337-0

Archives of Biochemistry and Biophysics

Volume 234, Issue 1, October 1984, Pages 161-166

https://doi.org/10.1016/0003-9861(84)90337-0 Get rights and content

Abstract

NADPH-cytochrome P-450 reductase and two purified isozymes of cytochrome P-450 have been incorporated into phospholipid vesicles by a cholate dialysis technique. The enzyme system reconstituted in this manner was catalytically active. The observed kinetics for substrate oxidation indicated that both enzymes were associated with the liposomal membranes, and were not simply entrapped in the interior of the vesicle. The N-demethylation of benzphetamine was measured in order to determine the effect of variations in the mole ratio between the two enzymes and between the lipid and the total enzyme on the observed steady-state kinetics. In addition, the kinetic isotope effects for the O-deethylation of 7-ethoxycoumarin were measured in order to compare these parameters to those previously observed in a reconstituted system [G. T. Miwa, and A. Y. H. Lu (1981) Arch. Biochem. Biophys.211, 454–458]. The results were all consistent with the association of the two proteins by lateral diffusion in the vesicle membrane. Moreover, the observed reduction in catalytic activity, as the enzymes were diluted in the vesicle membrane, can only be explained by the formation of a transient P-450-reductase complex, and not by the existence of a stable complex between the two proteins. These results provide compelling evidence for a mass action model for the interaction of these two enzymes in liposomal membranes.

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Citation Excerpt :
As shown in Fig. 2, the observed activity depends critically on the relative ratio of CPR to CYP3A4, indicating the tight binding of CPR to CYP3A4-ND with an estimated dissociation constant in the range of 10–50 nM. This is similar to those reported earlier for CYP3A4 [25] and other mammalian cytochromes P450 [26]. As the CPR:CYP3A4 molar ratio increases, the activity also increases, reaching a saturation at ∼10:1 molar ratio.
Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 °C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug–drug interaction studies.
Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners
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As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.
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The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b₅ from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1–P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b₅ stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b₅. The effects of cytochrome b₅ may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b₅ in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.
Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b<inf>5</inf> enzymes
2005, Archives of Biochemistry and Biophysics
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In the calculations involving estimates of the Kd values of the human P450 complexes with other proteins, we used the assumption that the stoichiometry of binding is 1:1. Although we do not have independent proof for this stoichiometry here, the literature does provide support from studies with purified enzymes (P450, NPR) and in membrane systems [56–58]. Both proteins aggregate in solution [59], and the addition of non-ionic detergents to favor monomers will either alter the apparent binding parameters, due to facilitated interaction in vesicles, or disrupt interaction of the P450s with the other proteins [60,61].
An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b₅ (b₅) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin–peroxidase complex was added, and protein–protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein–protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r² = 0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K_d value for the Pdx · PdR complex was 0.054 μM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K_d values (with the reductase) ranged between 0.005 and 0.1 μM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b₅ or apo-b₅ (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b₅, although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b₅, with P450 3A4 yielding the lowest K_d values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b₅ or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450 · substrate K_d values estimated from substrate-induced UV–visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b₅, with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.

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The association of cytochrome P-450 and NADPH-cytochrome P-450 reductase in phospholipid membranes

Abstract

Arch. Biochem. Biophys

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