Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5 enzymes

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 Kd value for the Pdx · PdR complex was 0.054 μM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the Kd 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 Kd 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 Kd 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.