INTERACTIONS AMONG P450 ENZYMES WHEN COMBINED IN RECONSTITUTED SYSTEMS - FORMATION OF A 2B4-1A2 COMPLEX WITH A HIGH-AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

The purpose of this study is to characterize the interactions among P4 50 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. P reviously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [C awley et al. (1995) Biochemistry 34, 1244-1247]. The goal of the curre nt study is to distinguish between two models to explain these interac tions: one model where substrate increases the affinity of one P450 en zyme for the reductase, and another model where substrate increases th e affinity of one P450 for the reductase through the formation of a 1A 2-2B4 complex. According to this model, the 1A2 moiety of 1A2-2B4 form s a high-affinity complex with reductase. Reductase, 1A2, and 2B4 were reconstituted with dilauroylphosphatidylcholine, and the effect of re ductase concentration on 7-pentoxyresorufin-O-dealkylation was examine d with 2B4-reductase and 1A2-reductase binary systems, and in ternary systems containing different 2B4:1A2 ratios. At subsaturating [reducta se], there was a dramatic inhibition of the 2B4-dependent activity in the ternary system as compared with the binary systems. These results are consistent with the formation of a ternary (reductase-1A2-2B4) com plex where the reductase is bound specifically to 1A2. At higher reduc tase concentrations where the reductase-binding sites on 1A2 become sa turated, the results are consistent with the formation of a quaternary complex in which reductase binds to both P450 enzymes (reductase-1A2- 2B4-reductase). Analogous experiments using the 1A2-preferred substrat e 7-ethoxyresorufin showed a stimulation of 7-ethoxyresorufin-O-deethy lation in the mixed reconstituted system, demonstrating that the high- affinity 2B4-1A2-reductase complex was functionally active and not mer ely an inhibitory complex.