The purpose of this study is to characterize the interactions among P450 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. Previously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [Cawley et al. (1995) Biochemistry 34, 1244-1247]. The goal of the current study is to distinguish between two models to explain these interactions: one model where substrate increases the affinity of one P450 enzyme for the reductase, and another model where substrate increases the affinity of one P450 for the reductase through the formation of a 1A2-2B4 complex. According to this model, the 1A2 moiety of 1A2-2B4 forms a high-affinity complex with reductase. Reductase, 1A2, and 2B4 were reconstituted with dilauroylphosphatidylcholine, and the effect of reductase concentration on 7-pentoxyresorufin-O-dealkylation was examined with 2B4-reductase and 1A2-reductase binary systems, and in ternary systems containing different 2B4:1A2 ratios. At subsaturating [reductase], 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) complex where the reductase is bound specifically to 1A2. At higher reductase concentrations where the reductase-binding sites on 1A2 become saturated, 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 substrate 7-ethoxyresorufin showed a stimulation of 7-ethoxyresorufin-O-deethylation in the mixed reconstituted system, demonstrating that the high-affinity 2B4-1A2-reductase complex was functionally active and not merely an inhibitory complex.

The goal of these studies was to demonstrate that one P450 isozyme can influence the function of another isozyme when combined in a reconstituted system. Benzphetamine and 7-pentoxyresorufin were both shown to be preferred substrates for P450 2B4 (LM2) as compared to P450 1A2 (LM4). However, these substrates exhibited different characteristics when examined in reconstituted systems containing reductase and both P450 isozymes. Whereas benzphetamine demethylation showed a small increase in catalytic activity when both P450 1A2 and 2B4 were present over the activities obtained in simple reconstituted systems, 7-pentoxyresorufin O-dealkylation (PROD) was dramatically inhibited when both isozymes were present. These results indicate that the functional interactions between P450s in complex reconstituted systems are dependent on the substrate present. Inhibition of PROD was also dependent on reductase levels, with the inhibitory effect being more pronounced at subsaturating reductase. Finally, these protein-protein interactions were shown to be dependent on the reductase concentration in the reconstituted system rather the P450 concentration, supporting the view that P450 1A2 is inhibiting the reaction by competing with P450 2B4 for reductase molecules.

Synopsis Previous studies have shown that the presence of one P450 enzyme can affect the function of another P450. The goal of this study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-cytochrome P450 reductase (POR) and a single P450. CYP2B4-, CYP2E1-, and CYP1A2-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behavior of CYP1A2 could be converted to a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical crosslinking and in cellular systems by bioluminescence resonance energy transfer (BRET). Crosslinking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2•CYP1A2 complexes that exhibit altered catalytic activity.