Heteromeric Complex Formation between CYP2E1 and CYP1A2:  Evidence for the Involvement of Electrostatic Interactions<sup>†</sup>

Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH−cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2−CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1−CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450−P450 complexes. The synergistic stimulation observed with the reductase−CYP1A2−CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2−CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2−CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.