Pivotal role of P450-P450 interactions in CYP3A4 allostery: The case of α-naphthoflavone

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes™ containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this systemwas dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450-P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes™. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.