Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into α-helix A‥ The site of modification was identified as Cys-64 with the help of CYP3A4 (C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and α-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of BADAN-modified enzyme is characterized by an S 50 of 18.2 ± 0.7, compared with the value of 2.2 ± 0.3 for the ANF-induced spin transition, thus revealing an additional low affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H 2 O 2 -dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer. KeywordsCytochrome P450 3A4; α-naphthoflavone; cooperativity; substrate binding site; BADAN; conformational heterogeneity A number of recent studies on the mechanisms of function and regulation of microsomal monooxygenase systems have focused on the importance of homo-and heterotropic cooperativity exhibited by various mammalian drug-metabolizing cytochromes P450 (1-8). Cooperative behavior has been reported for such enzymes as P450 1A2 (9), 2C9 (10-12), 2C5 (13), 2B6 (14), and 2B4 (15). However, the most prominent cases of P450 cooperativity are found in cytochrome P450 3A4 (CYP3A4), the principal drug-metabolizing enzyme in humans † This research was supported by NIH grant GM54995, Center grant ES06676, and research grant H-1458 from the Robert A. Welch NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 28. Published in final edited form as:Biochemistry. 2007 January 9; 46(1): 106-119. doi:10.1021/bi061944p. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (16). In addition to the homotropic cooperativity observed with a wide variety of substrates (1,4,(16)(17)(18), CYP3A4 also provides important examples of heterotropic activation (1...

SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) 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 based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. 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.

Background: Cytochrome P450 3A4 (CYP3A4) can bind several substrate molecules simultaneously and exhibits cooperativity. Results: Ligand binding in the active site is preceded by functionally important interactions at a distinct peripheral site. Conclusion: The mechanism of cooperativity involves a ligand-induced allosteric transition. Significance: Allosteric mechanism suggested by our results transforms the view of the grounds and significance of CYP3A4 cooperativity.