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.

Background: There are multiple cytochrome P450 species co-localized in the endoplasmic reticulum. Results: Membrane-bound cytochromes P450 3A4, 3A5, and 2E1 associate into heteromeric complexes, where the properties of the individual enzymes are considerably modified. Conclusion:The properties of the P450 ensemble cannot be predicted by summation of the properties of the individual enzymes. Significance: We disclose a mechanism of regulatory cross-talk between multiple P450 species through hetero-oligomerization.

We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55 ± 6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.

To establish a direct method to monitor substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding (S50 = 8.2 ± 1.3 µM, n = 2.3 ± 0.1). Fluorescence resonance energy transfer (FRET) experiments suggest the presence of at least two substrate binding sites with apparent KD values in the ranges of 0.1 – 0.3 and 6 – 9 µM. Similar to earlier observations with CYP3A4, increasing hydrostatic pressure does not cause either a complete dissociation of the substrate complexes or a displacement of the spin equilibrium towards the low-spin state. Rather, increased pressure enhances the cooperativity of the F7GA-induced spin shift, so that the Hill coefficient approaches 3 at 2 kbar. Lifetime FRET experiments revealed an important increase in the affinity of the enzyme for F7GA at elevated pressures, suggesting that the binding of the ligand induces a conformational transition associated with an important increase in protein hydration. This transition largely attenuates the solvent accessibility of the heme pocket and causes an unusual stability of the high-spin, substrate-bound enzyme at elevated pressures.

Functional cross-talk among human drug-metabolizing cytochromes P450 through their association is a topic of emerging importance. Here we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer (LRET) between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochromes P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by MDMA, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that pre-incubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 heterooligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3–0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.