Mechanism of Interactions of α-Naphthoflavone with Cytochrome P450 3A4 Explored with an Engineered Enzyme Bearing a Fluorescent Probe

Abstract

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...

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

“…One of the first pieces of evidence for the involvement of such changes was obtained in our studies with P450eryF, the beststudied bacterial enzyme known to exhibit cooperativity [32,33]. An evidence of a conformational transition in CYP3A4 caused by α-naphthoflavone (ANF), a prototypic heterotropic activator, was obtained in our recent work using site-directed incorporation of a fluorescent probe into a cysteine-depleted mutant bearing a fluorescent probe [34]. More recently, the use of pressure-perturbation spectroscopy allowed us to demonstrate unusual stabilization of the substrate-bound high-spin state and a prominent increase in the cooperativity of CYP3A4 at elevated pressure, suggesting that allosteric mechanisms involve decreased protein hydration [35].…”

“…Wild-type CYP3A4 and its cysteine-depleted mutant CYP3A4 (C58,C64) [34] were expressed as His-tagged proteins in E. coli TOPP3 cells and purified by chromatography on Ni-NTA resin followed by ion-exchange chromatography on Macro-Prep CM Support resin (Bio-Rad Laboratories, Hercules, CA) [34]. Modification of CYP3A4 (C58,C64) by BADAN was performed as described earlier, except for the final step where the removal of detergent and unreacted label was performed on Ni-NTA Agarose as opposed to CM Sepharose CL-6B [34].…”

“…Modification of CYP3A4 (C58,C64) by BADAN was performed as described earlier, except for the final step where the removal of detergent and unreacted label was performed on Ni-NTA Agarose as opposed to CM Sepharose CL-6B [34].…”

“…The major trend in much current research on CYP3A4 is to assess whether the "non-MichaelisMenten" behavior represents a manifestation of true allosteric regulation based on an effectorinduced conformational transition in the enzyme [29][30][31][32][33][34][35]. One of the first pieces of evidence for the involvement of such changes was obtained in our studies with P450eryF, the beststudied bacterial enzyme known to exhibit cooperativity [32,33].…”

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

“…In P450 studies it was first used to determine intramolecular distance between the heme and a fluorescent donor (FITC) attached to the N-terminal methionine of P450 LM2 [34], and later, to monitor conformational dynamics of CYP11A1 where the same fluorescence donor was selectively attached to a Lys-residue in the K-helix (close to SRS5) [35]. More recent work by Halpert and Davydov has revealed applicability of FRET to resolve multiple substrate binding sites in CYP107A1 (EryF) [36] and CYP3A4 [37].…”