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Fluorescent Photoaffinity Labeling of Cytochrome P450 3A4 by Lapachenole:  Identification of Modification Sites by Mass Spectrometry†

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Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115
Cite this: Biochemistry 2005, 44, 6, 1833–1845
Publication Date (Web):January 20, 2005
https://doi.org/10.1021/bi048228c
Copyright © 2005 American Chemical Society
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    Abstract

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    While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97−105) and VLQNFSFKPCK (positions 459−469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B−B‘ loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.

    †

     This work was supported by NIH Grant GM32165 (to S.D.N.) and the UW NIEHS-sponsored Center for Ecogenetics and Environmental Health:  NIEHS P30ES07033.

    ‡

     University of Washington.

    §

     Harvard Medical School.

    *

     To whom correspondence should be addressed:  telephone (206) 543-1419; fax (206) 685-3252; e-mail [email protected].

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    Deconvoluted spectra of P450 reductase and cytochrome b5 and ESI mass spectrum of the heme of CYP3A4 from LC/ESI MS analysis of the reconstituted CYP3A4 system treated with lapachenole under photolytic conditions (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

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