Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe

[3H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([3H]HBP) was synthesized and used to photoaffinity label P450Cam. The imidazole moiety of HBP anchors the compound in the P450Cam active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450Cam from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450Cam. Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B′/C loop region of P450Cam. The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.