Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4

Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fra gments that irreversibly bind to the protein. We have attempted to characte rize this process structurally, using purified, C-14-heme labeled, recombin ant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and prote olytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman s equencing, and mass spectrometric analyses]. The heme-peptide adducts isola ted after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the pepti des isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although t he precise residue(s) targeted remain to be identified, we have narrowed do wn the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although th e exact structures of the heme-modifying fragments and the nature of the ad duction remain to be established conclusively, the incremental masses of ap proximate to 302 and 314 Da detected by electrospray mass spectrometric ana lyses of the heme-modified peptides are consistent with a dipyrrolic heme f ragment comprised of either pyrrole ring A-D or B-C, a known soluble produc t of peroxidative heme degradation, as a modifying species.