HEME OXYGENASE ACTIVE-SITE RESIDUES IDENTIFIED BY HEME-PROTEIN CROSS-LINKING DURING REDUCTION OF CBRCL3

The reduction of CBrCl3 by the heme-heme oxygenase complex forms disso ciable and covalently bound heme products. No such products are formed with mesoheme in which the heme vinyl substituents are replaced by et hyl groups. The dissociable heme products are chromatographically simi lar but not identical to those obtained in the analogous reaction with myoglobin. Tryptic digestion of the heme-protein adduct and Edman seq uencing and mass spectrometric analysis of the heme-linked peptide ide ntify His-25, the proximal iron ligand, as the alkylated residue. Reac tion of CBrCl3 with the heme complexes of the T135V mutant and a Delta (221) C-terminal truncated protein yields heme-linked peptides in addi tion to that from the wild-type reaction. The sequence of the principa l labeled peptide from the T135V reaction, (205)TAFLLNIQLFEELQELLTHDTK (226), and the lability of the adduct suggest the heme is attached to one of the carboxylic acid residues. A carboxylic acid residue is prob ably also labeled in the modified peptide (49)LVMASLYHIYVALEEEIER(67) from the Delta(221) truncated protein. Thus, addition of the reductive ly generated trichloromethyl radical to a heme vinyl group produces a species that alkylates active-site residues. The changes in the alkyla ted residue caused by the Thr-135 mutation or truncation of the protei n places residues in the sequences 49-67 and 205-226 within the active site. Furthermore, this is the first demonstration that heme oxygenas e, like cytochrome P450, may catalyze the reductive metabolism of halo carbons and thus contribute to the toxicity of these agents.