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 dissociable and covalently bound heme products. No such products are formed with mesoheme in which the heme vinyl substituents are replaced by ethyl groups. The dissociable heme products are chromatographically similar but not identical to those obtained in the analogous reaction with myoglobin. Tryptic digestion of the heme-protein adduct and Edman sequencing and mass spectrometric analysis of the heme-linked peptide identify His-25, the proximal iron ligand, as the alkylated residue. Reaction of CBrCl3 with the heme complexes of the T135V mutant and a Delta221 C-terminal truncated protein yields heme-linked peptides in addition to that from the wild-type reaction. The sequence of the principal labeled peptide from the T135V reaction, 205TAFLLNIQLFEELQELLTHDTK226 , and the lability of the adduct suggest the heme is attached to one of the carboxylic acid residues. A carboxylic acid residue is probably also labeled in the modified peptide 49LVMASLYHIYVALEEEIER67 from the Delta221 truncated protein. Thus, addition of the reductively generated trichloromethyl radical to a heme vinyl group produces a species that alkylates active-site residues. The changes in the alkylated residue caused by the Thr-135 mutation or truncation of the protein places residues in the sequences 49-67 and 205-226 within the active site. Furthermore, this is the first demonstration that heme oxygenase, like cytochrome P450, may catalyze the reductive metabolism of halocarbons and thus contribute to the toxicity of these agents.
Wilks
A
A
Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center, University of California, San Francisco, California 94143-0446, USA.
Medzihradszky
K F
KF
Ortiz de Montellano
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001975:Bromotrichloromethane / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D006418:Heme; D006419:Heme Oxygenase (Decyclizing); D001975:Bromotrichloromethane