1) Horseradish peroxidase (HRP) was used as a comparative system for the inactivation of cytochrome P450 by a porphyrinogenic agent, 3,5- dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydroxy-pyridine (DDEP). The loss of heme during the inactivation of HRP was dependent on the duration of exposure to DDEP as well as the concentration of H2O2 and DDEP and paralleled the formation of the altered heme product. Ethylhydrazine (EH) however, was a more efficient ethyl radical-mediated inactivator of HRP by EH. The DDEP mediated inactivation of HRP in the presence of H2O formed only heme-C2H4OH (a substitution product of a C2H4OH moiety for a meso proton of the prosthetic heme), whereas both -meso-ethylheme adduct and heme-C2H4 OH were formed in the inactivation of HRP by EH. Perhaps the proximity of the heme to the ethyl radical may play a role in determining the nature of the heme products formed. A hypothetical reaction pathway for the formation of heme-C2H4OH is proposed. Ethyl radicals generated by one-electron oxidation of DDEP may be rapidly trapped by oxygen molecules to form ethylperoxy radicals, which may undergo isomerization by an intramolecular 1,4 H-atom shift to form ethylhydroperoxide radicals. The ethylhydroperoxide radicals may then react with heme to form heme-C2H4OH. (2) Mechanism of heme alteration in the reaction of myoglobin with H2O2 responsible for the ischemia- reperfusion injury was investigated. The peroxide mediated reaction differed from the coupled oxidation reaction of heme to biliverdin catalyzed by hemeoxygenase. Two major heme products were isolated by HPLC. Visible, mass and NMR spectrometry analyses revealed that the two major altered heme produts were monooxygenated and dihydroxylated hemes. H218O and H218O2 experiments indicated that the added oxygens derived water. Time courses for the formation of monooxygenated heme and covalent adduct to the apoprotein paralleled the loss of heme, and the formation of dihydroxylated heme paralleled the disappearance of monooygenated heme.
