In the presence of H2O2, horseradish peroxidase (HRP) catalyzes the one-electron oxidation of a porphyrinogenic agent, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), leading to formation of an ethyl radical, inactivation of the enzyme, and formation of an altered heme product. 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. The pseudo first order rate constant for the oxidation of DDEP by compound I of HRP at pH 7.4 was 0.07 min-1, and the maximal extent of heme loss was 35%. The altered heme product, which was isolated by reverse phase HPLC, was characterized by the use of mass and 1H NMR spectrometry as a substitution product of a C2H4OH moiety for a meso proton of the prosthetic heme [meso- (hydroxyethyl)heme]. The source of the oxygen in the C2H4OH moiety appeared not to be H2O2 or H2O as 18O was not incorporated in the heme adduct when H218O2 or H218O was used. The DDEP-mediated reaction did not form the expected δ-mesoethylheme adduct analogous to the ethyl radical-mediated inactivation of HRP by ethylhydrazine (EH) [Ator et al. (1987) J. Biol. Chem. 262, 14954–14960], However, we have found that meso-(hydroxyethyl)heme was formed in the EH-mediated reaction, albeit in apparently lower amounts than δ-mesoethylheme. Perhaps the proximity of the heme to the ethyl radical may play a role in determining the nature of the heme products formed.
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