Hydroxyl and alkoxyl radical production by oxidation products of metmyoglobin.

Abstract

The one-electron oxidation of a reduced nitroxide (2,2,6,6-tetramethyl-1,4-dihydroxypiperidine, TOLH), detected by ESR, was used to resolve and quantify oxidants arising from the reaction of heme proteins with hydroperoxides, including chelatable iron released subsequent to oxidative cleavage of the porphyrin ring. Released iron was distinguished from protein radicals and ferryl heme by analyzing TOLH oxidation in the presence of different chelating agents. Metmyoglobin (metMb) treatment with one mole of H2O2 per mole of heme produced protein-bound oxidants that oxidized about two molecules of TOLH per heme. Some of the oxidizing species responsible for TOLH oxidation were highly persistent (t1/2 for the decay was 3 hrs at 25 degrees C). Iron release, metMb bleaching and the catalysis of Fenton-type chemistry were compared in metMb solutions treated with tert-butyl hydroperoxide (tBH). Iron release required about five-fold higher hydroperoxide concentrations than did metMb bleaching. Alkoxyl and methyl radical production was catalyzed by iron released from metMb but not by protein-bound iron in oxidized metMb solutions treated with tBH and ascorbic acid. The results suggest that ascorbate-mediated hydroxyl and alkoxyl radical production by hydroperoxide-treated metMb is due to released iron and that the protein-bound non-heme iron that arises during bleaching is at most a weak Fenton reagent.