Oxidative damage to tissues by hydrogen peroxide and PUFA hydroperoxides has become a recurring theme as a mechansim for the induction of a variety of medical conditions including myocardial ischemia, cancer and aging. In addition to lipids, proteins are a target of such damage. Myoglobin, a heme protein that is ubiquitous in aerobic muscle tissues, has peroxidase activity that can result in oxidative damage to a variety of biological molecules, including itself and membrane lipids. The reaction between metmyoglobin and hydrogem peroxide produces both a ferryl-oxo heme and a globin-centered radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for the localization of the globin-centered radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with one another.
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