Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants.

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems. Copyright 1999 Academic Press.