Protective mechanisms against peptide and protein peroxides generated by singlet oxygen

Abstract

Reaction of certain amino acids, peptides, and proteins with singlet oxygen yields substrate-derived peroxides. Recent studies have shown that these species are formed within intact cells and can inactivate key cellular enzymes. This study examines potential mechanisms by which cells might remove or detoxify such peroxides. It is shown that catalase, horseradish peroxidase, and Cu/Zn superoxide dismutase do not react rapidly with these peroxides. Oxymyoglobin and oxyhemoglobin, but not the met (Fe3+) forms of these proteins, react with peptide but not protein, peroxides with oxidation of the heme iron. Glutathione peroxidase, in the presence of reduced glutathione (GSH) rapidly removes peptide, but not protein, peroxides, consistent with substrate size being a key factor. Protein thiols, GSH, other low-molecular-weight thiols, and the seleno-compound ebselen react, in a nonstoichiometric manner, with both peptide and protein peroxides. Cell lysate studies show that thiol consumption and peroxide removal occur in parallel; the stoichiometry of these reactions suggests that thiol groups are the major direct, or indirect, reductants for these species. Ascorbic acid and some derivatives can remove both the parent peroxides and radicals derived from them, whereas methionine and the synthetic phenolic antioxidants Probucol and BHT show little activity. These studies show that cells do not have efficient enzymatic defenses against protein peroxides, with only thiols and ascorbic acid able to remove these materials; the slow removal of these species is consistent with protein peroxides playing a role in cellular dysfunction resulting from oxidative stress.