A protector protein from Saccharomyces cerevisiae prevented the inactivation of enzyme and oxidative damage to protein and DNA caused by a thiol/Fe3+/O2 metal-catalyzed oxidation (MCO) system but not when thiol was replaced by ascorbate. In the presence of a reduced thiol such as dithiothreitol and reduced glutathione, however, the protector protein prevented inactivation of E. coli glutamine synthetase against a MCO system comprised of ascorbate and Fe3+. The protector protein also inhibited the fragmentation of protein, incorporation of carbonyl groups into protein, strand breaks in pBluescript plasmid DNA, and the formation of 8-hydroxydeoxyguanosine in calf thymus DNA when induced by either the thiol/Fe3+ system or the ascorbate/Fe3+ system supplemented with dithiothreitol. These results suggest that antioxidant activity of protector protein against a MCO system requires thiol as a reducing equivalent to restore its catalytic activity.