A 25-kDa antioxidant enzyme from yeast, identified as thioredoxin peroxidase (TPx), reduces hydrogen peroxide and alkyl hydroperoxides using thioredoxin, thioredoxin reductase, and NADPH. TPx is a dimer with two conserved cysteine residues, Cys47 and Cys170, which are essential for its peroxidase activity. The oxidized form of Cys47 is oxidized by peroxides and reacts with Cys170 of the other subunit to form an intermolecular disulfide. Mutant TPx proteins lacking either Cys47 or Cys170 lose thioredoxin-coupled peroxidase activity. The TPx disulfide is specifically reduced by thioredoxin, though it can also be reduced by small molecular thiol compounds. The yeast thioredoxin reductase gene was cloned and sequenced, showing 51% identity with the Escherichia coli enzyme. The 25-kDa protein, previously named thiol-specific antioxidant (TSA), is not an appropriate name as it can be reduced by thiols but not ascorbate. The 25-kDa protein is homologous to the Salmonella typhimurium alkylhydroperoxide reductase (AhpC), suggesting it may also act on peroxides. The reduction of the 25-kDa protein disulfide is likely achieved by an enzyme similar to AhpF. The 25-kDa protein may function as an antioxidant against both the ascorbate and thiol oxidation systems, with the previously observed specificity for the thiol system attributed to the ability of thiols to reduce the 25-kDa protein disulfide. The 25-kDa protein's antioxidant activity was supported by thioredoxin (Trx) and thioredoxin reductase (TR). In the presence of Trx, TR, and NADPH, the 25-kDa protein reduced H2O2 and alkylhydroperoxide. The yeast TR gene was cloned and sequenced, showing 51% identity with the Escherichia coli enzyme. The 25-kDa protein's peroxidase activity was confirmed, and it was shown to reduce peroxides with Trx as the immediate hydrogen donor. The 25-kDa protein's disulfide was reduced by thioredoxin, and its activity was essential for protecting glutamine synthetase against oxidative damage. The 25-kDa protein's structure and function were further characterized, revealing two conserved cysteine residues, Cys47 and Cys170, which are essential for its peroxidase activity. The 25-kDa protein's disulfide was reduced by thioreA 25-kDa antioxidant enzyme from yeast, identified as thioredoxin peroxidase (TPx), reduces hydrogen peroxide and alkyl hydroperoxides using thioredoxin, thioredoxin reductase, and NADPH. TPx is a dimer with two conserved cysteine residues, Cys47 and Cys170, which are essential for its peroxidase activity. The oxidized form of Cys47 is oxidized by peroxides and reacts with Cys170 of the other subunit to form an intermolecular disulfide. Mutant TPx proteins lacking either Cys47 or Cys170 lose thioredoxin-coupled peroxidase activity. The TPx disulfide is specifically reduced by thioredoxin, though it can also be reduced by small molecular thiol compounds. The yeast thioredoxin reductase gene was cloned and sequenced, showing 51% identity with the Escherichia coli enzyme. The 25-kDa protein, previously named thiol-specific antioxidant (TSA), is not an appropriate name as it can be reduced by thiols but not ascorbate. The 25-kDa protein is homologous to the Salmonella typhimurium alkylhydroperoxide reductase (AhpC), suggesting it may also act on peroxides. The reduction of the 25-kDa protein disulfide is likely achieved by an enzyme similar to AhpF. The 25-kDa protein may function as an antioxidant against both the ascorbate and thiol oxidation systems, with the previously observed specificity for the thiol system attributed to the ability of thiols to reduce the 25-kDa protein disulfide. The 25-kDa protein's antioxidant activity was supported by thioredoxin (Trx) and thioredoxin reductase (TR). In the presence of Trx, TR, and NADPH, the 25-kDa protein reduced H2O2 and alkylhydroperoxide. The yeast TR gene was cloned and sequenced, showing 51% identity with the Escherichia coli enzyme. The 25-kDa protein's peroxidase activity was confirmed, and it was shown to reduce peroxides with Trx as the immediate hydrogen donor. The 25-kDa protein's disulfide was reduced by thioredoxin, and its activity was essential for protecting glutamine synthetase against oxidative damage. The 25-kDa protein's structure and function were further characterized, revealing two conserved cysteine residues, Cys47 and Cys170, which are essential for its peroxidase activity. The 25-kDa protein's disulfide was reduced by thiore