Disulfide biochemistry in 2-Cys peroxiredoxin: Requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin

摘要

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys P) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys P. Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8 ? resolution of Tsa1 C47S in the decameric form [(α 2) 5] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1 C47S structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys P α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys P reactivity. Therefore, Tsa1 E50A and Tsa1 R146Q mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10 6 M - 1 s - 1. Remarkably, although Tsa1 E50A and Tsa1 R146Q were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.