Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics.

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.

Cytosolic thioredoxin peroxidase I and II are important defenses of yeast against organic hydroperoxide insult: catalases and peroxiredoxins cooperate in the decomposition of H2O2 by yeast.

The cytosolic thioredoxin peroxidase II (cTPxII/Tsa2p) from Saccharomyces cerevisiae shares 86% identity with the relatively well characterized cytosolic thioredoxin peroxidase I (cTPxI/Tsa1p). In contrast to cTPxI protein, cTPxII is not abundant and is highly inducible by peroxides. Here, we describe a unique phenotype for DeltacTPxII strain; these cells were highly sensitive to tert-butylhydroperoxide (TBHP) but presented resistance to H(2)O(2) in fermentative and respiratory conditions. In contrast, DeltacTPxI strain was very sensitive to both TBHP and H(2)O(2), whatever the carbon source present in the media. These differences in the response of mutant cells to the different kinds of peroxide insult could not be attributed to enzymatic properties of cTPxI and cTPxII since the recombinant proteins showed similar in vitro efficiencies (K(cat) /K(m)) in the removals of both kinds of peroxide. This specific sensitivity of DeltacTPxII cells to TBHP could not be related to the expression pattern of TSA2 (cytosolic thioredoxin peroxidase II gene) either, since this gene is highly inducible by both H(2)O(2) and TBHP when cells were grown in different conditions. Finally, peroxide-removing assays were performed and showed that catalase activity increased significantly only in DeltacTPxII cells, which appear to be related with the resistance of this strain to H(2)O(2). Taken together, present data indicate that cTPxII and cTPxI are key components of the yeast defense system against organic peroxide insult. In regard to the stress induced by H(2)O(2), catalases (peroxisomal and/or cytosolic) and cTPxII seemed to cooperate with cTPxI in the defense of yeast against this oxidant.