Deglutathionylation of 2-Cys peroxiredoxin is specifically catalyzed by sulfiredoxin.

Reversible protein glutathionylation plays a key role in cellular regulation and cell signaling and protects protein thiols from hyperoxidation. Sulfiredoxin (Srx), an enzyme that catalyzes the reduction of Cys-sulfinic acid derivatives of 2-Cys peroxiredoxins (2-Cys Prxs), has been shown to catalyze the deglutathionylation of actin. We show that deglutathionylation of 2-Cys Prx, a family of peroxidases, is specifically catalyzed by Srx. Using the ubiquitously expressed member of 2-Cys Prx, Prx I, we revealed the following. (i) Among its four Cys residues, Cys(52), Cys(83), and Cys(173) can be glutathionylated in vitro. Deglutathionylation with Cys mutants showed that Cys(83) and Cys(173) were preferentially catalyzed by Srx, with glutathionylated Srx as the reaction intermediate, whereas glutaredoxin I was more favorable for deglutathionylating Cys(52). (ii) Studies using site-directed mutagenesis coupled with binding and deglutathionylation activities revealed that Pro(174) and Pro(179) of Prx I and Tyr(92) of Srx are essential for both activities. Furthermore, relative to glutaredoxin I, Srx exhibited negligible deglutathionylation activity for glutathionylated cysteine and glutathionylated BSA. These results indicate that Srx is specific for deglutathionylating Prx I due to its favorable affinity for Prx I. To assess the biological relevance of these observations, we showed that Prx I is glutathionylated in A549 and HeLa cells under modest levels of H(2)O(2). In addition, the level of glutathionylated Prx I was substantially elevated in small interfering RNA-mediated Srx-knocked down cells, whereas the reverse was observed in Srx-overexpressing cells. However, glutathionylation of Prx V, not known to bind to Srx, was not affected by the change in Srx expression levels.