Glutaredoxin (Grx)-catalyzed deglutathionylation of protein–glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix [Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027–2033]. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency (kcat/KM) of Grx2 for protein deglutathionylation (due primarily to a decreased kcat), reflecting a higher pKa of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 [Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607–14613], hGrx2 catalyzes glutathione-thiyl radical (GS•) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS• serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower kcat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.

Glutaredoxin (GRx) is a thiol‐disulfide oxidoreductase that utilizes glutathione (GSH) to reduce protein‐glutathione mixed disulfides (protein‐SSG). GRx catalysis serves important roles in redox homeostasis and signal transduction (Shelton et. al., 2005). Mammalian cells have two GRx isoforms: GRx1, primarily in cytosol, and GRx2, primarily in mitochondria (Gladyshev et al., Lundberg et. al., 2001). GRx2 displays <35% sequence identity to GRx1, but it has analogous active site and glutathionyl stabilization motifs. Here we report GRx2 mimics GRx1 remarkably. Like GRx1, GRx2 displays ping‐pong kinetics ‐ the apparent KM and Vmax values for protein‐SSG and GSH change in proportion to concentration of the other substrate. The active site thiol pKa of GRx2 is 4.5, one pH unit higher than the corresponding pKa for GRx1 (3.5), predicting Grx2 would display 25% of GRx1 activity (Srinivasan et al. 1997). In fact, GRx2 activity is 10% of GRx1, suggesting another change in chemistry of catalysis besides the pKa difference. While GRx2 can couple to thioredoxin reductase (TRase) (Johansson et al. 2004), turnover by TRase is far less efficient than by GSH plus GSSG reductase (GRase), even when GRase and GSH are one‐tenth their typical concentrations, simulating oxidative stress. Thus, turnover by TRase is unlikely to support GRx2 activity in situ. Supported by NIH R01 AG 024413 & P01 AG 15885, and VA Merit Review (JJM).