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.

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.