Abstract
Glutaredoxins are oxidoreductases specialized in reducing glutathione-protein mixed disulfides. In the first step of deglutathionylation, glutaredoxins form a mixed disulfide with glutathione, releasing reduced peptide. The specificity of this reaction is based on the unusual amide linkage formed between the γ-carboxylate of the N-terminal glutamic acid and the α-amino group of the cysteine present in glutathione. In the second step of deglutathionylation, glutathione reduces the glutaredoxin-glutathione mixed disulfide. Here we show that the specificity of this second reaction for Escherichia coli Grx1, but not for human or yeast Grx1, also is based on the unusual γ-linkage present in glutathione. Mutating Tyr13, Thr58, and/or Asp74 to alanine in E. coli Grx1 results in the glutaredoxin-peptide mixed disulfide being thermodynamically favored over the glutaredoxin-glutathione mixed disulfide in the first step of the reaction. An increased propensity to form glutaredoxin-protein mixed disulfides was observed in vivo for these same mutants. Furthermore, we demonstrate that all mutations studied in Cys14, the C-terminal active site cysteine, abolish the specificity of E. coli Grx1 for glutathione over the corresponding tripeptide Glu-Cys-Gly, which has a normal peptide bond linking Glu-Cys instead of the γ-linkage present in glutathione, in the second step of deglutathionylation. Antioxid. Redox Signal. 11, 1819–1828.