The dithiol mechanism of class I glutaredoxins promotes specificity for glutathione as a reducing agent

• Published in: Redox biology 2024-12, Vol.78, p.103410, Article 103410
• Main Authors: Lang, Lukas, Reinert, Philipp, Diaz, Cedric, Deponte, Marcel
• Format: Article
• Language: English
• Subjects: Catalytic Domain; Disulfide; Disulfides - chemistry; Dithiol; Enzyme mechanism; Glutaredoxin; Glutaredoxins - chemistry; Glutaredoxins - metabolism; Glutathione; Glutathione - chemistry; Glutathione - metabolism; Glutathione Disulfide - chemistry; Glutathione Disulfide - metabolism; Humans; Oxidation-Reduction; Redox catalysis; Reducing Agents - chemistry; Research Paper; Stopped-flow kinetics; Substrate Specificity; Sulfhydryl Compounds - chemistry; Sulfhydryl Compounds - metabolism; Toluene - analogs & derivatives
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2024.103410

Class I glutaredoxins reversibly reduce glutathione- and nonglutathione disulfides with the help of reduced glutathione (GSH) using either a monothiol mechanism or a dithiol mechanism. The monothiol mechanism exclusively involves a single glutathionylated active-site cysteinyl residue, whereas the dithiol mechanism requires the additional formation of an intramolecular disulfide bond between the active-site cysteinyl residue and a resolving cysteinyl residue. While the oxidation of glutaredoxins by glutathione disulfide substrates has been extensively characterized, the enzyme-substrate interactions for the reduction of S-glutathionylated glutaredoxins or intramolecular glutaredoxin disulfides are still poorly characterized. Here we compared the thiol-specificity for the reduction of S-glutathionylated glutaredoxins and the intramolecular glutaredoxin disulfide. We show that S-glutathionylated glutaredoxins rapidly react with a plethora of thiols and that the 2nd glutathione-interaction site of class I glutaredoxins lacks specificity for GSH as a reducing agent. In contrast, the slower reduction of the partially strained intramolecular glutaredoxin disulfide involves specific interactions with both carboxylate groups of GSH at the 1st glutathione-interaction site. Thus, the dithiol mechanism of class I glutaredoxins promotes specificity for GSH as a reducing agent, which might explain the prevalence of dithiol glutaredoxins in pro- and eukaryotes. We compared the reduction of glutaredoxin disulfides and glutathionylated glutaredoxins revealing different specificities of both enzyme species for GSH as a reducing agent. [Display omitted] •Glutathionylated glutaredoxins (Grx) lack specificity for GSH as a reducing agent.•The Grx dithiol mechanism promotes specificity for GSH as a reducing agent.•The different specificities result from alternative glutathione interaction sites.•The extent of intramolecular disulfide bond formation determines GSH specificity.•Grx are optimized for glutathione recruitment while preventing inhibition by GSH.