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.

中文翻译：

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I 类戊二氧杂毒素的二硫醇机制促进了谷胱甘肽作为还原剂的特异性


I 类戊二氧还蛋白在还原型谷胱甘肽 （GSH） 的帮助下，使用单硫醇机制或二硫醇机制可逆地还原谷胱甘肽和非谷胱甘肽二硫化物。单硫醇机制仅涉及单个谷胱甘肽化活性位点半胱氨酰残基，而二硫醇机制需要在活性位点半胱氨酰残基和分离半胱氨酰残基之间额外形成分子内二硫键。虽然谷胱甘肽二硫键底物对谷二氧杂蛋白的氧化已被广泛表征，但用于还原 S-谷胱甘肽化戊二氧杂蛋白或分子内戊二氧英二硫化物的酶-底物相互作用仍然表征不佳。在这里，我们比较了 S-谷胱甘肽化戊二氧还蛋白和分子内戊二氧还蛋白二硫化物还原的巯基特异性。我们表明，S-谷胱甘肽化戊二氧还蛋白与大量的硫醇迅速反应，并且 I 类戊二氧杂蛋白的第 2 个谷胱甘肽相互作用位点对作为还原剂的 GSH 缺乏特异性。相比之下，部分应变的分子内戊二氧还素二硫化物的较慢还原涉及在第 1 个谷胱甘肽相互作用位点与 GSH 的两个羧酸盐基团的特异性相互作用。因此，I 类戊二氧还蛋白的二硫醇机制促进了 GSH 作为还原剂的特异性，这可能解释了二硫醇戊二氧还蛋白在原核生物和真核生物中的普遍性。