Deciphering the catalytic mechanism of redox-active glutaredoxins

谷氧还蛋白（Grx）在原核生物和真核生物生理和病理条件下的氧化还原和铁代谢中起着重要作用。大多数Grx属于两个主要的亚家族，I类或典型的Grx，这是谷胱甘肽依赖性硫醇：二硫化物氧化还原酶，和II类Grx或Grx样蛋白，这是在标准的氧化还原酶测定无活性，但作为铁传感器，是至关重要的谷胱甘肽依赖性铁硫簇的交付。尽管最近取得了重大进展，但仍然主要未知的是（i）谷胱甘肽化的I类Grx在还原半反应期间如何被还原型谷胱甘肽（GSH）精确还原，（ii）非谷胱甘肽二硫化物在氧化半反应期间如何被还原，（iii）为什么大多数I类Grx在催化半胱氨酸附近具有第二或甚至第三半胱氨酸残基，及（iv）I类Grx的活性是否受规管及如何受规管。在这里，我们希望在两个主要工作包中解决这些悬而未决的问题：1。破解Grx催化还原谷胱甘肽二硫化物·Grx对还原底物GSH的偏好来自哪里？·靠近反应中心的选定残基是否具有调节作用？2.解密Grx催化还原非谷胱甘肽二硫化物·I类Grx直接还原蛋白质二硫化物还是激活GSH还原？·蛋白质二硫键底物的氧化还原电位决定了机制吗？·替代性半胱氨酸在体内分解捕获的Grx二硫化物吗？

Glutaredoxins (Grx) play central roles in redox and iron metabolism in pro- and eukaryotes under physiological and pathophysiological conditions. Most Grx belong to two major subfamilies, class I or canonical Grx, which are glutathione-dependent thiol:disulfide oxidoreductases, and class II Grx or Grx-like proteins, which are inactive in standard oxidoreductase assays but serve as iron sensors and are crucial for the glutathione-dependent delivery of iron-sulfur clusters. Despite significant recent advances, it is still predominantly unknown (i) how glutathionylated class I Grx are exactly reduced by reduced glutathione (GSH) during the reductive half-reaction, (ii) how non-glutathione disulfides are reduced during the oxidative half-reaction, (iii) why most class I Grx have a second or even third cysteine residue in the proximity of the catalytic cysteine, and (iv) if and how the activity of class I Grx is regulated. Here we want to address these open questions in two major work packages: 1. Deciphering the Grx-catalyzed reduction of glutathione disulfides • Where does the preference of Grx for GSH as the reducing substrate come from? • Do selected residues close to the reaction center have a regulatory role? 2. Deciphering the Grx-catalyzed reduction of non-glutathione disulfides • Do class I Grx reduce protein disulfides directly or do they activate GSH for reduction? • Do the redox potentials of protein disulfide substrates determine the mechanism? • Do alternative cysteines resolve trapped Grx disulfides in vivo?