Deciphering the catalytic mechanism of redox-active glutaredoxins

Glutaredoxins （Grx）在原核和真核生物生理和病理生理条件下的氧化还原和铁代谢中起核心作用。大多数Grx属于两个主要的亚家族，I类或规范Grx，它们是谷胱甘肽依赖的硫醇：二硫氧化还原酶，以及II类Grx或Grx样蛋白，它们在标准的氧化还原酶检测中无活性，但作为铁传感器，对谷胱甘肽依赖的铁硫簇递送至关重要。尽管最近取得了重大进展，但主要仍不清楚(i)谷胱甘肽化的i类Grx在还原半反应中如何被还原性谷胱甘肽（GSH）还原，（ii）非谷胱甘肽二硫化物在氧化半反应中如何还原，（iii）为什么大多数i类Grx在催化半胱氨酸附近有第二个甚至第三个半胱氨酸残基，以及（iv） i类Grx的活性是否以及如何被调节。在这里，我们希望通过两个主要工作包来解决这些悬而未决的问题：Grx催化谷胱甘肽二硫化物的还原•Grx对谷胱甘肽作为还原底物的偏好来自哪里？•靠近反应中心的选定残基是否具有调节作用？2. Grx催化非谷胱甘肽二硫化物还原？ I类Grx是直接还原蛋白质二硫化物还是激活谷胱甘肽还原？•二硫蛋白底物的氧化还原电位是否决定其机制？•替代半胱氨酸能在体内分解被捕获的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?