Deciphering the catalytic mechanism of redox-active glutaredoxins

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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?