Redox properties and evolution of human glutaredoxins

Glutaredoxins (Grxs) are glutathione‐dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol‐disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C‐X‐Y‐C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be −232 and −221 mV, respectively, using a combination of redox buffers, protein–protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol−1 additional stability by linking the C‐terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be −317 mV and is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C‐G‐F‐S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins. Proteins 2007. © 2007 Wiley‐Liss, Inc.