Redox properties and evolution of human glutaredoxins

Abstract

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.

Mechanistic and Kinetic Details of Catalysis of Thiol-Disulfide Exchange by Glutaredoxins and Potential Mechanisms of Regulation

“…NMR structural analysis predicts that they are all solvent accessible (128), and thus susceptible to oxidative modification. In contrast, the two non-active-site cysteines of Grx2 (C28, C100) appear to be involved in a structural disulfide bond (60,112). Although these observations support the possibility of posttranslational redox regulation of Grx, little is known about the specific modifications or their relevance to redox homeostasis in vivo.…”

“…Colors indicate the species from which the Grx-SSG structure was determined kinetically (see earlier). Overviews of glutaredoxin proteins from different species can be found elsewhere (33,68,109,112); here, we review evidence for deglutathionylation activity in glutaredoxins from prototype organisms (see Table 1), with special emphasis on newly described Grx proteins, and putative glutaredoxin domains on multidomain proteins.…”

Effects of bpV(pic) and bpV(phen) on H9c2 cardiomyoblasts during both hypoxia/reoxygenation and H2O2-induced injuries

“…Thus, scavenging excessive ROS and restoring the reduction-oxidation (redox) balance in the body is an important strategy in inhibiting reperfusion injury, as the redox balance is the solid physiological condition in humans from birth (13,14), and, despite evolution, this balance has always been conserved in all organisms (15,16).…”

Glutaredoxin regulates vascular development by reversible glutathionylation of sirtuin 1

“…Two conserved additional cysteine residues characterize Grx2 as a vertebrate-specific enzyme (8,9). Human Grx2 is expressed in three different isoforms located in mitochondria (Grx2a) and cytosol/nucleus (Grx2b and Grx2c) (10).…”