Biochemical Characterization of Yeast Mitochondrial Grx5 Monothiol Glutaredoxin

Abstract

Grx5 is a yeast mitochondrial protein involved in ironsulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically characterized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of ؊175 mV was calculated for wild type Grx5. The pK a values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pK a . Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pK a value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.

Combining data from genomes, Y2H and 3D structure indicates that BolA is a reductase interacting with a glutaredoxin

“…Their exact function has not been elucidated. It has been suggested that they are involved in the deglutathionylation of protein-GS mixed sulfides [27,28]. For this process dithiol glutaredoxins only require their N-terminal cysteine thiol [29], which is the one cysteine that is conserved in mono-thiol glutaredoxins.…”

“…For this process dithiol glutaredoxins only require their N-terminal cysteine thiol [29], which is the one cysteine that is conserved in mono-thiol glutaredoxins. Indeed the best characterized member of this family, the mitochondrial protein Grx5 from yeast, is able to deglutathionylate proteins [27], and is involved in defense against oxidative stress [30].…”

Structure-Function Relationship of the Chloroplastic Glutaredoxin S12 with an Atypical WCSYS Active Site

“…Glutaredoxins (Grxs) 5 are GSH-or thioredoxin reductasedependent oxidoreductases involved in the maintenance of cellular redox homeostasis. When Grxs are recycled by GSH, the GSSG formed is in turn reduced by NADPH and glutathione reductase (GR), forming the GSH/Grx reducing system.…”

Reversible Sequestration of Active Site Cysteines in a 2Fe-2S-bridged Dimer Provides a Mechanism for Glutaredoxin 2 Regulation in Human Mitochondria

“…More recently glutaredoxin 5 (GLRX5), with only one cysteine residue in the active-site motif, has been characterized in the yeast Saccharomyces cerevisiae, with homologs found in organisms from bacteria to humans (6 -9). It is of particular interest that GLRX5 is targeted to mitochondria like GLRX2 and that GLRX5 participates in the biogenesis of iron-sulfur clusters (10,11). An alignment of these three human isoforms is provided in Fig.…”