Two Novel Monothiol Glutaredoxins from Saccharomyces cerevisiae Provide Further Insight into Iron-Sulfur Cluster Binding, Oligomerization, and Enzymatic Activity of Glutaredoxins

Abstract

Two novel monothiol glutaredoxins from yeast (ScGrx6 and ScGrx7) were identified and analyzed in vitro. Both proteins are highly suited to study structure-function relationships of glutaredoxin subclasses because they differ from all monothiol glutaredoxins investigated so far and share features with dithiol glutaredoxins. ScGrx6 and ScGrx7 are, for example, the first monothiol glutaredoxins showing an activity in the standard glutaredoxin transhydrogenase assay with glutathione and bis-(2-hydroxyethyl)-disulfide. Steady-state kinetics of ScGrx7 with glutathione and cysteine-glutathione disulfide are similar to dithiol glutaredoxins and are consistent with a ping-pong mechanism. In contrast to most other glutaredoxins, ScGrx7 and ScGrx6 are able to dimerize noncovalently. Furthermore, ScGrx6 is the first monothiol glutaredoxin shown to directly bind an iron-sulfur cluster. The cluster can be stabilized by reduced glutathione, and its loss results in the conversion of tetramers to dimers. ScGrx7 does not bind metal ions but can be covalently modified in Escherichia coli leading to a mass shift of 1090 +/- 14 Da. What might be the structural requirements that cause the different properties? We hypothesize that a G(S/T)x3 insertion between a highly conserved lysine residue and the active site cysteine residue could be responsible for the abrogated transhydrogenase activity of many monothiol glutaredoxins. In addition, we suggest an active site motif without proline residues that could lead to the identification of further metal binding glutaredoxins. Such different properties presumably reflect diverse functions in vivo and might therefore explain why there are at least seven glutaredoxins in yeast.

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

“…Glutaredoxins are widely considered the primary cellular catalysts for protein deglutathionylation (43,44). Notably, two glutaredoxins (Grx6 and Grx7) localize to the ER/Golgi region in yeast, and these enzymes have the potential to facilitate deglutathionylation of BiP (32,45). However, our preliminary data show no change in the kinetics of BiP-glutathione adduct reduction in the glutaredoxin-deficient grx6⌬ grx7⌬ strain (data not shown).…”

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

“…Iron-Sulfur Cluster Assembly-Another important point is that human Grx2 (SCSYC active site), poplar GrxC1 (YCGYC), S. cerevisiae Grx6 (TCSYS), and Trypanosoma brucei Grx1 (MCAYS) are able to bind an iron-sulfur cluster, most likely a [2Fe-2S] cluster (20,35,44,45). The crystal structures of Grx2 and GrxC1 in the holoform are similar and showed that the incorporation of the cluster requires the dimerization of the Grx and the presence of two external glutathione molecules (20,35,46).…”

Mono- and Dithiol Glutaredoxins in the Trypanothione-Based Redox Metabolism of Pathogenic Trypanosomes

“…With few exceptions, such as yeast Grx 6 (54,59) and trypanosomal 1-C-Grx1 (16, 27, Fleitas, Manta, and Comini, unpublished), 1-C-Grxs are monomeric proteins in the apo-state (13,18,25,28,47,59,69,82). Similarly to 2-C-Grxs, distinct isoforms of 1-C-Grxs are found in the cytosol, nucleus, mitochondria as well as plant chloroplast (reviewed in 86).…”

“…Similarly to 2-C-Grxs, distinct isoforms of 1-C-Grxs are found in the cytosol, nucleus, mitochondria as well as plant chloroplast (reviewed in 86). Except for yeast Grx6 and Grx7 (59,60), most 1-C-Grxs lack or have negligible classical disulfide reductase activity (18,26,27,43,69,82). Instead, the capability to coordinate ISCs appears to be a common feature for 1-C-Grxs (16,40,42,43,54,67,89) with yeast Grx7 being, so far, the only known exception (59,60).…”

“…Except for yeast Grx6 and Grx7 (59,60), most 1-C-Grxs lack or have negligible classical disulfide reductase activity (18,26,27,43,69,82). Instead, the capability to coordinate ISCs appears to be a common feature for 1-C-Grxs (16,40,42,43,54,67,89) with yeast Grx7 being, so far, the only known exception (59,60). The latter feature determines an evolutionary conserved and indispensable role of 1-C-Grxs in the biogenesis and assembly of iron-sulfur proteins (11,61) and other cell-specific regulatory functions such as the (in)activation of nuclear transcription factors (35,62,86).…”