The crystal structure of human GLRX5: iron–sulfur cluster co-ordination, tetrameric assembly and monomer activity

Abstract

Human GLRX5 (glutaredoxin 5) is an evolutionarily conserved thiol-disulfide oxidoreductase that has a direct role in the maintenance of normal cytosolic and mitochondrial iron homoeostasis, and its expression affects haem biosynthesis and erythropoiesis. We have crystallized the human GLRX5 bound to two [2Fe-2S] clusters and four GSH molecules. The crystal structure revealed a tetrameric organization with the [2Fe-2S] clusters buried in the interior and shielded from the solvent by the conserved β1-α2 loop, Phe⁶⁹ and the GSH molecules. Each [2Fe-2S] cluster is ligated by the N-terminal activesite cysteine (Cys⁶⁷) thiols contributed by two protomers and two cysteine thiols from two GSH. The two subunits co-ordinating the cluster are in a more extended conformation compared with iron-sulfur-bound human GLRX2, and the intersubunit interactions are more extensive and involve conserved residues among monothiol GLRXs. Gel-filtration chromatography and analytical ultracentrifugation support a tetrameric organization of holo-GLRX5, whereas the apoprotein is monomeric. MS analyses revealed glutathionylation of the cysteine residues in the absence of the [2Fe-2S] cluster, which would protect them from further oxidation and possibly facilitate cluster transfer/acceptance. Apo-GLRX5 reduced glutathione mixed disulfides with a rate 100 times lower than did GLRX2 and was active as a glutathione-dependent electron donor for mammalian ribonucleotide reductase.

Structural insights into the N-terminal GIY–YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts

“…In contrast, we found that in the AtGRXS16 dimer, 65% of the Cys residues in the NTD (SVPELCGSVK) and 66% of the Cys residues in the CTD (SAPQCGFSQR), as readouts as free thiols in these assays, did not form an intramolecular disulfide bond (Table S2), which is consistent with the fact that the Cys residue in the "CGFS" motif is essential for Fe-S cluster binding (Fig. S5C) (20,21,23). These results also suggest that AtGRXS16-NTD could bind to the Grx domain bridged by an intramolecular disulfide bond and inhibit the Grx activity in vivo.…”

Redox homeostasis and age‐related deficits in neuromuscular integrity and function

“…GRX1 is mainly localized in the cytosol but can be found in the MIS; it can be translocated into the nucleus and exported from the cell 159. GRX2 is expressed in the mitochondria,165 GRX3 in the cytosolic and nuclear compartment, and monothiol GRX5 has a mitochondrial translocation signal and shares the active site motif of GRX3 166. Evidence has also shown that the GRX system can also catalyse reversible protein glutathionylation,167 which is an important redox regulatory mechanism, and control the redox state of thiol groups168 in situations where the redox environment is being compromised.…”

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…The role of Grx5 as an Fe/S cluster transfer protein is supported by the crystal structure of the human homologue GLRX5 with a bridging [2Fe-2S] cluster (73). The definition for bacterial GrxD as a cluster "carrier protein" cannot be adopted for mitochondria to avoid confusion with "mitochondrial carrier proteins" involved in inner membrane transport of metabolites (74).…”