How Does Iron–Sulfur Cluster Coordination Regulate the Activity of Human Glutaredoxin 2?

Abstract

Human mitochondrial glutaredoxin (Grx2) was described as the first iron-sulfur protein from the thioredoxin superfamily of proteins. The [2Fe-2S] cluster was proposed to serve as redox sensor for the activation of Grx2 during oxidative stress. The authors have demonstrated that the iron-sulfur cluster is complexed by the two N-terminal active site thiols of two Grx2 monomers and two molecules of glutathione that are bound noncovalently to the proteins and in equilibrium with glutathione in solution. When reduced glutathione becomes the limiting factor for cluster coordination, the holo-Grx2 complex dissociates, yielding enzymatically active Grx2.

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…T(SH) 2 (27), T. brucei Tpx (19), T. cruzi TR (28), T. brucei glutathione peroxidase type tryparedoxin peroxidase (Px III) (29), T. brucei His 6 -peroxiredoxin type tryparedoxin peroxidase (TXNPx) (30), the small subunit of T. brucei RR (R2) (31), and His-tagged E. coli cysteine desulfurase (9) were prepared as described. The gene for the large subunit (R1) of RR was cloned in the modified pET vector described below, and the tag-free protein was purified by chromatography on two consecutive nickel-NT-Sepharose columns as outlined below for Grx1 and Grx2.…”

“…The absorption spectrum displayed, in addition to the maximum around 280 nm, peaks at 320 and 420 nm that were reminiscent of Grxs shown to coordinate [2Fe-2S] clusters (Fig. 6A) (7,9,11). Size exclusion chromatography revealed two peaks.…”

“…6A, inset), suggesting formation of a dimeric protein species with a bound FeS cluster. This finding was rather unexpected because T. brucei Grx1 contains the canonical CPYC active site, and the presence of the Pro had been hypothesized to prevent cluster incorporation in other Grxs (9,11). Unfortunately, the cluster was not stable upon further purification.…”

“…Important physiological functions are the reversible S-glutathionylation of proteins upon oxidative stress or redox signaling and the delivery of reducing equivalents for the synthesis of DNA precursors by ribonucleotide reductase (RR). Recently, human Grx2 and poplar GrxC1 have been shown to coordinate iron-sulfur clusters (7)(8)(9)(10)(11). As monomeric (apo) forms, the proteins are regular Grxs, whereas in the dimeric form with a [2Fe-2S] cluster that bridges the monomers, they are catalytically inactive.…”

“…Reconstitution and Analysis of FeS Grx Clusters-FeS cluster assembly was performed essentially as described (9). 50 -230 M Grx1 or Grx2 in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, 5 mM DTT was incubated at room temperature under argon atmosphere with Fe(NH 4 ) 2 (SO 4 ) 2 , cysteine, Gsp or GSH, cysteine desulfurase, and pyridoxal phosphate in a molar ratio of 1 (Grx):2:2.5:4:0.02:0.04 for at least 1 h. Subsequently, the reaction mixture was centrifuged for 2 min at 13,000 rpm and 4°C and immediately analyzed by UV-visible spectroscopy and gel filtration chromatography on a Superdex 75 10/300 column (GE Healthcare) in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0.…”