The E. coli Monothiol Glutaredoxin GrxD Forms Homodimeric and Heterodimeric FeS Cluster Containing Complexes

Abstract

Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homolog of E. coli BolA) in yeast, and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G. et al. (2008) Nature Methods 5, 789–795]. These data connected the only E. coli mono-Grx, GrxD, to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes, to ultimately uncover if either of these complexes perform functions linked to FeS cluster biosynthesis.

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…A physiological relevance of a Grx-Bol heterodimer has been established only for the yeast cytosolic monothiol glutaredoxin Grx3 and Bol2 (formerly known as Fra2), which are involved in iron homeostasis in the yeast cytosol (94 -96). Similar Fe/S cluster-containing Bol-Grx heterocomplexes have been observed in bacteria (BolA with GrxD or Grx4), plants (BolA1 and BolA2 with GrxS14 and GrxS17), and recently in humans (BOLA1 and BOLA3 with GLRX5) (93,97,98). Mitochondrial Bol1 proteins contain three conserved histidine residues, and Bol3 contains a histidine and a cysteine residue as candidates for coordination of the [2Fe-2S] cluster in the heterodimers with Grx5 (Fig.…”

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…The function of these holo-heterodimers is still not clear (6,9,50,51). The only characterized physiological role for a GrxBolA holo-heterodimer is in sensing the intracellular iron/Fe-S cluster status in yeast.…”

“…This cluster is ligated by two cysteines (one from Grx and one from a GSH molecule) and one histidine (invariant histidine of BolAs), but the fourth ligand is unknown (7,8). On the other hand, it was reported that Grx-BolA couples from various sources could also form apo-heterodimers (7,9). This is consistent with the observation that an in vivo Grx3/4-Fra2 interaction is found both in iron-replete and iron-depleted yeast cells (5).…”

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

“…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).…”