Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and CD, resonance Raman, and EPR spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans.

Monothiol glutaredoxins (Grxs) are proposed to function in Fe-S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe2S2]2+ cluster, and to accept [Fe2S2]2+ clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe2S2]2+ cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe-S cluster carrier proteins (NifIscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe2S2]2+ cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandii NifIscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe-S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe-S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe2S2]2+ cluster-bound heterodimeric Grx3/Fra2 complex can be formed by intact [Fe2S2]2+ cluster transfer from NifIscA. The significance of these results for Fe-S cluster biogenesis or repair and the cellular regulation of the Fe-S cluster status are discussed.

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The E. coli genome encodes members of both of these proteins families, namely the monothiol glutaredoxin Grx4, and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimers as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are DHAD, which contains an oxygen-labile [FeS] cluster, and spinach DHAD, which contains an oxygen-resistant [FeS] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism and resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from (). Our results indicated that DHAD can accommodate [FeS] and [FeS] clusters. However, only the [FeS] cluster-bound form is catalytically active. We found that the [FeS] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. cluster transfer and reconstitution reactions revealed that [FeS] cluster-containing NFU2 protein is likely the physiological cluster donor for maturation ofDHAD. In summary, DHAD binds either one [FeS] or one [FeS] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [FeS] cluster donor for DHAD maturation. This work represents the first characterization of recombinantDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.