Unlike thioredoxins, glutaredoxins are involved in ironsulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys 32 ) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter-or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

Regulation of iron metabolism in Saccharomyces cerevisiae is achieved at the transcriptional level by low (Aft1 and Aft2) and high iron-sensing (Yap5) transcription factors, and at the post-transcriptional level by mRNA-binding proteins (Cth1 and Cth2). In this review we highlight recent studies unveiling the critical role that iron-sulfur clusters play in control of Aft1/2 and Yap5 activity, as well as the complex relationship between iron homeostasis and thiol redox metabolism. In addition, new insights into the localization and regulation of Cth1/Cth2 have added another layer of complexity to the cell’s adaptation to iron deficiency. Finally, biophysical studies on subcellular iron speciation changes in response to environmental and genetic factors have further illuminated the elaborate control mechanisms required to manage iron bioavailability in the cell.

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.

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10 4 min ؊1 . We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis.

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.

Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes. However, some limitations arise from sample preparation requirements, complexity of individual techniques, or the necessity for combining multiple methods in order to achieve a complete investigation. In this chapter, we focus on the use of UV-visible circular dichroism spectroscopy as a fast and simple initial approach for investigating glutaredoxin Fe-S cluster-dependent interactions.

An elaborate cascade of iron‐sulfur cluster‐dependent cellular interactions are employed in the yeast S. cerevisiae in order to maintain adequate iron levels. Currently available data indicate that under iron deplete conditions, the two paralogous transcription factors, Aft1 and Aft2, are primarily localized in the nucleus, and activate the transcription of iron uptake and transport genes. Under iron replete conditions, Aft1/2 undergo conformational changes and nucleocytoplasmic shuttling upon interacting with CGFS monothiol glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, resulting in subsequent deactivation of the iron regulon. Iron‐sulfur clusters have been chosen by nature as signaling molecules to accomplish the inhibition of Aft2 (and presumably Aft1), as the dimerization occurs via direct ligation of a [2Fe‐2S] cluster acquired from the [2Fe‐2S] cluster‐bridged Grx3/4‐Bol2 heterodimer. However, the mechanistic details of iron regulation at the molecular level in the baker's yeast are not fully understood, and defining the functional interactions of each component in the iron signaling pathway remains to be elucidated. We are currently using complementary biophysical and molecular genetic methods to probe the iron‐sulfur cluster‐dependent interaction between [2Fe‐2S]2+‐Grx3/4 and Bol2, and [2Fe‐2S]2+‐Grx3/4‐Bol2 and Aft2 to gain a better understanding of their in vivo functions. The results indicate rapid interaction between [2Fe‐2S]2+‐Grx3 and Bol2, followed by a fast iron‐sulfur cluster transfer to Aft2, with second order rates above previously reported rates for reactions involving iron‐sulfur clusters. Furthermore, we are showing that mutations in the amino acid residues ligating the iron‐sulfur cluster in the [2Fe‐2S]2+‐Grx3‐Bol2 heterodimer, or in the CxC iron‐sulfur cluster binding motif of Aft2 have a significant impact on the rate of interaction and cluster transfer, respectively. Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism.Support or Funding InformationNational Institute of General Medical Sciences R01 (Grant GM100069 to CEO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.