Roles of glutaredoxin and thioredoxin-like Nfu proteins in iron-sulfur cluster biogenesis
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Iron-sulfur (Fe-S) clusters are one of the most ancient, ubiquitous and functionally versatile prosthetic groups in nature. Based on the organization of genes in bacterial operons, three highly conserved types of machinery for the biogenesis of Fe-S clusters have emerged, namely the NIF (Nitrogen Fixation), the ISC (Iron-Sulfur Cluster) and the SUF (Sulfur Mobilization) systems. However, recent studies have implicated roles in Fe-S cluster biogenesis for accessory proteins such as glutaredoxins and thioredoxin-like Nfu proteins that are not encoded by genes in nif, isc, or suf operons. This research project was designed to elucidate the role of dithiol and monothiol glutaredoxins and thioredoxin-like Nfu proteins in Fe-S cluster biogenesis. The approach involved structural and/or spectroscopic characterization of clusters assembled on plant dithiol and monothiol glutaredoxins and Azotobacter vinelandii NfuA protein coupled with in vitro studies of cluster transfer to physiologically relevant acceptor proteins and in vivo assessment of function via gene knock out or complementation studies. The A. vinelandii 2+ NfuA protein was shown to assemble a subunit-bridging [4Fe-4S]cluster which is capable of activating apo aconitase at physiologically relevant rates. Taken together with in vivo studies, which indicate impaired maturation of Fe-S proteins in strains inactivated for nfuA, the results indicate a role of NfuA as a class of intermediate [Fe-S] cluster carrier proteins in bacteria. Spectroscopic, mutagenesis and crystallographic studies of the cytosolic poplar dithiol glutaredoxin C1 (CGYC active site) have demonstrated that dithiol glutaredoxins can assemble a 2+novel glutathione-ligated [2Fe-2S] cluster at the subunit interface of the homodimer and have established the active-site sequence requirement for Fe-S cluster incorporation. The inability to 2+transfer the [2Fe-2S] clusters to apo forms of acceptor Fe-S proteins suggests that the cluster in dithiol glutaredoxins may play a role in sensing oxidative stress. The chloroplastic poplar monothiol glutaredoxin S14 (CGFS active site) was also shown to assemble a similar 2+glutathione-ligated [2Fe-2S] cluster at the subunit interface of the homodimer. However, in 2+contrast to dithiol glutaredoxins, kinetic studies indicate that the [2Fe-2S] cluster on glutaredoxin S14 is rapidly and stoichiometrically transferred intact to apo Synechocystis ferredoxin, a physiologically relevant acceptor protein. Moreover, in vivo studies showed that glutaredoxin S14 can complement the Fe-S cluster assembly deficiency in yeast mitochondrial monothiol glutaredoxin 5 (CGFS active-site) deletion mutants. These results indicate that monothiol glutaredoxins play a role as scaffold proteins involved in the assembly and/or delivery 2+of [2Fe-2S] clusters in mitochondria and chloroplasts.