SynopsisIscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/ SufA paralogs blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthesis pathway in E. coli cells under aerobic conditions and that addition of the ironbound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the cluster assembly in protein ThiC of the thiamin biosynthesis pathway, aconitase B of the citrate acid cycle, and endonuclease III of the DNA base excision repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin, and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogs are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions. Keywords aconitase; branched-chain amino acids; dihydroxyacid dehydratase; iron-sulfur clusters; IscA/SufA paralogs; thiamin IntroductionIron-sulfur clusters are one of the most ancient and ubiquitous redox centers in biology. They are involved in diverse physiological processes including respiratory electron transfer, nitrogen fixation, photosynthesis, biosynthesis of amino acids, thiamin, heme, biotin, and lipoic acid, DNA synthesis and repair, RNA modification, and the regulation of gene expression [1,2]. However, the mechanism underlying the iron-sulfur cluster assembly is still not fully understood [3]. The discovery of cysteine desulfurase (NifS) Experimental Mutant strains and cell growthThe E. coli deletion mutants in which iscA and sufA were in-frame deleted were previously constructed [28]. Each deletion in E. coli cells was confirmed by PCR as described in [28]. For cell growth analysis, overnight cell cultures grown in Luria-Bertani (LB) medium were washed twice with minimal medium containing glucose (0.2%) before inoculated on minimal medium plates or in liquid minimal medium at 37°C with aeration (250 rpm). Cell growth was recorded by measuring the optical density of cell culture at 600 nm. When indicated, minimal medium was supplemented with the three branched-chain amino acids ...

This study provides evidence for a role of RTN3 in inducing obesity and triglyceride accumulation and suggests that inhibiting the expression of RTN3 in fat tissue may be a novel therapeutic approach to treat obesity and hypertriglyceridemia.

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not the proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not the proteins without iron-sulfur clusters, are modified forming protein-bound DINCs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of “chelatable iron pool” in the wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of “chelatable iron pool” in cells.

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SYNOPSIS IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. Here we report the in vivo evidence demonstrating the iron binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1μM) in the M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralog SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions but not under anaerobic growth conditions. The results provide the in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.

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