755 research outputs found

    Function of IscA in biogenesis of Iron-sulfur clusters and repair of NO-modified iron-sulfur proteins

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    Iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups that function in diverse fundamental life processes. However, the biogenesis of iron-sulfur clusters in vivo is not a spontaneous process. Previous studies indicated that Fe-S clusters maybe synthesized by three major systems: the Nif, the ISC, and the SUF systems. Among these three systems, IscU, NifU are the scaffold proteins for the Fe-S clusters assembly. IscA and its paralog, SufA are proposed as an alternative iron-sulfur cluster assembly scaffold proteins. The cysteine desulfurases: IscS, NifS and SufS catalyze desulfurization of the L-cysteine and provide sulfide for Fe-S clusters assembly. However, the iron donor for the Fe-S clusters assembly remains poorly understood. In this research, we reported that IscA is a strong iron binding protein. Under physiological conditions, if only iron is available, iron will bind to IscA. The addition of L-cysteine to this iron-bound IscA mobilizes the iron center in IscA and transfer iron to IscU for the Fe-S cluster assembly. However, if both iron and sulfide are available, Fe-S clusters are preferred to be assembled in IscU. Under oxidative stress conditions, IscA fails to bind ferrous iron due to the oxidation of its iron binding thiolate groups. CyaY, an E. coli homology of Frataxin is able to bind iron under oxidative stress conditions and effectively alleviate the production of the deleterious hydroxyl free radicals. Nevertheless, unlike IscA, CyaY cannot function as an efficient iron donor for the Fe-S clusters assemlby due to its weak iron binding property. We also investigated the repair mechanism for the NO-modified aconitase B [4Fe-4S] clusters. We found that E. coli [4Fe-4S] aconitase B is readily converted to the protein-bound DNICs by NO in vitro and in vivo. L-cysteine and oxygen are required for decomposition of the protein-bound DNICs. We further demonstrated that a complete repair of the NO-modified aconitase B requires two sequential steps: decomposition of the protein-bound DNICs requires both L-cysteine and oxygen, and the reassembly of Fe-S clusters, which requires Fe-S clusters assembly machinery

    Interplay of IscA and IscU in biogenesis of iron-sulfur clusters

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    Increasing evidence suggests that sulfur in ubiquitous iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon isc-SUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. (2005) J. Biol. Chem. 280, 30432-30437). To further elucidate the function of IscA in biogenesis of iron-sulfur clusters, we evaluate the iron-sulfur cluster binding activity of IscA and IscU under physiologically relevant conditions. When equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS, L-cysteine and dithiothreitol, iron-sulfur clusters are assembled in IscU, but not in IscA, suggesting that IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contrast, when equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS and dithiothreitol but without L-cysteine, nearly all iron is bound to IscA. The iron binding in IscA appears to prevent the formation of the biologically inaccessible ferric hydroxide under aerobic conditions. Subsequent addition of L-cysteine efficiently mobilizes the iron center in IscA and transfers the iron for the iron-sulfur cluster assembly in IscU. The results suggest an intriguing interplay between IscA and IscU in which IscA acts as an iron chaperon that recruits free iron and delivers the iron for biogenesis of iron-sulfur clusters in IscU under aerobic conditions. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

    Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli

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    The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulphur enzyme essential for the BCAA biosynthesis, is completely inactivated in cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC). Fractionation of the cell extracts prepared from the NO-exposed cells reveals that a large number of different protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that l-cysteine may have an important role in repairing the NO-modified iron-sulphur proteins in aerobically growing E. coli cells. The results suggest that cellular deficiency to repair the NO-modified iron-sulphur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions. © 2008 The Authors

    Distinct iron binding property of two putative iron donors for the iron-sulfur cluster assembly: IscA and the bacterial frataxin ortholog CyaY under physiological and oxidative stress conditions

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    Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the ironsulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc
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