Characterization of iron binding in IscA, an ancient iron-sulphur cluster assembly protein.
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ABSTRACT:
SUBMITTER: Ding H
PROVIDER: S-EPMC1224081 | biostudies-other | 2004 Apr
REPOSITORIES: biostudies-other
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Project description:Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.
| S-EPMC4132641 | biostudies-literature
Project description:Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by l-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.
| S-EPMC3569480 | biostudies-literature
Project description:NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.
| S-EPMC2435133 | biostudies-literature
Project description:The biogenesis of iron-sulphur clusters requires the co-ordinated delivery of both iron and sulphur. It is now clear that sulphur in iron-sulphur clusters is derived from L-cysteine by cysteine desulphurases. However, the iron donor for the iron-sulphur cluster assembly still remains elusive. Our previous studies indicated that Escherichia coli IscA, a member of the iron-sulphur cluster assembly machinery, is an iron-binding protein that can provide iron for the iron-sulphur cluster assembly in a proposed scaffold IscU. To determine how the iron centre in IscA is transferred for the iron-sulphur cluster assembly in IscU, we explore the mobility of the iron centre in IscA. The UV-visible and EPR measurements show that L-cysteine, but not IscU, is able to mobilize the iron centre in IscA and make the iron available for the iron-sulphur cluster assembly in IscU. Other related biological thiols such as N-acetyl-L-cysteine or reduced glutathione have no effect on the iron centre of IscA, suggesting that L-cysteine is unique in mobilizing the iron centre of IscA. Nevertheless, L-cysteine alone is not sufficient to transfer the iron from IscA to IscU. Both L-cysteine and cysteine desulphurase (IscS) are required for the IscA-mediated assembly of iron-sulphur clusters in IscU. The results suggest that L-cysteine may have two distinct functions in the biogenesis of iron-sulphur clusters: to mobilize the iron centre in IscA and to provide sulphur via cysteine desulphurase (IscS) for the iron-sulphur cluster assembly in IscU.
| S-EPMC1180730 | biostudies-other
Project description:Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron-sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions.
| S-EPMC2982165 | biostudies-literature
Project description:Iron-sulphur (Fe-S) clusters are ensembles of iron and sulphide centres. They are found in all life forms and are important components of many enzymes involved in diverse cellular processes, including respiration, DNA synthesis or gene regulation. However, the increase in oxygen after the emergence of oxygenic photosynthesis created a threat to Fe–S proteins and, consequently, to the organisms relying on them. Therefore, bacteria have evolved mechanisms to maintain a precise intracellular iron concentration. A major role of IscR in R. sphaeroides iron dependent regulation was suggested in a bioinformatic study , which predicted a binding site in the upstream regions of several iron uptake genes. Most known IscR proteins have Fe-S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbour only one Cys residue and it was considered unlikely that they can ligate an Fe-S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe-S cluster status of the cell was analysed. The results provide evidence that R. sphaeroides IscR functions as transcriptional repressor of genes involved in iron metabolism by binding to the predicted DNA binding motif. Furthermore, IscR possesses a unique Fe-S cluster ligation scheme with only a single cysteine involved.
2015-02-03 | GSE65537 | GEO
Project description:A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron-sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron-sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.
| S-EPMC2447459 | biostudies-other
Project description:Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.
| S-EPMC6211773 | biostudies-other
Project description:Iron-sulphur (Fe-S) clusters are ensembles of iron and sulphide centres. They are found in all life forms and are important components of many enzymes involved in diverse cellular processes, including respiration, DNA synthesis or gene regulation. However, the increase in oxygen after the emergence of oxygenic photosynthesis created a threat to FeM-bM-^@M-^SS proteins and, consequently, to the organisms relying on them. Therefore, bacteria have evolved mechanisms to maintain a precise intracellular iron concentration. A major role of IscR in R. sphaeroides iron dependent regulation was suggested in a bioinformatic study , which predicted a binding site in the upstream regions of several iron uptake genes. Most known IscR proteins have Fe-S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbour only one Cys residue and it was considered unlikely that they can ligate an Fe-S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe-S cluster status of the cell was analysed. The results provide evidence that R. sphaeroides IscR functions as transcriptional repressor of genes involved in iron metabolism by binding to the predicted DNA binding motif. Furthermore, IscR possesses a unique Fe-S cluster ligation scheme with only a single cysteine involved. RNA samples collected from a control strain (wild type 2.4.1) and of the iscR deletion strain (2.4.1M-bM-^HM-^FiscR) were analyzed by two-color microarrays
2015-02-03 | E-GEOD-65537 | biostudies-arrayexpress
Project description:Staphylococcus aureus SufT is composed solely of the domain of unknown function 59 (DUF59) and has a role in the maturation of iron-sulphur (Fe-S) proteins. We report that SufT is essential for S. aureus when growth is heavily reliant upon lipoamide-utilizing enzymes, but dispensable when this reliance is decreased. LipA requires Fe-S clusters for lipoic acid (LA) synthesis and a ΔsufT strain had phenotypes suggestive of decreased LA production and decreased activities of lipoamide-requiring enzymes. Fermentative growth, a null clpC allele, or decreased flux through the TCA cycle diminished the demand for LA and rendered SufT non-essential. Abundance of the Fe-S cluster carrier Nfu was increased in a ΔclpC strain and a null clpC allele was unable to suppress the LA requirement of a ΔsufT Δnfu strain. Over-expression of nfu suppressed the LA requirement of the ΔsufT strain. We propose a model wherein SufT, and by extension the DUF59, is essential for the maturation of holo-LipA in S. aureus cells experiencing a high demand for lipoamide-dependent enzymes. The findings presented suggest that the demand for products of Fe-S enzymes is a factor governing the usage of one Fe-S cluster assembly factor over another in the maturation of apo-proteins.
| S-EPMC5161685 | biostudies-literature