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

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 iscSUA, 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.     

IscA mediates iron delivery for assembly of iron-sulfur clusters in IscU under the limited accessible free iron conditions

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.     

Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Assembly mechanism of [Fe2S2] cluster in ferredoxin from Acidithiobacillus ferrooxidans

Ferredoxin is a typical iron-sulfur protein that is ubiquitous in biological redox systems. This study investigates the in vitro assembly of a [Fe2S2] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins: IscA, IscS, and IscU. The spectra and MALDI-TOF MS results for the reconstituted ferredoxin confirm that the iron-sulfur cluster was correctly assembled in the protein. The inactivation of cysteine desulfurase by L-allylglycine completely blocked any [Fe2S2] cluster assembly in the ferredoxin in E. coli, confirming that cysteine desulfurase is an essential component for iron-sulfur cluster assembly. The present results also provide strong evidence that [Fe2S2] cluster assembly in ferredoxin follows the AUS pathway.     

Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.     

Iron-sulfur cluster biosynthesis: characterization of Escherichia coli CYaY as an iron donor for the assembly of [2Fe-2S] clusters in the scaffold IscU

The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use L-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Nevertheless, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported so far. In this paper we demonstrate for the first time a specific interaction between CyaY and IscS, a cysteine desulfurase participating in iron-sulfur cluster assembly. Analysis of the iron-loaded CyaY protein demonstrated a strong binding of Fe(3+) and a weak binding of Fe(2+) by CyaY. Biochemical analysis showed that the CyaY-Fe(3+) protein corresponds to a mixture of monomer, intermediate forms (dimer-pentamers), and oligomers with the intermediate one corresponding to the only stable and soluble iron-containing form of CyaY. Using spectroscopic methods, this form was further demonstrated to be functional in vitro as an iron donor during [Fe-S] cluster assembly on the scaffold protein IscU in the presence of IscS and cysteine. All of these results point toward a link between CyaY and [Fe-S] cluster biosynthesis, and a possible mechanism for the process is discussed.     

Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.     

嗜酸氧化亚铁硫杆菌ISC铁硫簇系统的合成

【目的】铁硫簇是最古老的一种氧化还原中心,它普遍存在于所有生命体内,在光合作用、呼吸作用和固氮作用这三个地球生命最基本的代谢途径中扮演着重要的角色。【方法】以嗜酸氧化亚铁硫杆菌(A.ferrooxidans ATCC 23270)基因组为模板,克隆表达其ISC铁硫簇组装的3个核心蛋白,IscS(半胱氨酸脱硫酶蛋白)、IscU(支架蛋白)和IscA(铁供体蛋白)。【结果】研究发现IscS能催化半胱氨酸脱硫,为铁硫簇的组装提供硫,支架蛋白IscU不具备结合铁的能力,IscA具有较强的铁结合能力。【结论】铁硫簇体外组装证明Fe-IscA在体外能将结合的铁传递给IscS,并在IscU上进行铁硫簇的组装。     

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from l-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2′-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5′-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess l-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.     

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.     

Characterization of iron-sulfur cluster assembly protein isca from Acidithiobacillus ferrooxidans

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes, but the mechanism of its function in the biogenesis of iron-sulfur cluster remains elusive. In this paper, we demonstrate that Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of 4·10²⁰ M^?1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form of IscA can be converted to iron-sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99, or Cys101 in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron-sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro. Taken together, these findings suggest that A. ferrooxidans IscA is not only an iron-sulfur protein, but also an iron binding protein that can act as an iron donor for biogenesis of iron-sulfur clusters.     

Expression,purification and characterization of an iron-sulfur cluster assembly protein,IscU, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe₂S₂] cluster could be assembled in apoIscU with Fe²⁺ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe²⁺ to synthesize [Fe₂S₂] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe₂S₂] cluster.     

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.     

Expression,purification and characterization of a cysteine desulfurase,IscS, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was 46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H₂S, depending on whether or not a reducing agent was added to the reaction mixture. Jia Zeng and Yanfei Zhang contributed equally to this work.     

Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells

Iron-sulfur (Fe-S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe-S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe-S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe-S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe-S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe-S cluster assembly are discussed.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Complementary roles of SufA and IscA in the biogenesis of iron-sulfur clusters in Escherichia coli

Biogenesis of iron-sulfur clusters requires a concerted delivery of iron and sulfur to target proteins. It is now clear that sulfur in iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. However, the specific iron donor for the iron-sulfur cluster assembly still remains elusive. Previous studies showed that IscA, a member of the iron-sulfur cluster assembly machinery in Escherichia coli, is a novel iron-binding protein, and that the iron-bound IscA can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU in vitro. However, genetic studies have indicated that IscA is not essential for the cell growth of E. coli. In the present paper, we report that SufA, an IscA paralogue in E. coli, may represent the redundant activity of IscA. Although deletion of IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA in E. coli results in a severe growth phenotype in minimal medium under aerobic growth conditions. Cell growth is restored when either IscA or SufA is re-introduced into the iscA-/sufA- double mutant, demonstrating further that either IscA or SufA is sufficient for their functions in vivo. Purified SufA, like IscA, is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU in the presence of a thioredoxin reductase system which emulates the intracellular redox potential. Site-directed mutagenesis studies show that the SufA/IscA variants that lose the specific iron-binding activity fail to restore the cell growth of the iscA-/sufA- double mutant. The results suggest that SufA and IscA may constitute the redundant cellular activities to recruit intracellular iron and deliver iron for the iron-sulfur cluster assembly in E. coli.     

Expression, Purification, and Characterization of an Iron Chaperon Protein CyaY from Acidithiobacillus ferrooxidans

CyaY is the bacterial homolog of frataxin, proposed to be involved in the assembly of iron-sulfur clusters. While, the physiological iron donor for the iron-sulfur clusters assembly remains controversial. In this study, the gene of CyaY from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The CyaY protein can bind ferric iron and serve as an iron donor for the biogenesis of iron-sulfur clusters on the scaffold protein IscU in the presence of IscS and L-cysteine in vitro.     

Role of conserved cysteines in mediating sulfur transfer from IscS to IscU

The role of the three conserved cysteine residues on Azotobacter vinelandii IscU in accepting sulfane sulfur and forming a covalent complex with IscS has been evaluated using electrospray-ionization mass spectrometry studies of variants involving individual cysteine-to-alanine substitutions. The results reveal that IscS can transfer sulfur to each of the three alanine-substituted forms of IscU to yield persulfide or polysulfide species, and formation of a heterodisulfide covalent complex between IscS and Cys(37) on IscU. It is concluded that S transfer from IscS to IscU does not involve a specific cysteine on IscU or the formation of an IscS-IscU heterodisulfide complex.