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.     

Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron complexes formed in Escherichia coli cells under nitric oxide stress

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 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 proteins without iron-sulfur clusters, are modified, forming protein-bound DNICs. 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 the "chelatable iron pool" in 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 the chelatable iron pool in cells.     

Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein

Frataxin is an iron binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron-sulfur cluster and heme biosynthesis. There is a high degree of structural homology for frataxin proteins from diverse sources, and all possess an anionic surface defined by acidic residues. In the human protein these residues principally lie on a surface defined by the alpha1 helix and beta1 sheet and the impact of multiple substitutions of these carboxylate residues on iron binding is described. Full-length human frataxin has previously been shown to undergo self-cleavage to produce a truncated form both in vitro and in vivo. This truncated protein has been shown to bind approximately seven iron centers that are presumably associated with the acidic patch. Relative to this native protein, the stoichiometry decreases according to the number and sites of mutations. Nevertheless, the iron-dependent binding affinity of each frataxin derivative to the iron-sulfur cluster scaffold protein ISU is found to be similar to that of native frataxin, as defined by isothermal titration calorimetry experiments, requiring only one iron center to promote nanomolar binding. While frataxins from various cell types appear to bind differing numbers of iron centers, the physiologically relevant number of bound irons appears to be small, with significantly higher binding affinity following complex formation with partner proteins (micromolar compared with nanomolar binding). By contrast, in reconstitution assays for frataxin-promoted [2Fe-2S](2+) cluster assembly on ISU, one derivative does display a modestly lower reconstitution rate. The overall consensus from these data is to consider a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets.     

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.     

An in vivo method for characterization of protein interactions within sulfur trafficking systems of E. coli

Sulfur trafficking systems are multiprotein systems that synthesize sulfur-containing cofactors such as iron-sulfur clusters. The sulfur is derived enzymatically from cysteine and transferred between nucleophilic cysteine residues within proteins until incorporation into the relevant cofactor. As these systems are poorly understood, we have developed an in vivo method for characterizing these interactions and have applied our method to the SUF system of Escherichia coli, which is responsible for iron-sulfur cluster biogenesis under oxidative stress and iron limitation. Proteins that interact covalently with SufE were trapped in vivo, purified, and identified by mass spectrometry. We identified SufE-SufS and SufE-SufB interactions, interactions previously demonstrated in vitro, indicating that our method has the ability to identify physiologically relevant interactions. The sulfur acceptor function of SufE is likely due to the low pK(a) of its active site C51, which we determined to be 6.3 ± 0.7. We found that SufE interacts with several Fe-S cluster proteins, further supporting the validity of the method, and with tryptophanase, glutaredoxin-3, and glutaredoxin-4, possibly suggesting a role for these enzymes in iron-sulfur biogenesis by the SUF system. Our results indicate that this method could serve as a general tool for the determination of sulfur trafficking mechanisms.     

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

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 iron-sulfur 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.     

Coming into view: eukaryotic iron chaperones and intracellular iron delivery

Eukaryotic cells contain hundreds of metalloproteins, and ensuring that each protein receives the correct metal ion is a critical task for cells. Recent work in budding yeast and mammalian cells has uncovered a system of iron delivery operating in the cytosolic compartment that involves monothiol glutaredoxins, which bind iron in the form of iron-sulfur clusters, and poly(rC)-binding proteins, which bind Fe(II) directly. In yeast cells, cytosolic monothiol glutaredoxins are required for the formation of heme and iron-sulfur clusters and the metallation of some non-heme iron enzymes. Poly(rC)-binding proteins can act as iron chaperones, delivering iron to target non-heme enzymes through direct protein-protein interactions. Although the molecular details have yet to be explored, these proteins, acting independently or together, may represent the basic cellular machinery for intracellular iron delivery.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments

 Cysteine is the ubiquitous ligand of iron-sulfur clusters in proteins, although chemical models have indicated that functional groups other than thiolates can coordinate iron in iron-sulfur compounds. Only a small number of naturally occurring examples of hydroxyl, histidinyl or carboxyl coordination have been clearly established but many others are suspected. Quite a few site-directed mutagenesis experiments have been aimed at replacing the cysteine ligands of iron-sulfur centers by other amino acids in various systems. The available data set shows that substituting one ligand, even by another functional residue, is very often destabilizing enough to impair cluster assembly; in some cases, the apoprotein cannot even be detected. One for one replacements have been demonstrated, but they have been so far almost exclusively confined to clusters with no more than one or two iron atoms. In contrast, changes of the cluster nuclearity or recruitment of free cysteine residues seem preferred ways for proteins containing larger clusters to cope with removal of a ligand, rather than using coordinating amino acids bearing different chemical functions. Furthermore, the possibility of replacing cysteines by other residues as ligands in iron-sulfur proteins does not uniquely depend on the ability of the cluster to accept other kinds of coordination than cysteinate; other factors such as the local flexibility of the polypeptide chain, the accessibility of the solvent and the electronic distribution on the active centers may also play a prominent role.     

铁硫蛋白的生物合成及相关疾病

铁硫蛋白是以铁硫簇为辅基,相对分子质量较小的一类蛋白质．它广泛存在于各种生物体内,参与电子传递、能量代谢以及基因表达调控等重要生理过程．其生物合成过程复杂,并且从细菌到人类高度保守．在真核细胞内,铁硫蛋白的组装由线粒体铁硫簇组装系统（mitochondrial iron sulfur cluster assembly system,mitochondrial ISC assembly system）和细胞质铁硫簇组装器（cytosolic iron sulfur cluster assembly,CIA）完成．研究发现,铁硫蛋白的合成异常可导致弗里德赖希共济失调(friedreich ataxia,FRDA)、遗传性肌病和铁粒幼细胞性贫血等多种罕见疾病,这些疾病严重影响个体的生活质量和寿命．因此,深入了解铁硫蛋白的结构和生物合成过程,对研究其生物学功能与相关疾病的诊断和治疗有重要意义．     

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.     

NMR studies of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins. Direct assignment of beta-cysteinyl carbon NMR resonances and further proton NMR assignments of cysteinyl and aromatic resonances.

Ferredoxins are proteins which contain iron and inorganic sulfide and are capable of electron transport. They are found in a wide range of organisms, from anaerobic bacteria, to plants and mammals. Although NMR spectroscopy has been used to study ferredoxins since the 1970s, little important structural or biochemical information has resulted from these investigations. The major difficulty has been the effect of the paramagnetic iron-sulfur clusters on the peptide resonances, hindering nuclear Overhauser effect (NOE) studies and causing broad line widths. These effects are most pronounced on resonances arising from the nuclei closest to the iron-sulfur center. Unfortunately, these are likely to be the most interesting nuclei, as they report the events and geometry in the vicinity of the active sites. In this paper, the first direct assignment of beta-cysteinyl 13C resonances for any iron-sulfur protein is reported for the spectrum of Pseudomonas putida ferredoxin. These resonances are of special significance, as they arise from the atoms on the protein closest to the iron centers, with the exception of the directly bound cysteinyl sulfur atoms. In addition, cysteinyl and ring system 1H NMR resonance assignments are made for the spectra of P. putida ferredoxin and Azotobacter vinelandii ferredoxin I.     

Insights into properties and energetics of iron-sulfur proteins from simple clusters to nitrogenase

Some of the principal physical features of iron-sulfur clusters in proteins are analyzed, including metal-ligand covalency, spin polarization, spin coupling, valence delocalization, valence interchange and small reorganization energies, with emphasis on recent spectroscopic and theoretical work. The current state of structural, spectroscopic, and computational knowledge for the iron-sulfur clusters in the nitrogenase iron and iron-molybdenum proteins is examined by comparison and contrast to 'simpler' ironclusters. The differing interactions of the nitrogenase iron and iron-molybdenum clusters compared with those of other iron-sulfur clusters with the protein and solvent environment are also explored.     

Tetranuclear and binuclear iron-sulfur clusters in succinate dehydrogenase: a method of iron quantitation by formation of paramagnetic complexes.

Soluble succinate dehydrogenase contains 8 atoms of iron, 8 atoms of acid labile sulfur and one covalently bound FAD per molecule; however, the distribution of iron and sulfur has not been well established. An iron counting method was devised in which electron spin resonance detectable complexes containing one iron each were formed with NO and cysteine and complex formation was measured during the gradual dissociation of the iron-sulfur clusters. In addition, a method described by Cammack was used to provide independent evidence. Both methods point to the existence of two binuclear clusters and one tetranuclear iron-sulfur cluster in the succinate dehydrogenase molecule.     

Linear three-iron centres are unlikely cluster degradation intermediates during unfolding of iron-sulfur proteins

Recent studies on the chemical alkaline degradation of ferredoxins have contributed to the hypothesis that linear three-iron centres are commonly observed as degradation intermediates of iron-sulfur clusters. In this work we assess the validity of this hypothesis. We studied different proteins containing iron-sulfur clusters, iron-sulfur centres and di-iron centres with respect to their chemical degradation kinetics at high pH, in the presence and absence of exogenous sulfide, to investigate the possible formation of linear three-iron centres during protein unfolding. Our spectroscopic and kinetic data show that in these different proteins visible absorption bands at 530 and 620 nm are formed that are identical to those suggested to arise from linear three-iron centres. Iron release and protein unfolding kinetics show that these bands result from the formation of iron sulfides at pH 10, produced by the degradation of the iron centres, and not from rearrangements leading to linear three-iron centres. Thus, at this point any relevant functional role of linear three-iron centres as cluster degradation intermediates in iron-sulfur proteins remains elusive.     

Iron sources forming nitrosyl complexes in animal tissues

Treatment of perfused mouse liver with nitric oxide does not change the intensities of ESR signals of iron-sulfur proteins characteristic of this tissue. Proceeding from this evidence and also from the ratio between the iron content in these proteins and dinitrosyl iron complexes (complexes 2.03) formed in the liver when it contacts with NO, it is concluded that iron-sulfur proteins are not involved in the formation of complexes 2.03. It seems that only the loosely bound form of non-heme iron-free iron is involved in this process.     

An iron-sulfur cluster in the family 4 uracil-DNA glycosylases

The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.     

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.