Structural insight into poplar glutaredoxin C1 with a bridging iron-sulfur cluster at the active site

Glutaredoxins are glutathione-dependent enzymes that function to reduce disulfide bonds in vivo. Interestingly, a recent discovery indicates that some glutaredoxins can also exist in another form, an iron-sulfur protein [Lillig, C. H., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 8168-8173]. This provides a direct connection between glutaredoxins and iron-sulfur proteins, suggesting a possible new regulatory role of iron-sulfur clusters along with the new functional switch of glutaredoxins. Biochemical studies have indicated that poplar glutaredoxin C1 (Grx-C1) is also such a biform protein. The apo form (monomer) of Grx-C1 is a regular glutaredoxin, and the holo form (dimer) is an iron-sulfur protein with a bridging [2Fe-2S] cluster. Here, we report the structural characterizations of poplar Grx-C1 in both the apo and holo forms by NMR spectroscopy. The solution structure of the reduced apo Grx-C1, which is the first plant Grx structure, shows a typical Grx fold. When poplar Grx-C1 forms a dimer with an iron-sulfur cluster, each subunit of the holo form still retains the overall fold of the apo form. The bridging iron-sulfur cluster in holo Grx-C1 is coordinated near the active site. In addition to the iron-sulfur cluster linker, helix alpha3 of each subunit is probably involved in the direct contact between the two subunits. Moreover, two glutathione molecules are identified in the vicinity of the iron-sulfur cluster and very likely participate in cluster coordination. Taken together, we propose that the bridging [2Fe-2S] cluster is coordinated by the first cysteine at the glutaredoxin active site from each subunit of holo Grx-C1, along with two cysteines from two glutathione molecules. Our studies reveal that holo Grx-C1 has a novel structural and iron-sulfur cluster coordination pattern for an iron-sulfur protein.     

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.     

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.     

Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.     

Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon. The product of one member of this operon, IscA, has been overexpressed, purified, and characterized. It can assemble an air-sensitive [2Fe-2S] cluster as shown by UV-visible and resonance Raman spectroscopy. The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the [2Fe-2S] holoferredoxin. These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation. This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.     

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.     

铁硫簇的生物合成

铁硫簇在细胞的生物学过程中起着重要的作用,可参与电子传递、代谢控制和基因调节等过程。研究显示铁硫簇具有多样性,它的合成依赖于ISC和SUF系统,固氮酶中还需要NIF系统的参与。ISC系统由iscSUA-hscBA-fdx基因串编码,合成的是一类“管家”蛋白,适于在正常条件下表达。SUF系统由基因串sufABCDSE编码,常在恶劣环境如氧化应激和铁饥饿条件下表达。NIF系统由nifSU基因编码,适于固氮酶(厌氧条件下起作用)铁硫簇的合成。     

Elimination of the disulfide bridge in the Rieske iron-sulfur protein allows assembly of the [2Fe-2S] cluster into the Rieske protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex

The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the [2Fe-2S] iron-sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis. These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.     

The crystal structure and reaction mechanism of Escherichia coli 2,4-dienoyl-CoA reductase

Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP+ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA thioesters.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.     

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO)

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.     

A HEAT-repeats containing protein,IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue,IscA2

IscA homologues are involved in iron-sulfur cluster biosynthesis. In the non-nitrogen-fixing cyanobacterium Synechocystis PCC 6803, there are two IscA homologues, SLR1417 and SLR1565 (designated IscA1 and IscA2), of which only IscA2 exists as a protein complex with the HEAT-repeat-containing protein, SLR1098 (IaiH). We observed that the absorption spectrum of the recombinant IscA2/IaiH complex resembles that of IscA2 alone, although it is sharper. In the presence of dithiothreitol, the [2Fe-2S] cluster of IscA2 alone, but not of the IscA2/IaiH complex, became reductively labile upon the addition of sodium dithionite. This implies that the IscA2 moiety of the [2Fe-2S] cluster is stabilized by the presence of IaiH. The [2Fe-2S] cluster of the IscA2/IaiH complex was destabilized by sodium dithionite in the absence of dithiothreitol, suggesting that the in vivo stability of the iron-sulfur cluster in the IscA2/IaiH complex is influenced by the redox state of cellular thiols. When any one of three conserved cysteine residues in IscA2, potential ligands for the [2Fe-2S] cluster, was replaced with serine, the amount of assembled [2Fe-2S] cluster and protein complex was significantly reduced in E. coli cells. The cysteine mutated IscA2/IaiH complexes that were present all contained a [2Fe-2S]-like cluster suggesting that the assembly of a stable iron-sulfur cluster bound to IscA2 is required for efficient and stable complex formation. Truncated IaiH proteins were analyzed using the yeast two-hybrid assay to identify the essential domain of IaiH that interacts physically with IscA2. At least 2 of the 5 N-terminal HEAT repeats of IaiH were found to be required for interaction with IscA2.     

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.     

Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation

The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s(4)U) and 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s(2)C) and N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm(5)s(2)U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms(2)io(6)A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms(2)io(6)A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.     

CpSufE activates the cysteine desulfurase CpNifS for chloroplastic Fe-S cluster formation

CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.     

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.     

The presence of the iron-sulfur protein (subunit V) of complex III in mitochondria of heme-deficient yeast cells lacking iron-sulfur clusters detectable by electron paramagnetic resonance

The presence of subunit V, the iron-sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5-aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 K Da) of the iron-sulfur protein was observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against subunit V and either the sensitive immuno-transfer technique or immunoprecipitation from dodecylsulfate-solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme-deficient cells. Electron paramagnetic resonance spectroscopy revealed the absence of iron-sulfur signals due to clusters S-1, S-2 and S-3 of succinate dehydrogenase or to Rieske's iron-sulfur cluster of complex III in mitochondria from the heme-deficient cells. The lack of iron-sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.     

Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis

The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.