Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.     

Biochemical characterization of iron-sulfur cluster assembly in the scaffold IscU of Escherichia coli

Iron-sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron-sulfur cluster biosynthesis. In this study, we cloned and purified iron-sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron-sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1: 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron-sulfur cluster assembly; the cluster was well assembled at pH 7.6–8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron-sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe-2S] cluster-bound IscU under the optimal laboratory conditions.     

Three-dimensional structure and determinants of stability of the iron-sulfur cluster scaffold protein IscU from Escherichia coli

The highly conserved protein, IscU, serves as the scaffold for iron-sulfur cluster (ISC) assembly in the ISC system common to bacteria and eukaryotic mitochondria. The apo-form of IscU from Escherichia coli has been shown to populate two slowly interconverting conformational states: one structured (S) and one dynamically disordered (D). Furthermore, single-site amino acid substitutions have been shown to shift the equilibrium between the metamorphic states. Here, we report three-dimensional structural models derived from NMR spectroscopy for the S-state of wild-type (WT) apo-IscU, determined under conditions where the protein was 80% in the S-state and 20% in the D-state, and for the S-state of apo-IscU(D39A), determined under conditions where the protein was ~95% in the S-state. We have used these structures in interpreting the effects of single site amino acid substitutions that alter %S = (100 × [S])/([S] + [D]). These include different residues at the same site, %S: D39V > D39L > D39A > D39G ≈ WT, and alanine substitutions at different sites, %S: N90A > S107A ≈ E111A > WT. Hydrophobic residues at residue 39 appear to stabilize the S-state by decreasing the flexibility of the loops that contain the conserved cysteine residues. The alanine substitutions at positions 90, 107, and 111, on the other hand, stabilize the protein without affecting the loop dynamics. In general, the stability of the S-state correlates with the compactness and thermal stability of the variant.     

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.     

Iron-sulfur cluster biosynthesis. Thermatoga maritima IscU is a structured iron-sulfur cluster assembly protein

Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU from Thermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. M?ssbauer (delta = 0.29 +/- 0.03 mm/s, DeltaE(Q) = 0.58 +/- 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% alpha-helix, 13.1% antiparallel beta-sheet, 11.3% parallel beta-sheet, 20.2% beta-turn, and 19.1% other at 20 degrees C, with negligible spectral change observed at 70 degrees C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in 1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritima IscU-mediated reconstitution of human apoferredoxin.     

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.     

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.     

Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA

IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold-[FeS] complexes to apoprotein acceptors have provided experimental support for this hypothesis, but investigations to date have yielded only single-cluster transfer events. We describe an in vitro assay system that allows for real-time monitoring of [FeS] cluster formation using circular dichroism spectroscopy and use this to investigate de novo [FeS] cluster formation and transfer from Escherichia coli IscU and IscA to apo-ferredoxin. Both IscU and IscA were found to be capable of multiple cycles of [2Fe2S] cluster formation and transfer suggesting that these scaffold proteins are capable of acting catalytically. Kinetic studies further showed that cluster transfer exhibits Michaelis-Menten behavior indicative of complex formation of holo-IscU and holo-IscA with apoferredoxin and consistent with a direct [FeS] cluster transfer mechanism. Analysis of the dependence of the rate of cluster transfer, however, revealed enhanced efficiency at low ratios of scaffold to acceptor protein suggesting participation of a transient, labile scaffold-[FeS] species in the transfer process.     

SufA from Erwinia chrysanthemi. Characterization of a scaffold protein required for iron-sulfur cluster assembly

SufA is a component of the recently discovered suf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress. The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized. It is a homodimer with the ability to assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by M?ssbauer spectroscopy. These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator. Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide. We propose the function of SufA is that of a scaffold protein for [Fe-S] cluster assembly and compare it to IscA, a member of the isc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes. Mechanistic and physiological implications of these results are also discussed.     

The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.     

Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.     

Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis.     

Crystal structure of IscA, an iron-sulfur cluster assembly protein from Escherichia coli

IscA, an 11 kDa member of the hesB family of proteins, binds iron and [2Fe-2S] clusters, and participates in the biosynthesis of iron-sulfur proteins. We report the crystal structure of the apo-protein form of IscA from Escherichia coli to a resolution of 2.3A. The crystals belong to the space group P3(2)21 and have unit cell dimensions a=b=66.104 A, c=150.167 A (alpha=beta=90 degrees, gamma=120 degrees ). The structure was solved using single-wavelength anomalous dispersion (SAD) phasing of a selenomethionyl derivative, and the IscA model was refined to R=21.4% (Rfree=25.4%). IscA exists as an (alpha1alpha2)2 homotetramer with the (alpha1alpha2) dimer comprising the asymmetric unit. Cys35, implicated in Fe-S cluster assembly, is located in a central cavity formed at the tetramer interface with the gamma-sulfur atoms of residues from the alpha1 and alpha2' monomers (and alpha1'alpha2) positioned close to one another (approximately equal 7 A). C-terminal residues 99-107 are disordered, and the exact positions of Cys99 and Cys101 could not be determined. However, computer modeling of C-terminal residues in the tetramer suggests that Cys99 and Cys101 in the alpha1 monomer and those of the alpha1' monomer (or alpha2 and alpha2') are positioned sufficiently close to coordinate [2Fe-2S] clusters between the two dimers, whereas this is not possible within the (alpha1alpha2) or (alpha1'alpha2') dimer. This symmetrical arrangement allows for binding of two [2Fe-2S] clusters on opposite sides of the tetramer. Modeling further reveals that Cys101 is positioned sufficiently close to Cys35 to allow Cys35 to participate in cluster assembly, formation, or transfer.     

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.     

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.     

Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance

A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.     

Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.     

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.