Mechanistic characterization of sulfur transfer from cysteine desulfurase SufS to the iron-sulfur scaffold SufU in Bacillus subtilis

Iron–sulfur cluster biosynthesis in Gram-positive bacteria is mediated by the SUF system. The transfer of sulfide from the cysteine desulfurase SufS to the scaffold protein SufU is one of the first steps within the assembly process. In this study, we analyzed the interaction between Bacillus subtilis SufS and its scaffold SufU. The activity of SufS represents a Ping-Pong mechanism leading to successive sulfur loading of the conserved cysteine residues in SufU. Cysteine 41 of SufU is shown to be essential for receiving sulfide from SufS, while cysteines 66 and 128 are needed for SufS/SufU interaction. In conclusion, we present the first step-by-step model for loading of the essential scaffold component SufU by its sulfur donor SufS.

Structured summary

SufS and SufUbind by molecular sieving(View interaction)SufSbinds to SufS by molecular sieving(View interaction)SufS and SufUredox react by enzymatic study (View Interaction 1, 2, 3, 4, 5)SufUphysically interacts with SufS by pull down (View Interaction 1, 2)     

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron–sulfur (Fe–S) clusters in Bacillus subtilis is mediated by the SUF‐like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe–S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe–S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe–S cluster assembly.     

Enterococcus faecalis SufU scaffold protein enhances SufS desulfurase activity by acquiring sulfur from its cysteine-153

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.     

SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.     

The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli

The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo.     

Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.     

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.     

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation,metabolism, and survival in vivo

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.     

SufU Is an Essential Iron-Sulfur Cluster Scaffold Protein in Bacillus subtilis

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium''s genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to ∼1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.Iron-sulfur (Fe/S) clusters are one of the most ubiquitous and versatile cofactors employed by nature for catalyzing a variety of redox reactions or for serving as redox sensors in a broad range of regulatory processes (10). Iron and sulfide are toxic for the cells in concentrations needed for spontaneous chemical Fe/S protein maturation. Hence, cells have developed complex biosynthesis machineries which are essential in vivo to assemble Fe/S proteins. Three phylogenetically distinct biosynthesis systems have been found in bacteria: ISC (iron-sulfur cluster), SUF (sulfur mobilization), and NIF (nitrogen fixation) (9, 11, 24). The ISC machinery is the most widely distributed bacterial Fe/S cluster biogenesis system and is also present in eukaryotes (31). In Escherichia coli a second system for Fe/S cluster assembly, SUF, is induced under conditions of iron limitation and/or oxidative stress, thus replacing the housekeeping ISC system for assembly of Fe/S proteins. In contrast, SUF was found as the exclusive Fe/S biogenesis system in mycobacteria (22) and Enterococcus faecalis (39) and hence may also serve as a constitutive system. Furthermore, SUF is present in plastids of green plants, resembling the situation found in their cyanobacterial ancestors (25, 53). The NIF system is responsible for the dedicated maturation of the complex Fe/S protein nitrogenase involved in nitrogen fixation, e.g., in Azotobacter vinelandii. Some NIF genes are associated with anaerobic or microaerobic growth in Helicobacter pylori and Entamoeba histolytica (24).Common principles for Fe/S protein assembly in each system have been defined (30). The de novo assembly of an Fe/S cluster occurs on scaffold proteins which transiently bind the Fe/S cluster before transfer to target apoproteins. Cysteine desulfurases such as IscS and SufS serve as sulfur donors, which acquire sulfur from free l-cysteine by pyridoxal-5′-phosphate-dependent desulfuration. The sulfur is transiently bound in the form of a persulfide to an active-site cysteine of the desulfurase and is subsequently transferred to the scaffold protein. Several SUF systems contain SufE, which specifically forms a complex with the cysteine desulfurase SufS (36, 42). SufE enhances SufS activity significantly and assists the sulfur transfer to scaffold proteins. In this case the persulfide is transiently bound to SufE and not to the desulfurase. Recent studies show that the E. coli cysteine desulfurase CsdA is able to complement the SUF system and interacts with SufE if SufS is inactivated (50). A general iron donor involved in Fe/S cluster assembly is not known yet; however, frataxin homologs in prokaryotes and eukaryotes are postulated to deliver iron to the scaffold protein IscU in the ISC system (5, 9, 31, 34).Several components have been suggested to act as scaffold proteins. U-type scaffold proteins such as bacterial NifU, IscU, and eukaryotic Isu1 preferentially bind [2Fe-2S] clusters. However, the assembly of [4Fe-4S] clusters was described to proceed by reductive coupling of two [2Fe-2S] clusters that bind successively to an IscU dimer (1). A-type scaffolds like bacterial SufA or IscA can bind [2Fe-2S] clusters in their monomeric state and were found to be involved in the maturation of [4Fe-4S] proteins such as aconitase (17, 32, 47). Overproduced IscA was also shown to bind mononuclear iron which could be used for Fe/S cluster assembly on IscU in vitro.The ISC system characteristically contains the molecular chaperone pair HscA and HscB that are involved in Fe/S cluster transfer from the IscU scaffold protein to the target proteins. The Hsp70-type HscA specifically binds to a highly conserved LPPVK motif located near the third strictly conserved cluster-binding cysteine in IscU. Specific IscU-HscA complex formation was found to be necessary and sufficient to stimulate the ATPase activity of HscA (12, 21, 48). The SUF system, in contrast, does not contain HscA and HscB chaperones in the suf gene cluster. Instead it comprises the SufBCD proteins. The SufC protein has intrinsic ATPase activity and forms a complex with SufB, a putative scaffold protein, and SufD (29). The precise molecular function of the ATP-hydrolyzing SufBCD complex is not yet clear.The best-characterized SUF system from E. coli contains the gene cluster sufABCDSE (5). However, many bacteria, in particular members of the phylum Firmicutes, contain a different suf gene cluster encoding sufCDSUB, which has been studied so far only by bioinformatic approaches (39, 46). While SufS and SufBCD in the two gene clusters appear to be similar proteins, SufE is lacking in most Firmicutes and also in the mycobacterial and Thermotoga maritima SUF machineries (22, 24). The additionally present SufU shares similarities with IscU of the ISC system (39). The protein contains all three conserved cysteine residues involved in Fe/S cluster association and yet characteristically lacks the LPPVK motif, consistent with the absence of HscA and HscB proteins in sufCDSUB species.In this study, we made use of the Gram-positive bacterium Bacillus subtilis to initiate functional analysis of the sufCDSUB genes in Fe/S cluster biosynthesis by genetic and biochemical approaches. In particular, since no functional information is available for SufU, we tested its putative role as an Fe/S scaffold protein. SufU was found to be crucial for cell viability and for Fe/S-dependent enzyme activities in crude cell lysates. In vitro cluster reconstitution with recombinant SufU indicated that SufU binds a labile Fe/S cluster which can be transferred to apo-Leu1 in a fast and efficient way, fully activating its catalytic function as a [4Fe-4S] cluster-dependent isopropylmalate isomerase. The B. subtilis SufU was found to activate the desulfurase activity of purified B. subtilis SufS. Our results suggest that SufS and the SufU scaffold protein closely act together in the Fe/S cluster biogenesis in B. subtilis.     

Structural Changes during Cysteine Desulfurase CsdA and Sulfur Acceptor CsdE Interactions Provide Insight into the trans-Persulfuration

In Escherichia coli, three cysteine desulfurases (IscS, SufS, and CsdA) initiate the delivery of sulfur for various biological processes such as the biogenesis of Fe-S clusters. The sulfur generated as persulfide on a cysteine residue of cysteine desulfurases is further transferred to Fe-S scaffolds (e.g. IscU) or to intermediate cysteine-containing sulfur acceptors (e.g. TusA, SufE, and CsdE) prior to its utilization. Here, we report the structures of CsdA and the CsdA-CsdE complex, which provide insight into the sulfur transfer mediated by the trans-persulfuration reaction. Analysis of the structures indicates that the conformational flexibility of the active cysteine loop in CsdE is essential for accepting the persulfide from the cysteine of CsdA. Additionally, CsdA and CsdE invoke a different binding mode than those of previously reported cysteine desulfurase (IscS) and sulfur acceptors (TusA and IscU). Moreover, the conservation of interaction-mediating residues between CsdA/SufS and CsdE/SufE further suggests that the SufS-SufE interface likely resembles that of CsdA and CsdE.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants

Summary. Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.     

Rechargeable Iron–Sulfur Battery without Polysulfide Shuttling

Sulfur represents one of the most promising cathode materials for next‐generation batteries; however, the widely observed polysulfide dissolution/shuttling phenomenon in metal–sulfur redox chemistries has severely restricted their applications. Here it is demonstrated that when pairing the sulfur electrode with the iron metal anode, the inherent insolubility of iron sulfides renders the shuttling‐free nature of the Fe–S electrochemical reactions. Consequently, the sulfur electrode exhibits promising performance for Fe²⁺ storage, where a high capacity of ≈1050 mAh g^?1, low polarization of ≈0.16 V as well as stable cycling of 150 cycles are realized. The Fe–S redox mechanism is further revealed as an intriguing stepwise conversion of S₈ ? FeS₂ ? Fe₃S₄ ? FeS, where a low volume expansion of ≈32.6% and all‐solid‐state phase transitions facilitate the reaction reversibility. This study suggests an alternative direction to exploit sulfur electrodes in rechargeable transition metal–sulfur batteries.     

Endogenous Inhibitors for Pollen Germination and Pollen Tube Elongation in Pinus densiflora SIEB,et Zucc.

Sulfur metabolism in Cephalosporium acremonium was investigated using a mutant, 8650⁺/ OAH^?/SeMe^R, which could not convert cysteine or inorganic sulfur to methionine. The production of cephalosporin by the mutant depended on the amount of S-sulfocysteine in a chemically defined medium supplemented with a low level of methionine sufficient to support optimal growth. S-Sulfocysteine was detected in an extract of cells grown in the presence of sodium thiosulfate and l-serine. Furthermore, an NADPH-linked reduction of S-sulfocysteine to cysteine was demonstrated in a cell-free extract. These facts suggest that S-sulfocysteine is a direct precursor in cysteine biosynthesis in C. acremonium and an alternative pathway involving the compound is one of the most important ones in cephalosporin C production by this fungus.     

Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase

Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.     

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

Trichomonas vaginalis is a protozoan parasite of humans that is able to synthesize cysteine de novo using cysteine synthase but does not produce glutathione. In this study, high pressure liquid chromatography analysis confirmed that cysteine is the major intracellular redox buffer by showing that T. vaginalis contains high levels of cysteine (∼600 μm) comprising more than 70% of the total thiols detected. To investigate possible mechanisms for the regulation of cysteine levels in T. vaginalis, we have characterized enzymes of the mercaptopyruvate pathway. This consists of an aspartate aminotransferase (TvAspAT1), which transaminates cysteine to form 3-mercaptopyruvate (3-MP), and mercaptopyruvate sulfurtransferase (TvMST), which transfers the sulfur of 3-MP to a nucleophilic acceptor, generating pyruvate. TvMST has high activity with 3-MP as a sulfur donor and can use several thiol compounds as sulfur acceptor substrates. Our analysis indicated that TvMST has a k_cat/K_m for reduced thioredoxin of 6.2 × 10⁷ m⁻¹ s⁻¹, more than 100-fold higher than that observed for β-mercaptoethanol and cysteine, suggesting that thioredoxin is a preferred substrate for TvMST. Thiol trapping and mass spectrometry provided direct evidence for the formation of thioredoxin persulfide as a product of this reaction. The thioredoxin persulfide could serve a biological function such as the transfer of the persulfide to a target protein or the sequestered release of sulfide for biosynthesis. Changes in MST activity of T. vaginalis in response to variation in the supply of exogenous cysteine are suggestive of a role for the mercaptopyruvate pathway in the removal of excess intracellular cysteine, redox homeostasis, and antioxidant defense.     

Parsing redox potentials of five ferredoxins found within Thermotoga maritima

Most organisms contain multiple soluble protein‐based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron–sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe₄S₄]¹⁺ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side‐by‐side fashion for the first time. While a single [Fe₄S₄]‐cluster bearing Fd (TM0927) has a potential of ?420 mV, the other four 2x[Fe₄S₄]‐bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).