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Cysteine-persulfide (Cys-SSH) is a cysteine whose sulfhydryl group is covalently bound to sulfur (sulfane sulfur). Cys-SSH and its glutathione (GSH) counterpart (GSSH) have been recognized as redox regulators, some of which were previously ascribed to cysteine and GSH. However, the production of Cys-SSH and GSSH is not well understood. Here, we show that 3-mercaptopyruvate sulfurtransferase (3MST) produces Cys-SSH and GSSH together with the potential signaling molecules hydrogen per- and tri-sulfide (H2S2 and H2S3). Cys-SSH and GSSH are produced in the brain of wild-type mice but not in those of 3MST-KO mice. The levels of total persulfurated species in the brain of 3MST-KO mice are less than 50% of that in the brain of wild-type mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST showed that Cys-SSH and GSSH were produced in the presence of physiological concentrations of cysteine and glutathione, while those with longer sulfur chains, Cys-SSnH and GSSnH, were produced in the presence of lower than physiological concentrations of cysteine and glutathione. The present study provides new insights into the production and physiological roles of these persulfurated species as well as the therapeutic targets for diseases in which these molecules are involved.

Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 atoms of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S](2+) cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. For BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S](2+) cluster in BioB must be reassembled, a process that could be conducted in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70 class molecular chaperone, whose yeast homologue has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in Saccharomyces cerevisiae. In this work, we show that in Escherichia coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S](2+) cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner, and a high-affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB-HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein-protein interaction.

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well-characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other γ-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3-mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.

To understand sulfur oxidation in thioauto-trophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox)(soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.

Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO 3 2− ), thiosulfate (S 2 O 3 2− ), tetrathionate (S 4 O 6 2− ), and elemental sulfur (S 0 ). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis , however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC , a gene cluster required for S 0 and S 2 O 3 2− respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA ) were identified in the genomes of Shewanella strains that reduce S 0 and S 2 O 3 2− yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA , the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S 0 or S 2 O 3 2− as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S 2 O 3 2− terminal reductase whose end products (sulfide [HS − ] or SO 3 2− ) participate in an intraspecies sulfur cycle that drives S 0 respiration.

Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe2+. Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe2+-loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.

The major mechanism of removing cyanide from the body is its enzymatic conversion by a sulfurtransferase, e.g. rhodanese, to the less toxic thiocyanate in the presence of a sulfur donor. Earlier results demonstrated that externally administered encapsulated rhodanese significantly enhances the in vivo efficacy of the given sulfur donor. Present studies are focused on liposomal carrier systems encapsulating rhodanese. Physicochemical properties, e.g. membrane rigidity, size distribution, surface potential, osmolarity, and viscosity, were determined for various liposomal lipid compositions and hydrating buffers to establish in vitro stability and in vivo fate. Lipid composition was also optimized to achieve maximum encapsulation efficiency.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

Hydrogen sulfide (H(2)S) is a synaptic modulator as well as a neuroprotectant. Currently, pyridoxal-5'-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) is thought to be the major H(2)S-producing enzyme in the brain. We recently found that brain homogenates of CBS-knockout mice, even in the absence of PLP, produce H(2)S at levels similar to those of wild-type mice, suggesting the presence of another H(2)S-producing enzyme. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) in combination with cysteine aminotransferase (CAT) produces H(2)S from cysteine. In addition, 3MST is localized to neurons, and the levels of bound sulfane sulfur, the precursor of H(2)S, are greatly increased in the cells expressing 3MST and CAT but not increased in cells expressing functionally defective mutant enzymes. These data present a new perspective on H(2)S production and storage in the brain.

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC. 2.8.1.1) is a ubiquitous enzyme present in all living organisms, from bacteria to humans, and plays a central role in cyanide detoxification. The purpose of this investigation was to determine and compare the pattern of distribution of rhodanese in different tissues of adult Japanese quail, partridge, and pigeon. In the three species studied, rhodanese was present in all tissues albeit in different amounts. The highest activity of rhodanese in Japanese quail was observed in kidney and proventriculus followed by liver and heart. In partridge, the highest activity of the enzyme was observed in heart, kidney, and proventriculus. In pigeon, the highest activity of the enzyme was observed in liver, kidney, and heart. Inter-species differences were seen in the level of enzyme in different tissues. The results of this study may indicate the involvement of rhodanese in cyanide detoxification in tissues that have greater potential to be exposed to higher levels of cyanide and also involvement of rhodanese in functions other than cyanide detoxification in other tissues.

Although Pelobacter species are closely related to Geobacter species, recent studies suggested that Pelobacter carbinolicus may reduce Fe(III) via a different mechanism because it lacks the outer-surface c -type cytochromes that are required for Fe(III) reduction by Geobacter sulfurreducens . Investigation into the mechanisms for Fe(III) reduction demonstrated that P. carbinolicus had growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. Unlike G. sulfurreducens , transcript levels for cytochrome genes did not increase in P. carbinolicus during growth on Fe(III). P. carbinolicus could use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests that P. carbinolicus reduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed for Geobacter species.

Sodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur compound isolated from garlic, induces apoptosis in a number of cancer cells. The molecular mechanism of action of sodium 2-propenyl thiosulfate has not been completely clarified. In this work we investigated, by in vivo and in vitro experiments, the effects of this compound on the expression and activity of rhodanese. Rhodanese is a protein belonging to a family of enzymes widely present in all phyla and reputed to play a number of distinct biological roles, such as cyanide detoxification, regeneration of iron–sulfur clusters and metabolism of sulfur sulfane compounds. The cytotoxic effects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated by flow cytometry and DNA fragmentation and by monitoring the progressive formation of mobile lipids by NMR spectroscopy. Sodium 2-propenyl thiosulfate was also found to induce inhibition of the sulfurtransferase activity in tumor cells. Interestingly, in vitro experiments using fluorescence spectroscopy, kinetic studies and MS analysis showed that sodium 2-propenyl thiosulfate was able to bind the sulfur-free form of the rhodanese, inhibiting its thiosulfate:cyanide-sulfurtransferase activity by thiolation of the catalytic cysteine. The activity of the enzyme was restored by thioredoxin in a concentration-dependent and time-dependent manner. Our results suggest an important involvement of the essential thioredoxin–thioredoxin reductase system in cancer cell cytotoxicity by organo-sulfane sulfur compounds and highlight the correlation between apoptosis induced by these compounds and the damage to the mitochondrial enzymes involved in the repair of the Fe–S cluster and in the detoxification system.

An intermolecular disulfide bond serves as a thioredoxin-dependent redox-sensing switch for the regulation of the enzymatic activity of 3-mercaptopyruvate sulfurtransferase (MST, EC.2.8.1.2). A cysteine residue on the surface of each subunit was oxidized to form an intersubunit disulfide bond so as to decrease MST activity, and thioredoxin-specific conversion of a dimer to a monomer increased MST activity. Further, a low redox potential sulfenate was reversibly formed at a catalytic site cysteine so as to inhibit MST, and thioredoxin-dependent reduction of the sulfenate restored the MST activity. Concludingly, MST partly contributes to the maintenance of cellular redox homeostasis via exerting control over cysteine catabolism.

Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.

Thiosulfate oxidation and mixotrophic growth with succinate or methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium oryzae CBMB20, which was recently characterized and reported as a novel species isolated from rice. Methylobacterium oryzae was able to utilize thiosulfate in the presence of sulfate. Thiosulfate oxidation increased the protein yield by 25% in mixotrophic medium containing 18.5 mmol·L–1of sodium succinate and 20 mmol·L–1of sodium thiosulfate on day 5. The respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfur and sulfite. Thiosulfate was predominantly oxidized to sulfate and intermediate products of thiosulfate oxidation, such as tetrathionate, trithionate, polythionate, and sulfur, were not detected in spent medium. It indicated that bacterium use the non-S4intermediate sulfur oxidation pathway for thiosulfate oxidation. Thiosulfate oxidation enzymes, such as rhodanese and sulfite oxidase activities appeared to be constitutively expressed, but activity increased during growth on thiosulfate. No thiosulfate oxidase (tetrathionate synthase) activity was detected.

Purpose: Identify the roles of sulfide oxidase and rhodanese in sulfide detoxification in rat colonic mucosa. Results: Gel filtration of colonic mucosa and purified bovine rhodanese showed that rhodanese and sulfide oxidizing activities resided in different proteins. In the presence of cyanide, rhodanese shifted the major mucosal metabolite of sulfide from thiosulfate to thiocyanate. The purported ability of purified rhodanese to metabolize sulfide reflects: (a) contamination with a sulfide oxidase, and (b) the spontaneous conversion of sulfide to thiosulfate during storage; rhodanese then catalyzes the conversion of this thiosulfate to thiocyanate. Conclusions: Rhodanese does not metabolize sulfide. The rate-limiting step in sulfide detoxification is oxidation by a sulfide oxidase to thiosulfate. Rhodanese then converts this thiosulfate to thiocyanate, but this reaction does not increase the rate of sulfide detoxification. The recent use of rhodanese activity as a surrogate for the rate that colonic mucosa detoxifies sulfide is inappropriate.

Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa . The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA . Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a Δ rhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA . Wild-type P. aeruginosa outcompetes the Δ rhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration.

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys247, in the active site and Cys154 and Cys263 on the surface of MST. The corresponding cysteine to Cys263 is conserved in mammalian MSTs, and Cys154 is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at ∼92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys154 and Cys154 and between Cys263 and Cys263, or asymmetrical via an intersubunit disulfide bond between Cys154 and Cys263. Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys35 was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys32 of E. coli Trx reacted with the redox-active cysteines, Cys154 and Cys263, by forming an intersubunit disulfide bond and a sulfenyl Cys247. A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.

The design of 3D scaffolds is a crucial step in the field of regenerative medicine. Scaffolds should be degradable and bioresorbable as well as display good porosity, interconnecting pores, and topographic features; these properties favour tissue integration and vascularization. These requirements could be fulfilled by hybrid hydrogels using a combination of natural and synthetic components. Here, the mechanical and biological properties of a polyethylene glycol-fibrinogen hydrogel (PFHy) are improved in order to favour the proliferation and differentiation of human Sca-1(pos) cardiac progenitor cells (hCPCs). PFHys are modified by embedding air- or perfluorohexane-filled bovine serum albumin microbubbles (MBs) and characterized. Changes in cell morphology are observed in MBs-PFHys, suggesting that MBs could enhance the formation of bundles of cells and influence the direction of the spindle growth. The properties of MBs as carriers of active macromolecules are also exploited. For the first time, enzyme-coated MBs have been used as systems for the production of hydrogen sulfide (H2 S)-releasing scaffolds. Novel H2 S-releasing PFHys are produced, which are able to improve the growth of hCPCs. This novel 3D cell-scaffold system will allow the assessment of the effects of H2 S on the cardiac muscle regeneration with its potential applications in tissue repair.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.

Hydrogen sulfide (H2S) has been recognized as a signaling molecule as well as a cytoprotective molecule. H2S modulates neurotransmission, regulates vascular tone, protects various tissues and organs, regulates inflammation, induces angiogenesis, and detects cellular oxygen levels. H2S is produced from l-cysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) together with cysteine aminotransferase (CAT). Recently, a novel pathway for the production of H2S from d-cysteine was identified, involving d-amino acid oxidase (DAO) together with 3MST. Sulfuration (also called sulfhydration), which adds sulfur atoms to the cysteine residues of target proteins to modify protein activity, has been extensively studied as a mode of H2S action. Recently, hydrogen polysulfides (H2Sn, where n = 3–7; n = 2 is termed as persulfide) have been found to sulfurate target proteins in the brain, including transient receptor potential ankyrin 1 (TRPA1) channels, Kelch-like ECH-associating protein 1 (Keap1), and phosphatase and tensin homolog (PTEN), much more potently than H2S. The physiological stimuli that trigger the production of H2S and polysulfides, and the mechanisms maintaining their local levels, remain unknown. Understanding the regulation of H2Sn (including H2S) production, and the specific stimuli that induce their release, will provide new insight into the biology of H2S and will provide novel avenues for therapeutic development in diseases involving H2S-related substances.

Background Cystathionine γ‐lyase, cystathionine β‐synthase, and 3‐mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H 2 S). Functions of H 2 S are mediated by several targets including ion channels and signaling proteins. Nuclear factor‐erythroid 2‐related factor 2 is responsible for the expression of antioxidant response element–regulated genes and is known to be upregulated by H 2 S. We examined the levels of H 2 S, H 2 S‐producing enzymes, and nuclear factor‐erythroid 2‐related factor 2 activation status in skeletal muscle obtained from critical limb ischemia ( CLI ) patients. Methods and Results Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ‐lyase, cystathionine β‐synthase, and 3‐mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H 2 S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor‐erythroid 2‐related factor 2 activation as well as antioxidant proteins, such as Cu, Zn‐superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. Conclusions The data demonstrate that H 2 S bioavailability and nuclear factor‐erythroid 2‐related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H 2 S‐producing enzymes may contribute to the pathogenesis of CLI .

Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2 S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), synthesizes H2 S in the presence of the substrate 3-mercaptopyruvate (3-MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non-lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2 S donors, the most active of which was diallyl trisulfide (DATS). The main pro-apoptotic mechanisms involved were suppression of nuclear factor-κB activity and inhibition of AKT and extracellular signal-regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l-cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l-cysteine/CSE/H2 S pathway is involved in melanoma progression.

Plants and animals have evolved various potential mechanisms to surmount the adverse effects of heavy metal toxicity. Plants possess low molecular weight compounds containing sulfhydryl groups (-SH) that actively react with toxic metals. For instance, glutathione (γ-Glu-Cys-Gly) is a sulfur-containing tripeptide thiol and a substrate of cysteine-rich phytochelatins (γ-Glu-Cys)2-11-Gly (PCs). Phytochelatins react with heavy metal ions by glutathione S-transferase in the cytosol and afterwards they are sequestered into the vacuole for degradation. Furthermore, heavy metals induce reactive oxygen species (ROS), which directly or indirectly influence metabolic processes. Reduced glutathione (GSH) attributes as an antioxidant and participates to control ROS during stress. Maintenance of the GSH/GSSG ratio is important for cellular redox balance, which is crucial for the survival of the plants. In this context, sulfurtransferases (Str), also called rhodaneses, comprise a group of enzymes widely distributed in all phyla, paving the way for the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors, at least in vitro. The best characterized in vitro reaction is the transfer of a sulfane sulfur atom from thiosulfate to cyanide, leading to the formation of sulfite and thiocyanate. Plants as well as other organisms have multi-protein families (MPF) of Str. Despite the presence of Str activities in many living organisms, their physiological role has not been clarified unambiguously. In mammals, these proteins are involved in the elimination of cyanide released from cyanogenic compounds. However, their ubiquity suggests additional physiological functions. Furthermore, it is speculated that a member of the Str family acts as arsenate reductase (AR) and is involved in arsenate detoxification. In summary, the role of Str in detoxification processes is still not well understood but seems to be a major function in the organism. DAAD

H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine β-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter.

Hydrogen sulfide is rapidly emerging as a key physiological mediator and potential therapeutic tool in numerous areas such as acute and chronic inflammation, neurodegenerative and cardiovascular disease, diabetes, obesity and cancer. However, the vast majority of the published studies have employed crude sulfide salts such as sodium hydrosulfide (NaSH) and sodium sulfide (Na2S) as H2S “donors” to generate H2S. Although these salts are cheap, readily available and easy to use, H2S generated from them occurs as an instantaneous and pH-dependent dissociation, whereas endogenous H2S synthesis from the enzymes cystathionine γ-lyase, cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase is a slow and sustained process. Furthermore, sulfide salts are frequently used at concentrations (e.g. 100 μM to 10 mM) far in excess of the levels of H2S reported in vivo (nM to low μM). For the therapeutic potential of H2S is to be properly harnessed, pharmacological agents which generate H2S in a physiological manner and deliver physiologically relevant concentrations are needed. The phosphorodithioate GYY4137 has been proposed as “slow-release” H2S donors and has shown promising efficacy in cellular and animal model diseases such as hypertension, sepsis, atherosclerosis, neonatal lung injury and cancer. However, H2S generation from GYY4137 is inefficient necessitating its use at high concentrations/doses. However, structural modification of the phosphorodithioate core has led to compounds (e.g. AP67 and AP105) with accelerated rates of H2S generation and enhanced biological activity. In this review, the therapeutic potential and limitations of GYY4137 and related phosphorodithioate derivatives are discussed.

3-mercaptopyruvate sulfurtransferase (MST) transfers sulfur from 3-mercaptopyruvate (3MP) or thiosulfate to a sulfur acceptor. We developed MST-knockout (MST-KO) mice to clarify the physiological role of MST. They showed increased anxiety-like behaviors. In the prefrontal cortex and hippocampus, levels of serotonin (5-hydroxytryptamin, 5-HT) and/or serotonin metabolite, 5-hydroxyindoleacetic acid were higher in MST-KO mice than in wild-type mice and further 5-HT2A receptor mRNA was increased. Experiments using MST-KO mouse revealed that MST produces hydrogen polysulfides from 3MP and sodium sulfide and hydrogen trisulfide was also enzymatically produced.

The rat 3-mercaptopyruvate sulfurtransferase (MST), a multifunctional enzyme, has redox-sensing molecular switches, a catalytic Cys247 and two cysteines, Cys154 and Cys263, on the outer surface of the enzyme. These switches are reduced or oxidized according to the redox state of their surrounding environment and require a redox active cysteine in thioredoxin (Trx) to interact with MST. Recently, MST has been demonstrated to be involved in the production of possible signaling molecules such as hydrogen sulfide, polysulfides, and/or sulfur oxides. However, the relationship between the production of signaling molecule(s) and action of these redox-sensing molecular switches has not been clarified, and a precise investigation of this relationship is underway.

Archaea domain is comprised of many versatile taxa that often colonize extreme habitats. Here, we report the discovery of strictly anaerobic extremely halophilic euryarchaeon, capable of obtaining energy by dissimilatory reduction of elemental sulfur using acetate as the only electron donor and forming sulfide and CO2 as the only products. This type of respiration has never been observed in hypersaline anoxic habitats and is the first example of such metabolic capability in the entire Archaea domain. We isolated and cultivated these unusual organisms, selecting one representative strain, HSR2, for detailed characterization. Our studies including physiological tests, genome sequencing, gene expression, metabolomics and [(14)C]-bicarbonate assimilation assays revealed that HSR2 oxidized acetate completely via the tricarboxylic acid cycle. Anabolic assimilation of acetate occurred via activated glyoxylate bypass and anaplerotic carboxylation. HSR2 possessed sulfurtransferase and an array of membrane-bound polysulfide reductase genes, all of which were expressed during the growth. Our findings suggest the biogeochemical contribution of haloarchaea in hypersaline anoxic environments must be reconsidered.

A cystine-catabolizing enzyme, 3-mercaptopyruvate sulfurtransferase catalyzes the trans-sulfuration reaction of mercaptopyruvate or thiosulfate to thiol-containing compounds or cyanide. During the catalytic process, persulfide is formed at the catalytic site cysteine residue and a sulfur-acceptor substrate donates the outer sulfur of the persulfide to form a new persulfide molecule. Subsequently, the molecule can be reduced by thioredoxin to form hydrogen sulfide. The enzyme is regulated by redox changes via two redox-sensing molecular switches consisting redox-sensitive cysteine residues. One switch is the catalytic cysteine in itself, which is oxidized to form a cysteine-sulfenate resulting in inhibition of catalytic activity. The sulfenate can be reduced by thioredoxin resulting in restoration of the activity. The redox potential of sulfenate is lower than that of glutathione and greater than that of thioredoxin. The other switch involves cysteine residues positioned on the surface of the enzyme. The oxidation the intermolecular disulfide linkage at these cysteine residues, leading to dimer formation, inhibits enzyme activity. On the other hand, reduction-associated monomer formation increases catalytic activity. Thioredoxin reduces the disulfide bond more effectively than dithiothreitol, although the specificity mechanism has not been identified. Congenital defects in this enzyme result in, mercaptolactate-cysteine disulfiduria associated with or without mental retardation. However, the pathogenesis has not been identified. Because 3-mercaptopyruvate sulfurtransferase serves as a cellular antioxidative protein, the other biological functions related to the inhabitant disease are being investigated.

Hydrogen sulfide (H2S) is a biologically active gas that is synthesized naturally by three enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). These enzymes are constitutively present in a wide array of biological cells and tissues and their expression can be induced by a number of disease states. It is becoming increasingly clear that H2S is an important mediator of a wide range of cell functions in health and in disease. This review therefore provides an overview of the biochemical and molecular regulation of H2S synthesizing enzymes both in physiological conditions and their modulation in disease states with particular focus on their regulation in asthma, atherosclerosis and diabetes. The importance of small molecule inhibitors in the study of molecular pathways, the current use of common H2S synthesizing enzyme inhibitors and the relevant characteristics of mice in which these enzymes have been genetically deleted will also be summarized. With a greater understanding of the molecular regulation of these enzymes in disease states, as well as the availability of novel small molecules with high specificity targeted towards H2S producing enzymes, the potential to regulate the biological functions of this intriguing gas H2S for therapeutic effect can perhaps be brought one step closer.

The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase) is an ubiquitous enzyme with activity in all living organisms, from bacteria to man. It has been suggested that this enzyme plays a central role in cyanide detoxification. The purpose of this investigation was to determine rhodanese activities in different tissues of human and compare them with those found in domestic animals. Normal human tissues were obtained from consenting patients who had undergone surgery after confirmation of nonneoplastic condition. Tissue extracts were prepared from liquid nitrogen-frozen samples and rhodanese specific activity and activity per gram tissue was measured by determination of enzymatically formed thiocyanate. The highest activity of rhodanese was found in kidney, followed by liver, brain, lung, muscle, and stomach. Other tissues studied did not show significant rhodanese activity. The results obtained in this study were compared with previously reported information on domestic animals. Human liver contains lower rhodanese activity compared with ruminants and nonruminants, except for dog which has comparable hepatic activity with human. Human kidney contains significant activity and the value is higher than those of camels, pigs, dogs, and chickens and is comparable with those of goats. The results of this study might indicate the involvement of rhodanese in cyanide detoxification in tissues which might be more exposed to cyanide due to higher blood supply to these tissues. On the other hand, rhodanese might perform other functions in human organs which are specific to these tissues.

Sulfur is a functionally important element of living matter. Incorporation into biomolecules occurs by two basic strategies. Sulfide is added to an activated acceptor in the biosynthesis of cysteine, from which methionine, coenzyme A and a number of biologically important thiols can be constructed. By contrast, the biosyntheses of iron sulfur clusters, cofactors such as thiamin, molybdopterin, biotin and lipoic acid, and the thio modification of tRNA require an activated sulfur species termed persulfidic sulfur (R-S-SH) instead of sulfide. Persulfidic sulfur is produced enzymatically with the IscS protein, the SufS protein and rhodanese being the most prominent biocatalysts. This review gives an overview of sulfur incorporation into biomolecules in prokaryotes with a special emphasis on the properties and the enzymatic generation of persulfidic sulfur as well as its use in biosynthetic pathways.

Hydrogen sulfide (H2S) is the endogenously produced gasotransmitter involved in the regulation of nervous system, cardiovascular functions, inflammatory response, gastrointestinal system and renal function. Together with nitric oxide and carbon monoxide, H2S belongs to a family of gasotransmitters. H2S is synthesized from L-cysteine and/or L-homocysteine by cystathionine β-synthase, cystathionine γ-lyase and cysteine aminotransferase together with 3-mercaptopyruvate sulfurtransferase. Significant progress has been made in recent years in our understanding of H2S biochemistry, signaling mechanisms and physiological role. H2S-mediated signaling may be accounted for not only by the intact compound but also by its oxidized form, polysulfides. The most important signaling mechanisms include reaction with protein thiol groups to form persulfides (protein S-sulfhydration), reaction with nitric oxide and related species such as nitrosothiols to form thionitrous acid (HSNO), nitrosopersulfide (SSNO(-)) and nitroxyl (HNO), as well as reaction with hemoproteins. H2S is enzymatically oxidized in mitochondria to thiosulfate and sulfate by specific enzymes, sulfide:quinone oxidoreductase, persulfide dioxygenase, rhodanese and sulfite oxidase. H2S donors have therapeutic potential for diseases such as arterial and pulmonary hypertension, atherosclerosis, ischemia-reperfusion injury, heart failure, peptic ulcer disease, acute and chronic inflammatory diseases, Parkinson's and Alzheimer's disease and erectile dysfunction. The group of currently available H2S donors includes inorganic sulfide salts, synthetic organic slow-releasing H2S donors, H2S-releasing non-steroidal antiinflammatory drugs, cysteine analogs, nucleoside phosphorothioates and plant-derived polysulfides contained in garlic. H2S is also regulated by many currently used drugs but the mechanism of these effects and their clinical implications are only started to be understood.

The recently described anaerobic moderately halophilic bacterium Halanaerobium congolense has been shown to reduce thiosulfate and sulfur—but not sulfate—into sulfide. When cultivated in the presence of thiosulfate as terminal electron acceptor, H. congolense possesses a highly active thiosulfate:cyanide sulfur-transferase activity (rhodanese-like enzyme). A gene library of H. congolense (DSM 11287T) was constructed, and a 3.1-kb Sau3A DNA that encompassed a thiosulfate:cyanide sulfur-transferase-encoding gene was isolated in Escherichia coli. This fragment contains 2 orfs, which were separately subcloned in E. coli. The 900-bp gene encoding the rhodanese-like protein was named rdlA. RdlA differs from other known rhodanese-like proteins by having two potential catalytic sites, one N-terminal and one C-terminal, both harboring a cysteine. The two putative active sites are preceded by a highly-conserved region of unknown function. Closely related genes were also characterized in other thiosulfate-reducing non-sulfate-reducing anaerobes belonging to phylogenetically distant microorganisms, thus suggesting that RdlA is of importance in the mechanism of thiosulfate reduction by numerous members of the domain Bacteria.

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.

A bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (EC 2.8.1.2), is inactivated by 3-chloropyruvate, an analog of 3-mercaptopyruvate serving as a sulfur-donor and -acceptor substrate. To elucidate a reaction mechanism of the enzyme, the inactivation kinetic studies using 3-chloropyruvate were carried out. However, 3-chloropyruvate cannot be mixed with 3-mercaptopyruvate, 2-mercaptoethanol and thiosulfate because these substrates decompose 3-chloropyruvate. Thus, 3-mercaptopyruvate sulfurtransferase was incubated with 3-chloropyruvate, and then the remaining activity was measured separately in the assay system containing 3-mercaptopyruvate and 2-mercaptoethanol. The inactivation kinetics was analyzed by Kitz and Wilson method (J. Biol. Chem. 237 (1962) 3245-3248). The inactivation of mercaptopyruvate sulfurtransferase by 3-chloropyruvate proceeded in one-on-one manner and exhibited pseudo first-order kinetics with k(inact) = 0.068 +/- 0.003 min(-1) and K(I) = 4.0 +/- 0.2 mM (n = 3, mean +/- S.D.). Further, SH titration using DTNB revealed that MST was inactivated by 3-chloropyruvate in a 1:1 stoichiometry. Site-directed mutagenesis for binding sites of 3-mercaptopyruvate (Arg(187)-->Gly or Arg(196)-->Gly) (J. Biol. Chem. 271 (1996) 27395-27401) did not critically affect the inactivation. These findings suggest that 3-chloropyruvate behaves as an affinity label and directly tags the catalytic site, Cys(247). An ESI-LC/Q-TOF mass spectrometric study suggests that a pyruvate adduct is formed at Cys(247), which mimics a reaction intermediate.

IscA/SufA proteins belong to complex protein machineries which are involved in iron-sulfur cluster biosynthesis. They are defined as scaffold proteins from which preassembled clusters are transferred to target apoproteins. The experiments described here demonstrate that the transfer reaction proceeds in two observable steps: a first fast one leading to a protein-protein complex between the cluster donor (SufA/IscA) and the acceptor (biotin synthase), and a slow one consisting of cluster transfer leading to the apoform of the scaffold protein and the holoform of the target protein. Mutation of cysteines in the acceptor protein specifically inhibits the second step of the reaction, showing that these cysteines are involved in the cluster transfer mechanism but not in complex formation. No cluster transfer from IscA to IscU, another scaffold of the isc operon, could be observed, whereas IscU was shown to be an efficient cluster source for cluster assembly in IscA. Implications of these results are discussed.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is located at the wobble position of the anticodons of tRNALys, tRNAGlu, and tRNA1Gln. Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm5se2U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm5se2U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His6 tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm5se2U synthesis is shown to be dependent on 2-selenouridine synthase, SePO3, and tRNA. Finally, we demonstrate that the conserved Cys97 (but not Cys96) in the rhodanese sequence motif Cys96-Cys97-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

The Azotobacter vinelandii rhodanese is a 31kDa sulfurtransferase protein that catalyzes the transfer of sulfur atom from thiosulfate to cyanide in the detoxification process from cyanide and is able to insert sulfur atom in the iron-sulfur cluster. A study of the uniformly 15N isotopic labeling by high resolution NMR, before obtaining the backbone sequential assignment, has been carried out. The sulfur loaded and the sulfur discharged forms of the enzyme show very similar HSQC spectra with a good spectral dispersion. Few resonances show changes in chemical shift between the two forms. Relaxation parameters T(1), T(2) and 1H-15N NOE of all amide nitrogen atoms, as well as isotope exchange kinetics, show that the two forms exhibit the same global correlation time and hydrodynamic properties. In parallel, essential dynamics studies show that formation and discharging of catalytic cysteine persulfide group has no significant impact on the overall conformation of the protein. These results, taken together, give a clearcut answer to the question if the catalytic mechanism of the enzyme involves a change in the conformation and/or in the mutual orientation of the two domains. On the contrary these results clearly indicate that upon the catalytic mechanism the two domains of the protein behave as a unique fold.

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions.

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.

Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.