Cys92, Cys101, Cys197, and Cys203 Are Crucial Residues for Coordinating the Iron–Sulfur Cluster of RhdA from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron–sulfur clusters. But so far, there were no articles to confirm the existence of iron–sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV–Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron–sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron–sulfur cluster binding.     

Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.     

Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins

Details are provided for a reproducible procedure for determination of labile sulfide in iron-sulfur (Fe-S) proteins in the range of 1 to 3 nmol. Analyses are also presented on the most stable Fe-S protein so far reported. In this case denaturation with guanidine.HCl was used in the presence of dithiothreitol. The values obtained then also include any sulfane sulfur (S0) present.     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.     

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.     

The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form

After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.     

The origin of sulfur in biotin

The comparative ability of Escherichia coli K-12 hpb lambda-, a biotin overproducing strain, to incorporate 35S from isotopically labeled L-methionine, L-cystine, and the sulfane sulfur of thiocystine was determined. Comparison of the specific activity of sulfur in biotin produced by the organism with that of the 35S-labeled amino acids demonstrates that the sulfur of cystine is transferred to biotin with an efficiency of at least 75%, that the sulfur of methionine does not contribute to biotin significantly, and that the sulfane sulfur of thiocystine contributes approximately one-third of the sulfur to newly synthesized biotin and is essentially equivalent in utilization to that of the other two sulfur atoms in the molecule.     

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.     

The level of sulfane sulfur in the fungus <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> wild type and mutant strains

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.     

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.     

Latest news about the sulfurtransferase protein family of higher plants

Sulfurtransferases/rhodaneses (Str) comprise a group of enzymes widely distributed in all phyla which catalyze in vitro the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. The best characterized Str is bovine rhodanese (EC 2.8.1.1) which catalyses in vitro 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 contain many proteins carrying a typical rhodanese pattern or domain forming multi-protein families (MPF). Despite the presence of Str activities in many living organisms, the physiological role of the members of this MPF has not been established unambiguously. While in mammals these proteins are involved in the elimination of toxic cyanogenic compounds, their ubiquity suggests additional physiological functions. In plants, Str are localized in the cytoplasm, mitochondria, plastids, and nucleus. Str probably also transfer reduced sulfur onto substrates as large as peptides or proteins. Several studies in different organisms demonstrate a protein–protein interaction with members of the thioredoxin MPF indicating a role of Str in maintenance of the cellular redox homeostasis. The increased expression of several members of the Str MPF in various stress conditions could be a response to oxidative stress. In summary, data indicate that Str are involved in various essential metabolic reactions.     

Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.     

Mobilization of sulfane sulfur from cysteine desulfurases to the <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> sulfurtransferase RhdA

Mobilization of the l-cysteine sulfur for the persulfuration of the rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the functional results about rhodanese-like proteins and RhdA itself, it is suggested that RhdA-dependent modulation of l-cysteine levels must deal with a redox-related process.     

Potential therapeutic advantage of ribose-cysteine in the inhibition of astrocytoma cell proliferation

It has been observed that astrocyte and astrocytoma cells differ in their response to d-ribose-l-cysteine (RibCys) in the culture medium. RibCys, a prodrug of l-cysteine, elevates the level of cysteine and glutathione in both astrocytoma and astrocyte cultures. It also affects the activity of two sulfurtransferases, 3-mercaptopyruvate sulfurtransferase and rhodanese, involved in the metabolism of sulfane sulfur-containing compounds and in consequence exerts an effect on the level of sulfane sulfur. Under conditions, in which the raised level of sulfane sulfur was accompanied by an elevated activity of 3-mercaptopyruvate sulfurtransferase, the proliferation of the human astrocytome U373 line was decreased. The experiments were simultaneously performed with murine astrocytes to compare the behavior of normal cells under similar conditions. In murine astrocytes, RibCys was capable of increasing cellular proliferation, and was accompanied by a diminished level of sulfane sulfur and unchanged activity of the two sulfurtransferases. Thus, RibCys might offer a therapeutic advantage in the inhibition of astrocytoma cell proliferation. Besides, in the absence of oxidative stress, measured as the ratio of GSH/GSSG, the obtained results confirm that the fall in the level of sulfane sulfur is associated with increasing proliferation of cells, whereas a rise in the level causes a decrease in the proliferation of U373 cells.     

The glial Gomori-positive material is sulfane sulpfur

The Gomori-positive glia are periventricular astrocytes with abundant cytoplasmic granular material, predominantly occupying a periventricular site in the brain. These granular inclusions are strongly stained with chrome hematoxylin in the Gomori's method as well as exhibit red autofluorescence and non-enzymatic peroxidase activity. The glial Gomori-positive material (GGPM) granules are positive in the performic acid Alcian blue method indicating the presence of protein-bound sulfur, what has been shown by our previous studies. The number of cells containing glial Gomori-positive granules dropped after administration of cyanide and increased under the influence of sulfane sulfur donor (diallyl disulfide). This suggests, that sulfur of these granules is a sulfane sulfur, possibly in the form of protein-bound cysteine persulfide. Sulfane sulfur is labile, reactive sulfur atom covalently bound to another sulfur atom. In this paper we present evidence that GGPM exhibit affinity to cyanolysis and its stainability in Gomori's method is due to the presence of protein-bound sulfane sulfur. The biological role of the Gomori-positive glia connected with protective properties of sulfane sulfur has been discussed.     

The lack of rhodanese RhdA affects the sensitivity of Azotobacter vinelandii to oxidative events

The rhdA gene of Azotobacter vinelandii codes for RhdA, a rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe-S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the present study we analysed the defects of the rhdA-null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner-Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe-4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys(230) thiol.