Rhodanese-Mediated sulfur transfer to succinate dehydrogenase.

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied. Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re-loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low-molecular-weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate. Sulfur release from rhodanese appears to depend on the presence of - SH groups in the acceptor protein. Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavorprotein. The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation. The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.     

Methods for in situ visualization and assay of sulfurtransferases

The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied. Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm). This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations. This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase. In addition, a refinement of the thiosulfate reductase assay technique is reported.     

Transfer of persulfide sulfur from thiocystine to rhodanese.

THiocystine (bis-[2-amino-2-carboxyethyl]trisulfide) is a natural substrate for rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1). Analogs of thiocystine were prepared by eliminating the carboxyl or amino group or by lengthening the carbon chain. Of these only homothiocystine (bis-[2-amino-2-carboxypropyl]trisulfide) had appreciable activity as a substrate. At pH 8.6, the optimum for rhodanese, transfer of sulfane sulfur to cyanide in the presence of rhodanese was nonspecific. Only the sulfane sulfur of 35S-labeled thiocystine was transferred to rhodanese. Thus, thiocystine and thiosulfate both produce a rhodanese persulfide as a stable intermediate in sulfur transfer.     

Isolation and characterization of a prokaryotic sulfurtransferase

A sulfurtransferase has been purified to apparent homogeneity from the prokaryote Acinetobacter calcoaceticus lwoffi by conventional protein fractionation techniques. Steady-state kinetic studies of the enzyme revealed that its formal mechanism varies with the acceptor substrate employed. With inorganic thiosulfate as the sulfane sulfur-donor substrate and cyanide anion as the acceptor, the enzyme was shown to catalyze the reaction by a double displacement mechanism like that of mammalian rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1). In contrast, with a thiol as the acceptor substrate at relatively high concentrations, the reaction proceeds by a single displacement mechanism, reminiscent of catalysis by another sulfur-transferase, thiosulfate reductase, glutathione-dependent (EC 2.8.1.3). When dithiothreitol is the acceptor substrate, the enzyme cycles through both the single and double displacement pathways, with the flux through each depending differentially on the concentration of dithiothreitol employed. In view of both the relaxed acceptor substrate specificity and the corresponding variability of formal mechanism, the more general name of sulfane sulfurtransferase is proposed for this bacterial enzyme.     

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.     

Contact versus energy transfer fluorescence quenching in the sulfur substituted form of the enzyme rhodanese: a study using cesium ion resolved emission spectra.

The intrinsic fluorescence of the enzyme rhodanese (EC 2.8.1.1) can be resolved into separate contributions from solvent accessible and solvent inaccessible tryptophan residues by comparing spectra run in 2 $\text{M}$ NaCl with those run in the quenching solution, 2 $\text{M}$ CsCl. Both these classes of tryptophan residues are quenched when sulfur is transferred to rhodanese forming a sulfur substituted enzyme which is an intermediate in the catalytic cycle. This observation is consistent with a non-radiative energy transfer mechanism for quenching as opposed to a mechanism requiring direct contact between the bound sulfur and an active site tryptophan. Therefore, the data supports the hypothesis that the primary stabilizing influence in forming the substituted enzyme intermediate is a persulfide bond between an active site sulfhydryl group and the transferred sulfur.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.     

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.     

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.     

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.     

Menadione effect on l-cysteine desulfuration in U373 cells

The non-cytotoxic concentration (20 microM) of menadione (2-methyl-1,4-naphthoquinone), after 1 h of incubation, leads to loss of the activity of rhodanese by 33%, 3-mercaptopyruvate sulfurtransferase by 20%, as well as the level of sulfane sulfur by about 23% and glutathione by 12%, in the culture of U373 cells, in comparison with the control culture. Reactive oxygen species generated by menadione oxidize sulfhydryl groups in active centers of the investigated enzymes, inhibiting them and saving cysteine for glutathione synthesis. A decreased sulfane sulfur level can be correlated with an oxidative stress.     

Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate

ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S(2-) rather than S(0).     

Rhodanese-thioredoxin system and allyl sulfur compounds

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.     

A chaperone-mimetic effect of serum albumin on rhodanese

Reactivation of denatured rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was found to be aided by the presence of serum albumin. Both the rate and the extent of reactivation of the urea-denatured enzyme were optimal at low rhodanese and moderate serum albumin concentrations. Similarly, stabilization of the sulfurtransferase activity of rhodanese that had been partially unfolded at 40°C was aided by the presence of serum albumin. All the observations are in accord with a model in which enzyme that has been partially refolded from the urea-denatured state or partially unfolded thermally interacts directly with serum albumin in a way that prevents rhodanese self-association. Serum albumin thus acts as a molecular chaperone in these systems.     

Sulfurtransferases and the content of cysteine, glutathione and sulfane sulfur in tissues of the frog Rana temporaria

L-cysteine desulfuration was examined in tissues of Rana temporaria, in October and January. The activities of 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine gamma-lyase (CST) and rhodanese were primarily concentrated in frog liver and kidney. The values of CST and rhodanese activity, as well as sulfane sulfur compounds levels fell in the range characteristic of rat. For each of the investigated tissues changes noted in the enzymatic activities and in the level of glutathione (GSH), protein-bound cysteine (PbCys) and sulfane sulfur compounds were dependent on the month in which the determination was performed, and on the character of the tissue. In such tissues as the liver or gonads, high GSH levels and high activities of MPST (in the liver) or MPST and rhodanese (in the gonads) seemed to accompany protein biosynthesis during hibernation. PbCys, the level of which was consequently diminished in all tissues in January, compensated the absence of exogenous cysteine. A significantly reduced GSH level in the brain in January seemed to be correlated with decreased requirements of the tissue for this important natural antioxidant at diminished thyroid hormones levels in the serum and minimal oxygen consumption during the hibernation. In the kidney, the possible participation of sulfane sulfur compounds in detoxification processes requires elucidation, similarly as in protection against cellular oxidative stress at extremely low levels of GSH.     

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii: a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The modality of the enzymatic mechanism is then confined to a surface interaction, including dynamics of water molecules buried in the tertiary structure. Thus, investigations have been carried out by fluorescence, circular dichroism, and nuclear magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated enzyme and the sulfur-free form indicated that small changes occur. Fluorescence quenching studies have been performed to evaluate the conformational changes during catalysis using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by (15)N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe(2)S(2) iron-sulfur cluster in sulfur-deprived ferredoxins.     

Effect of mercury ions on cysteine metabolism in Xenopus laevis tissues

The effect of mercury ions on the level of cysteine, glutathione, sulfane sulfur, and on the activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase in brain, heart muscle, liver, kidneys, testes and skeletal muscle of adult Xenopus laevis was investigated. Frogs of both sexes were exposed for 7 or 14 days to 1.353mgL(-1) (ppm) of mercury chloride (HgCl(2)) dissolved in water. The activity of the investigated enzymes participating in cysteine metabolism depends on cysteine in their active sites. Mercury ions can bind to -SH groups and, therefore, lower the activity of enzymes and change the level of sulfane sulfur, a product of l-cysteine desulfuration. The effect of mercury was found to depend on the time of exposure and the kind of tissue. In the liver, the main site of glutathione biosynthesis, the ratio of GSH to GSSG was essentially unchanged. The total glutathione level was decreased after 7 days of exposure to mercury, similarly as the activity of rhodanese. Sulfane sulfur levels were significantly increased after a shorter duration, while they decreased after a longer time of exposure. The kidney, brain and testes were able to enhance the level of GSH, probably thanks to high γ-glutamyltranspeptidase activity. These tissues showed an increased value of GSH/GSSG ratio during the shorter exposure to mercury. The activity of sulfurtransferases was decreased, especially after the longer exposure to mercury. In the heart and skeletal muscle, the level of GSH, sulfane sulfur, and the activity of the investigated sulfurtransferases was diminished after 14 days of exposure to Hg. It can be concluded that the main mechanism of toxic Hg activity is generation of reactive oxygen species in cells due to depleted GSH level, and a decreased sulfurtransferases activity either by blocking or oxidation of their -SH groups, what in consequence results in a diminished sulfane sulfur levels in tissues, especially the heart and testes.     

Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese.

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.     

Persulfide properties of thiocystine and related trisulfides

Thiocystine (bis[2-amino-2-carboxyethyl]trisulfide) functions as a persulfide in transferring its sulfane sulfur to thiophilic acceptors. This occurs by formation of a reactive intermediate, thiocysteine (alanine hydrogen disulfide). In the absence of an acceptor sulfur is released in elemental form. Thiocystine is relatively stable in the pH range of 2–9. However, its conversion to unstable thiocysteine is accelerated by sulfhydryl compounds, rhodanese, or reagents that cleave sulfur-sulfur bonds to yield sulfhydryl groups. Since thiocystine has been detected in biological systems, it is proposed that in provides a storage form of sulfane sulfur. Trisulfides related to thiocystine show qualitatively similar properties.