New crystalline derivatives of bovine liver rhodanese

Mitochondrial bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase) has been crystallized in the form deprived of the transferable sulfur. The essential condition for crystallization was the removal of oxygen. Crystals of the sulfur-free enzyme are isomorphous with the previously characterized crystals of the sulfur-substituted enzyme. The new crystal species can react with either thiosulfate or selenosulfate to form the catalytic intermediate and, subsequently, with cyanide to form the corresponding product. Furthermore, the enzyme active site can be alkylated by iodoacetic acid.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

Chromogenic substrates for sulfurtransferases

The azo dye 4-(dimethylamino)-4'-azobenzene (DAB) thiosulfonate anion can serve as a sulfur-donor substrate for rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) and for thiosulfate reductase (EC unassigned) with cyanide anion and GSH, respectively, as acceptor substrates. In either case, the dye product is DAB sulfinate, which differs substantially in light absorption at 500 nm. Moreover, DAB sulfinate can serve as a sulfur-acceptor substrate for rhodanese with either inorganic thiosulfate or a colorless thiosulfonate anion as donor, and this reaction provides a second chromogenic assay procedure.     

Immunohistochemical localization of rhodanese

Summary The role of rhodanese in the detoxication of acute cyanide exposure is controversial. The debate involves questions of the availability of rhodanese to cyanide in the peripheral circulation. Blood-borne cyanide will distribute to the brain and may induce lesions or even death. The present study addresses the dispute by determining the distribution of rhodanese in tissues considered to have the highest rhodanese activity and thought to serve as major detoxication sites. The results indicate that rhodanese levels are highest in (1) hepatocytes that are in close proximity to the blood supply of the liver (2) epithelial cells surrounding the bronchioles (a major entry route for gaseous cyanide) and (3) proximal tubule cells of the kidney (serving to facilitate cyanide detoxication and elimination as thiocyanate). Rhodanese activity in the brain is low compared with liver and kidney (Mimoriet al., 1984; Drawbaugh & Marrs, 1987); the brain is not considered to be a major site of cyanide detoxication. The brain, however, is the target for cyanide toxicity. In this study our goal was also to differentiate the distribution of rhodanese in an area of the brain. We found that the enzyme level is highest in fibrous astrocytes of the white matter. Cyanide-induced brain lesions may thus occur in areas of the brain lacking sufficient sites for detoxication.     

Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures.

The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation. The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state. Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C. Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP. A recovery of about 80% was achieved after 1 h of the addition of those components. Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60. The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction. However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature. The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures. In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.     

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.     

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.     

Reducing sugars can induce the oxidative inactivation of rhodanese.

The enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is inactivated on incubation with reducing sugars such as glucose, mannose, or fructose, but is stable with non-reducing sugars or related polyhydroxy compounds. The enzyme is inactivated with (ES) or without (E) the transferable sulfur atom, although E is considerably more sensitive, and inactivation is accentuated by cyanide. Inactivation of E is accompanied by increased proteolytic susceptibility, a decreased sulfhydryl titer, a red-shift and quenching of the protein fluorescence, and the appearance of hydrophobic surfaces. Superoxide dismutase and/or catalase protect rhodanese. Inactive enzyme can be partially reactivated during assay and almost completely reactivated by incubation with thiosulfate, lauryl maltoside, and 2-mercaptoethanol. These results are similar to those observed when rhodanese is inactivated by hydrogen peroxide. These observations, as well as the cyanide-dependent, oxidative inactivation by phenylglyoxal, are explained by invoking the formation of reactive oxygen species such as superoxide or hydrogen peroxide from autooxidation of alpha-hydroxy carbonyl compounds, which can be facilitated by cyanide.     

Production of rhodanese by bacteria present in bio-oxidation plants used to recover gold from arsenopyrite concentrates

Considerably larger quantities of cyanide are required to solubilize gold following the bio-oxidation of gold-bearing ores compared with oxidation by physical-chemical processes. A possible cause of this excessive cyanide consumption is the presence of the enzyme rhodanese. Rhodanese activities were determined for the bacteria most commonly encountered in bio-oxidation tanks. Activities of between 6.4 and 8.2 micromol SCN min(-1) mg protein(-1) were obtained for crude enzyme extracts of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus caldus, but no rhodanese activity was detected in Leptospirillum ferrooxidans. Rhodanese activities 2-2.5-fold higher were found in the total mixed cell mass from a bio-oxidation plant. T. ferrooxidans synthesized rhodanese irrespective of whether it was grown on iron or sulphur. With a PCR-based detection technique, only L. ferrooxidans and T. caldus cells were detected in the bio-oxidation tanks. As no rhodanese activity was associated with L. ferrooxidans, it was concluded that T. caldus was responsible for all of the rhodanese activity. Production of rhodanese by T. caldus in batch culture was growth phase-dependent and highest during early stationary phase. Although the sulphur-oxidizing bacteria were clearly able to convert cyanide to thiocyanate, it is unlikely that this rhodanese activity is responsible for the excessive cyanide wastage at the high pH values associated with the gold solubilization process.     

The inactivation of rhodanese by nitrite and inhibition by other anions in vitro

Cyanide detoxification in mammals occurs, in part, by sulfur transfer by rhodanese to form the less toxic thiocyanate. Thiosulfate and nitrite are often used in combination for the treatment of cyanide intoxication. This report shows that nitrite can inhibit the rate of sulfur transfer by rhodanese in vitro. Nitrate, chloride, sulfate, and acetate were also examined as inhibitors. Inhibition by nitrite appeared to be more complex than for the other anions tested. Closer examination showed that nitrite can inactivate the sulfur-free rhodanese. Our observation leads to the suggestion that, in vivo, either rhodanese is maintained in its more stable sulfur-substituted form or cellular compartmentalization prevents inactivation by nitrite.     

Distribution of rhodanese in tissues of goat (Capra hircus)

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 is to determine and compare rhodanese activity in different tissues of adult male and female goats (Capra hircus). The results showed that the specific activity of rhodanese in different tissues was significantly different (P<0.05). The highest activity of rhodanese was in epithelium of rumen, followed by epithelia of reticulum and omasum and liver. No significant difference was observed when tissues of male and female goats were compared. The lowest specific activity of rhodanese was observed in spleen, urinary bladder, lymph node, ovary, skeletal muscle and pyloric muscle of abomasum. The results of this study may indicate the involvement of rhodanese in cyanide detoxification in goat tissues that have greater potential to be exposed to higher levels of cyanide.     

Improved Assay for Rhodanese in Thiobacillus spp

Rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) catalyzes the conversion of thiosulfate and cyanide to thiocyanate and sulfite. Conventional rhodanese assays colorimetrically measure the formation of one or the other of the products. These assays suffer from the fact that there is significant nonbiological formation of these products in addition to the enzymatically catalyzed reaction. In the present report, we describe a modified procedure for assaying rhodanese in which a separate boiled control was prepared for each assay trial. The boiled control corrected for the nonbiological contributions to product formation.     

The inhibition of rhodanese by lipoate and iron-sulfur proteins

A study was made on the effects of DL-dihydrolipoate, lipoate and iron-sulfur proteins on the activity of rhodanese (EC 2.8.1.1) with dihydrolipoate or cyanide as acceptors. DL-Dihydrolipoate inactivates rhodanese, lipoate does not, and the opposite occurs with the sulfur-free form of the transferase. The observed effects vary with the sulfane sulfur acceptor from rhodanese (i.e., dihydrolipoate or cyanide) and depend on intramolecular oxidation of the catalytic sulfhydryl or on formation of a mixed disulfide with dihydrolipoate. Thiosulfate protects against inactivation by reloading the active-site cysteine with persulfide sulfur. The inhibition of sulfur transfer by iron-sulfur proteins appears related to the amount of native iron-sulfur structure interacting with rhodanese. The implications of the results for a possible biological role of rhodanese are considered.     

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.     

Spectral differences between rhodanese catalytic intermediates unrelated to enzyme conformation

Circular dichroism (CD) spectra and UV absorption spectra of two obligatory intermediates in rhodanese catalysis were compared. A broad CD band between 250 and 287 nm increased in a manner stoichiometrically related to the content of enzyme-bound persulfide. Titration of a sample of sulfur-substituted rhodanese (ES) with either cyanide or sulfite gave a stoichiometry that is consistent with one persulfide/molecule of rhodanese (Mr = 33,000). This result agrees with that determined by x-ray crystallography and a method based on quenching of intrinsic fluorescence. Cyanolysis of the persulfide in ES is accompanied by a decrease of UV absorption in the region between 250 and 300 nm. Cyanide titrations followed by the change in absorbance at 263, 272, and 292 nm gave the expected stoichiometry. The magnitude of the difference between the far UV-CD spectra of E and ES found here is smaller than reported previously. This variability suggests that the differences in the secondary structure of these intermediates may not be obligatorily related to the cyanolysis of the persulfide. This view is compatible with recent evidence which suggested that E and ES may be made different by structural relaxation events that occur outside of the catalytic cycle. Furthermore, the methods developed here will be useful in studies on the stability of the catalytic persulfide that has been suggested to be central in the mechanism of several enzymes important in sulfur metabolism.     

Cyanide catabolizing enzymes in Trichoderma spp.

An investigation was made into the occurrence and distribution of the enzymes involved in HCN catabolism in different strains of the fungus Trichoderma. Three enzymes, cyanide hydratase, rhodanese and β-cyanoalanine synthase were studied. All the strains showed a high capacity to degrade cyanide via both the cyanide hydratase and rhodanese pathways. β-Cyanoalanine synthase, however, was not observed in any of the strains. The enzyme activities were found in varying levels in each of the Trichoderma strains. Experiments conducted with cyanide addition to the medium to assess whether the enzymes were induced in the presence of cyanide failed to show any statistically significant increase. This suggests a constitutive nature of both the enzymes in all the selected strains of Trichoderma used in this study.     

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.     

Alternative sulphur donors for detoxification of cyanide in the chicken.

1. Urinary excretion of thiocyanate by hens after dosage with cyanide was studied over 3 hr periods during which various sulphur sources were infused. 2. With 20 mumoles cyanide, endogenous sulphur supplies appeared to be almost sufficient. 3. With 45 mumoles cyanide, thiocyanate excretion was doubled with 90 mumoles of sulphur donor. Higher doses of mercaptopyruvate were also effective but not rhodanese substrates (thiosulphate or methanethiosulphonate): they interfered with thiocyanate excretion and may also have suppressed its formation. 4. Mercaptopyruvate and rhodanese substrates also differed in their effects on blood cyanide concentration and on the excretion of isotope from radiolabelled cyanide.     

Distribution of rhodanese in different parts of the urogenital systems of sheep at pre- and post-natal stages

The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase) 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 is to determine and compare rhodanese activity in different parts of urogenital systems of male and female sheep fetuses at 2.5, 3, 3.5, 4, 4.5, and 5 months of age. The highest activity of rhodanese in male fetus was in kidney cortex, followed by medulla of the kidney. No significant difference was observed in other organs. In female fetus, the highest activity was in kidney cortex followed by oviduct and medulla of kidney. The enzyme activity of tissues increased with age. There was no significant difference (P > 0.05) between male and female fetuses in levels of rhodanese activity of different tissues except in urinary bladder at 2.5 and 3 months and in urethra at 4.5 months of age. The results of this study might indicate the involvement of rhodanese in cyanide detoxification in tissues which are more exposed to cyanide. On the other hand, rhodanese might perform other functions which are specific in these tissues.