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.     

Microbes and microbial enzymes for cyanide degradation

Cyanide is an important industrial chemical produced on a grand scale each year. Although extremely toxic to mammalian life, cyanide is a natural product generated by fungi and bacteria, and as a result microbial systems have evolved for the degradation of cyanide to less toxic compounds. The enzymes which utilize cyanide as a substrate can be categorized into the following reaction types: substitution/addition, hydrolysis, oxidation, and reduction. Each of these categories is reviewed with respect to the known biochemistry and feasibility for use in treatment of cyanide containing wastes.     

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.     

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.     

Rhodanese distribution in porcine (Sus scrofa) tissues

The enzyme rhodanese (thiosulfate/cyanide sulfurtransferase) is an ubiquitous enzyme and its activity is present in all living organisms from bacteria to man. Evidence has been accumulated to indicate that this enzyme plays a central role in cyanide detoxification. A comparison was made of rhodanese activity in different tissues of young male and adult male and female pig (Sus scrofa). The highest activity of rhodanese was in liver and kidney cortex of all animals. Among the remaining tissues examined, the kidney medulla and the stomach epithelium tended to have higher levels than other tissues, although this was not significant (P>0.05). The rhodanese activity of heart ventricle tissue of 6-month-old male animals was higher than 7-week-old male animals (P<0.05), and 6-month-old male animals had higher rhodanese activity in lung tissue, compared to 6-month-old female pigs (P<0.05). Medulla and spleen of younger male animals exhibited higher levels of activity (P<0.10) compared to older male pigs. The results of this study may indicate the involvement of rhodanese in cyanide detoxification in pig tissues, which have greater potential to be exposed to higher levels of cyanide.     

The current and future applications of microorganism in the bioremediation of cyanide contamination

Inorganic cyanide and nitrile compounds are distributed widely in the environment, chiefly as a result of anthropogenic activity but also through cyanide synthesis by a range of organisms including higher plants, fungi and bacteria. The major source of cyanide in soil and water is through the discharge of effluents containing a variety of inorganic cyanide and nitriles. Here the fate of cyanide compounds in soil and water is reviewed, identifying those factors that affect their persistence and which determine whether they are amenable to biological degradation. The exploitation of cyanides by a variety of taxa, as a mechanism to avoid predation or to inhibit competitors has led to the evolution in many organisms of enzymes that catalyse degradation of a range of cyanide compounds. Microorganisms expressing pathways involved in cyanide degradation are briefly reviewed and the current applications of bacteria and fungi in the biodegradation of cyanide contamination in the field are discussed. Finally, recent advances that offer an insight into the potential of microbial systems for the bioremediation of cyanide compounds under a range of environmental conditions are identified, and the future potential of these technologies for the treatment of cyanide pollution is discussed.     

Microbial destruction of cyanide wastes in gold mining: process review

Microbial destruction of cyanide and its related compounds is one of the most important biotechnologies to emerge in the last two decades for treating process and tailings solutions at precious metals mining operations. Hundreds of plant and microbial species (bacteria, fungi and algae) can detoxify cyanide quickly to environmentally acceptable levels and into less harmful by-products. Full-scale bacterial processes have been used effectively for many years in commercial applications in North America. Several species of bacteria can convert cyanide under both aerobic and anaerobic conditions using it as a primary source of nitrogen and carbon. Other organisms are capable of oxidizing the cyanide related compounds of thiocyanate and ammonia under varying conditions of pH, temperature, nutrient levels, oxygen, and metal concentrations. This paper presents an overview of the destruction of cyanide in mining related solutions by microbial processes.     

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.     

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.     

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.     

A reexamination of the postulated charge transfer interactions at the active site of the enzyme rhodanese.

Spectral and kinetic studies of the interaction of N-methylnicotinamide chloride and nicotinamide with the enzyme thiosulphate sulphurtransferase (thiosulphate: cyanide sulfurtransferase, EC 2.8.1.1) (also known as rhodanese) have been performed and compared with previous inhibition data obtained with N-1-(4-pyridyl)pyridinium chloride (NPP). Like NPP both N-methylnicotinamide chloride and nicotinamide are competitive inhibitors of rhodanese with respect to the substrate thiosulfate. Rhodanese binding of N-methylnicotinamide chloride gives rise to no charge transfer absorbtion band. In addition, the free energy of interaction (deltaG0) of NPP with rhodanese is approximately equal to the sum of the individual deltaG0 values of MNA and NA. These compounds are analogous to the two halves of the NPP structure. We conclude that NPP and N-methylnicotinamide chloride are not bound via a charge transfer mechanism. The major stabilizing influence appears to be an ionic interaction with an anionic enzyme site with accessory apolar stabilization. It is postulated that the ionized active site sulfhydryl group in rhodanese could provide the ionic site.     

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.     

Cyanide detoxification by microbial consortia of natural-industrial complexes of gold heap leaching

Microorganisms that have adapted not only to high concentrations of pollutants but also to environmental conditions develop in autochthonous microbial communities of natural-industrial complexes of gold heap leaching. The biotechnological potential and diversity of autochthonous microbial communities involved in cyanide detoxification was evaluated by the example of a deposit situated in the Sakha (Yakutia) Republic. Under the zoning conditions of the ore heap, the biological component had a greater impact on cyanide destruction than chemical transformation. Metabolically active representatives of a microbial consortium are capable of surviving developed under these conditions. Phylotypes of the genus Serratia and family Alcaligenaceae that are capable of cyanide destruction and are potentially promising for the detoxification of wastes of gold heap leaching were revealed.     

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.     

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.     

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.     

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.     

Comparative studies on the distribution of rhodanese in different tissues of domestic animals.

1. The activity of rhodanese in different tissues of some domestic animals was measured. 2. Rhodanese was present in all tissues studied. 3. The activity of rhodanese in most tissues of sheep was higher than other animals studied. 4. In sheep and cattle the epithelium of rumen, omasum and reticulum were the richest sources of rhodanese. Significant activity of rhodanese was also present in liver and kidney. 5. In camel the liver contained the highest level of rhodanese followed by lung and rumen epithelium. Camel liver contained a third of the activity of sheep liver. 6. Equine liver had a third of the activity of sheep liver. Other tissues showed low levels of rhodanese activity. 7. Dog liver contained only 4% of the activity of sheep liver. In this animal, brain was the richest source of rhodanese. 8. The results are discussed in terms of efficacy of different tissues of animals in cyanide detoxification.     

微生物共培养在新活性化合物挖掘中的研究进展

微生物次级代谢产物结构新颖、活性多样,一直以来都是药物及其先导化合物的重要来源。但近年来在普通微生物纯培养中发现的新活性物质不断减少,重复发现逐渐增加;而微生物共培养因更加接近微生物生长的自然环境,在药物及其先导物挖掘方面发挥着越来越重要的作用。本文分析了近5年来利用真菌vs真菌、真菌vs细菌共培养挖掘新活性化合物的研究现状,以及研究中存在的问题。     

Structural rearrangements of the two domains of Azotobacter vinelandii rhodanese upon sulfane sulfur release: essential molecular dynamics, 15N NMR relaxation and deuterium exchange on the uniformly labeled protein

The Azotobacter vinelandii rhodanese is a 31 kDa 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 T₁, T₂ 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.