Evidence for the existence of rhodanese (thiosulfate:cyanide sulfurtransferase) in plants: preliminary characterization of two rhodanese cDNAs from Arabidopsis thaliana

The existence of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) in plants has been highly controversial. We have isolated and characterized for the first time in plants two cDNAs encoding rhodanese isoforms in Arabidopsis thaliana, AtRDH1 and AtRDH2. Both cDNAs contained a full-length open reading frame, the expression of which increased the rhodanese activity of transgenic yeast. AtRDH1 protein was mitochondrial, while AtRDH2 was cytosolic. AtRDH1 and AtRDH2 genes originated from the duplication of a large genomic region in chromosome 1 which took place before the appearance of the Arabidopsis genus. Our results confirm the existence of rhodanese in plants.     

A physical characterization of sulfane sulfurtransferase

The bacterial enzyme sulfane sulfurtransferase has been studied using spectroscopic techniques. The enzyme was characterized in terms of its near-UV absorption spectrum, molar ellipticity, intrinsic fluorescence spectra and the effects of general and ionic quenching reagents upon its fluorescence. Fluorescence model studies are consistent with sulfane sulfurtransferase having only a single tryptophan residue, which accounts for its low UV absorption coefficient and suggested that this residue is at least partially exposed to solvent. Second derivative absorption spectroscopy studies revealed that most of the bacterial enzyme's tyrosine residues are exposed to solvent. Unlike the better known sulfurtransferase, bovine liver rhodanese, sulfane sulfurtransferase does not undergo a detectable increase in quantum yield when shifting from the sulfur-containing covalent enzyme intermediate to the free enzyme form (which lacks sulfur) during catalysis. CD studies suggest that sulfane sulfurtransferase has a significantly higher proportion of alpha-helix than rhodanese. The renaturation of sulfane sulfurtransferase denatured in 6 M guanidine was shown to be rapid and complete provided that the enzyme had not been oxidized while in the denatured state. Sulfane sulfurtransferase, like rhodanese, catalyzes the transfer of sulfur from thiosulfate to cyanide via a persulfide intermediate, and displays remarkably similar kinetics in this process (Aird, B.A., Heinrikson, R.L. and Westley, J. (1987) J. Biol. Chem 262, 17327-17335). In light of this, the results of the structural studies with sulfane sulfurtransferase are compared and contrasted to data from similar experiments with rhodanese in hopes that they would provide insight about which phenomena observed with rhodanese are intrinsic to the process of transferring sulfur atoms.     

Interaction of rhodanese with intermediates of oxygen reduction

Cyanide-promoted inactivation of the enzyme rhodanese [thiosulfate sulfurtransferase (EC 2.8.1.1)] in the presence of ketoaldehydes is caused by reduced forms of molecular oxygen generated during autoxidation of the reaction products. The requirement of both catalase and superoxide dismutase to prevent rhodanese inactivation indicates that hydroxyl radical could be the most efficient inactivating agent. Rhodanese, also in the less stable sulfur-free form, shows a different sensitivity towards oxygen activated species. While the enzyme is unaffected by superoxide radical, it is rapidly inactivated by hydrogen peroxide. The extent of inactivation depends on the molar ratio between sulfur-free enzyme and oxidizing agent. Fully inactive enzyme is reactivated by reduction with its substrate thiosulfate.     

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.     

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.     

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.     

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).     

Identification of putative sulfurtransferase genes in the extremophilic Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins

Eight nucleotide sequences containing a single rhodanese domain were found in the Acidithiobacillus ferrooxidans ATCC 23270 genome: p11, p14, p14.3, p15, p16, p16.2, p21, and p28. Amino acids sequence comparisons allowed us to identify the potentially catalytic Cys residues and other highly conserved rhodanese family features in all eight proteins. The genomic contexts of some of the rhodanese-like genes and the determination of their expression at the mRNA level by using macroarrays suggested their implication in sulfur oxidation and metabolism, formation of Fe-S clusters or detoxification mechanisms. Several of the putative rhodanese genes were successfully isolated, cloned and overexpressed in E. coli and their thiosulfate:cyanide sulfurtransferase (TST) and 3-mercaptopyruvate/cyanide sulfurtransferase (MST) activities were determined. Based on their sulfurtransferase activities and on structural comparisons of catalytic sites and electrostatic potentials between homology- modeled A. ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST) and SseA (MST) proteins, two of the rhodanese-like proteins (P15 and P16.2) could clearly be defined as TSTs, and P14 and P16 could possibly correspond to MSTs. Nevertheless, several of the eight A. ferrooxidans rhodanese-like proteins may have some different functional activities yet to be discovered.     

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.     

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.     

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.     

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

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.     

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.     

On the molecular weight of thiosulfate sulfurtransferase.

Bovine liver thiosulfate sulfurtransferase (rhodanese) (EC 2.8.1.1) HAS BEEN REPORTED TO EXIST IN SOLUTION IN A RAPID, PH-dependent equilibrium between monomeric and dimeric forms of molecular weights 18 500 and 37 000 (Volini, M., DeToma, F. and Westley, J. (1967), J. Biol. Chem. 242, 5220). We have reinvestigated the proposed dissociation using sodium dodecylsulfate-polyacrylamide gel electrophoresis. The smallest rhodanese species observed has a molecular weight around 35 000, which is not reduced by severe denaturing conditions, including alkylation in 8 M guanidine-HCl or dialysis against 2% sodium dodecylsulfate and 5% mercaptoethanol. After limited CNBr cleavage, intermediate products of greater than 18 500 molecular weight are formed. The apparent molecular weight of these intermediate fragments is not changed by addition of mercaptoethanol. The total apparent molecular weights of the CNBr fragments after exhaustive cleavage is approx. 45 000 plus or minus 15 000. These results are not consistent with a monomer molecular weight of approx. 18 500 for thiosulfate sulfurtransferase.     

The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanese and phosphatase enzyme families

Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.     

Oxidative inactivation of the enzyme rhodanese by reduced nicotinamide adenine dinucleotide

The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is inactivated with a half-time of approximately 3 min when incubated with 50 mM NADH. NAD+, however, has virtually no effect on the activity. Inactivation can be prevented by the inclusion of the substrate thiosulfate. The concentration of thiosulfate giving half-protection is 0.038 mM. In addition, NADH, but not NAD+, is a competitive inhibitor with respect to thiosulfate in the catalyzed reaction (Ki = 8.3 mM). Fluorescence studies are consistent with a time-dependent oxidation of NADH in the presence of rhodanese. The sulfur-free form of rhodanese is more rapidly inactivated than the sulfur-containing form. Spectrophotometric titrations show that inactivation is accompanied by the loss of two free SH groups per enzyme molecule. Inactivation is prevented by the exclusion of air and the inclusion of EDTA (1 mM), and the enzyme activity can be largely protected by incubation with superoxide dismutase or catalase. Rhodanese, inactivated with NADH, can be reactivated by incubation with the substrate thiosulfate (75 mM) for 48 h or more rapidly, but only partially, by incubating with 180 mM dithiothreitol. It is concluded that, in the presence of rhodanese, NADH can be oxidized by molecular oxygen and produce intermediates of oxygen reduction, such as superoxide and/or hydrogen peroxide, that can inactivate the enzyme with consequent formation of an intraprotein disulfide. In addition, NADH, but not NAD+, can reversibly bind to the active site region in competition with thiosulfate. These data are of interest in view of x-ray studies that show structural similarities between rhodanese and nucleotide binding proteins.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

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.     

The "rhodanese" fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic rhodaneses. In particular, conformational variation of the rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.