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.     

Inhibition of the catalytic activity of rhodanese by S-nitrosylation using nitric oxide donors

Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The –SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (–S–SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of –SH groups.

The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese.

Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5 mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5 mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case.

A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors.     

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.     

Active site modifications quench intrinsic fluorescence of rhodanese by different mechanisms

Beef liver rhodanese can be modified covalently at the active site (Cys-247) either reversibly or irreversibly by sulfur, selenium, iodoacetate, and hydrogen peroxide. Each derivative shows an intrinsic fluorescence lower than that of the free enzyme. The reaction of rhodanese with iodoacetate or hydrogen peroxide is time-dependent and accompanied by enzyme inactivation, by the loss of one or two sulfhydryl groups, respectively, by quenching and bathochromic shift of fluorescence, and by an absorbance perturbation in the near UV. The latter findings are indicative for a displacement of some tryptophyl side chains from hydrophobic to hydrophilic environment. The fluorescence decays of the various rhodanese derivatives can be fitted by a double-exponential function with two lifetimes: a shorter one of 1-1.7 ns and a longer one of 2.8-4.6 ns. The S-loaded and Se-loaded rhodanese samples have proportionally shorter lifetimes and lower quantum yields. No such proportionality was observed for the iodoacetate-treated and for the hydrogen peroxide treated enzyme. These findings indicate that two different quenching mechanisms are operating in rhodanese derivatives, a long-range energy transfer from tryptophan to persulfide (or sulfoselenide) group and a static quenching accompanying a conformational change of the protein after modification of the active site.     

Properties of an Escherichia coli rhodanese

A rhodanese enzyme of less than 20,000 molecular weight has been purified from Escherichia coli. The enzyme is accessible to substrates upon addition of whole cells to standard assay mixtures. This rhodanese has a Stokes radius of 17 A which for a globular protein corresponds to a molecular weight close to 14,000. It undergoes autoxidation to a polymeric form which is probably an inert dimer. Enzyme inactivated by oxidation can be reactivated by millimolar concentrations of cysteine. Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur by way of a double displacement mechanism with formation of a covalent enzyme-sulfur intermediate. The turnover number for the enzyme-catalyzed reaction, with thiosulfate as donor substrate and cyanide ion as the sulfur acceptor, is 260 s-1. This value corresponds to a catalytic efficiency 60% of that measured for a previously characterized bovine liver enzyme of more than twice the molecular weight. Furthermore, KmCN is 24 mM which is 2 orders of magnitude higher than the value observed previously for the bovine enzyme. Evidence from chemical inactivation studies implicates an essential sulfhydryl group in the enzyme activity. It is proposed that this group is the site of substrate-sulfur binding in the obligatory enzyme-sulfur intermediate. Furthermore, a cationic site important for binding of the donor thiosulfate is tentatively identified from anion inhibition studies. Tests of alternate acceptor substrates indicate that the physiological dithiol, dihydrolipoate, is a more efficient acceptor than cyanide ion for the enzyme-bound sulfur. Of possibly greater physiological significance, it has been found that the enzyme catalyzes the formation of iron-sulfur centers. Other work indicates the E. coli rhodanese is subject to catabolite repression and suggests a physiological role for the enzyme in aerobic energy metabolism.     

Activation of porcine heart mitochondrial malate dehydrogenase by zero valence sulfur and rhodanese.

Incubation of malate dehydrogenase with thiosulfate and rhodanese lead to an increase of dehydrogenasic activity. Selenosulfate, elemental sulfur and elemental selenium were shown similarly able to activate this protein. The activation is limited to the presence of SH groups on the protein. Experiments with ³⁵S demonstrated the direct transfer of zero valence sulfur from rhodanese to malate dehydrogenase. It is proposed that this activation could be a mechanism of enzyme activity modulation in vivo.     

Polymorphic Variants of Human Rhodanese Exhibit Differences in Thermal Stability and Sulfur Transfer Kinetics

Rhodanese is a component of the mitochondrial H₂S oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306A→C, has an allelic frequency of 1% and results in an E102D substitution in the encoded protein. The second polymorphism, 853C→G, has an allelic frequency of 5% and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfur transfer reactions. The Asp-102 and Ala-285 variants are more stable than wild-type rhodanese and exhibit k_cat/K_m,CN values that are 17- and 1.6-fold higher, respectively. All three rhodanese forms preferentially catalyze sulfur transfer from GSSH to sulfite, generating thiosulfate and glutathione. The k_cat/K_m,sulfite values for the variants in the sulfur transfer reaction from GSSH to sulfite were 1.6- (Asp-102) and 4-fold (Ala-285) lower than for wild-type rhodanese, whereas the k_cat/K_m,GSSH values were similar for all three enzymes. Thiosulfate-dependent H₂S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.     

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound,inactivated, and reactivated by GroEL

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.     

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

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

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.     

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.     

A study of the apolar interaction of the enzyme rhodanese with octyl substituted agarose gel

The accessibilities of sites on the surface of the enzyme rhodanese for binding to macromolecular apolarity have been measured for the two forms of the enzyme related to obligatory catalytic intermediates: the free enzyme, E and the sulfur substituted enzyme, ES. This study was done using a micromethod developed for this purpose which allows facile assessment of the apolar binding of proteins to commercially available beads of cross-linked agarose on which hydrophobic groups have been immobilized. The results indicate that the enzyme rhodanese can bind to macromolecular apolarity and that there is considerably more binding of the E form than the ES form. The fact that the binding is relatively slow implicates a protein conformational change in the rate limiting binding step. In fact, there is a large increase in the binding when the temperature is raised from 23° to 40° which correlates with previous results showing a conformational change in rhodanese over the same temperature range. These results in comparison with other solution studies and with x-ray studies are consistent with a model for rhodanese which has an apolar active site and a mechanism for catalysis that includes a conformational change.     

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.     

The differential functional stability of various forms of bovine liver rhodanese

Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.     

The structure of bovine liver rhodanese. II. The active site in the sulfur-substituted and the sulfur-free enzyme.

X-ray studies at 2.5 Å resolution show that the active site of bovine liver rhodanese is a depression between the two domains. In sulfur-substituted rhodanese the density of the essential Cys247 corresponds with that of a persulfide. Both sulfur atoms are interacting via hydrogen bonds with several peptide NH and side-chain OH groups. One side of the active site pocket contains mainly hydrophylic, the other side mainly hydrophobic residues. None of these hydrophylic or hydrophobic groups appears to interact strongly with the persulfide.Crystals of the sulfur-substituted enzyme were treated with cyanide, a sulfur acceptor. Subsequent difference Fourier studies show that the extra sulfur atom has been removed. Only minor conformational differences appear to exist between the two rhodanese species studied. These are a movement of the Sγ atom of Cys247 and some rearrangement of solvent molecules near the active site.The combination of these observations with the results of experiments performed by other investigators suggest a mechanism for sulfur transfer by rhodanese in which the thiol group of Cys247 is the essential nucleophile, whereas the positive charges on Arg186 and Lys249 act in various ways as “electrophilic assistants”. The transition state and the persulfide in the sulfur-substituted enzyme are stabilized by several hydrogen bonds.     

An unusual tandem-domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium?hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In?vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell.     

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.     

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.     

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.     

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

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