Persulfide Sulfur Research Articles

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers

AbstractProtein persulfides (R‐S‐SH) have emerged as a common post‐translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur–sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N‐ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R‐S‐S‐Alk) rapidly convert into the corresponding thioethers (R‐S‐Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N‐t‐butyl‐iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R‐S‐S‐Alk) to tautomerize into the thiosulfoxide (R‐(S=S)‐Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.

Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe–S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆yck2) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe–S clusters within mitochondria.

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria: COORDINATED USE OF PERSULFIDE SULFUR AND IRON AND REQUIREMENTS FOR GTP, NADH, AND ATP

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [(35)S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-(35)S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the (35)S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.

Frataxin or a mutant Fe‐S cluster scaffold protein with frataxin‐bypassing ability directly stimulates mitochondrial cysteine desulfurase by exposing substrate‐binding sites (578.4)

Fe‐S cluster biogenesis in mitochondria is essential for cell survival. During the process, the cysteine desulfurase Nfs1 binds the substrate cysteine, removing sulfur from the substrate and forming a persulfide on the active site cysteine in a process requiring the accessory protein Isd11. The persulfide sulfur is then transferred to the Isu scaffold, where it combines with iron to form the Fe‐S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how. We find that yeast frataxin (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate‐binding sites. This novel function of frataxin does not require iron, Isu1 or Isd11. The subsequent persulfide formation is independent of frataxin but is strictly dependent on Isd11. Importantly, a point mutation in Isu1 is able to rescue Fe‐S cluster synthesis in yeast mitochondria lacking Yfh1. In agreement with this finding, the Isu1 suppressor is found to mimic the frataxin effects on Nfs1, explaining the bypassing activity. We propose that Nfs1 undergoes at least two conformational changes for optimum activity. One change is mediated by frataxin/Yfh1 interaction and promotes substrate binding. A second change is mediated by Isd11 and induces persulfide formation.Friedreich's ataxia is an incurable neurodegenerative disease associated with frataxin deficiency. A small molecule or an Isu1 suppressor mimic capable of exposing the “buried” cysteine‐binding sites of the cysteine desulfurase perhaps could be developed into a drug for treating the disease.Grant Funding Source: Supported by NIH grants RO1AG030504 to D.P. and R37DK053953 to A.D.

Pseudomonas aeruginosa PA1006 Is a Persulfide-Modified Protein That Is Critical for Molybdenum Homeostasis

A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) “top-down” analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein’s persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe-S cluster synthesis.

Cysteine desulfurases abstract sulfur from the substrate cysteine, generate a covalent persulfide on the active site cysteine of the enzyme, and then donate the persulfide sulfur to various recipients such as Fe-S clusters. In Saccharomyces cerevisiae, the Nfs1p protein is the only known cysteine desulfurase, and it forms a complex with Isd11p (Nfs1p·Isd11p). Both of these proteins are found primarily in mitochondria and both are essential for cell viability. In the present study we show, using the results of experiments with isolated mitochondria and purified proteins, that Isd11p is required for the cysteine desulfurase activity of Nfs1p. Whereas Nfs1p by itself was inactive, the Nfs1p·Isd11p complex formed persulfide and was active as a cysteine desulfurase. In the absence of Isd11p, Nfs1p was able to bind the substrate cysteine but failed to form a persulfide. Addition of Isd11p allowed Nfs1p with bound substrate to generate a covalent persulfide. We suggest that Isd11p induces an activating conformational change in Nfs1p to bring the bound substrate and the active site cysteine in proximity for persulfide formation. Thus mitochondrial Nfs1p is different from bacterial cysteine desulfurases that are active in the absence of accessory proteins. Isd11p may serve to regulate cysteine desulfurase activity in mitochondria.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors

Cysteine desulfurases are pyridoxal 5'-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron-sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.

Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

Cysteine is the major sulfur donor for thio cofactors in bacterial and eukaryotic systems. The first step in sulfur mobilization involves a PLP-dependent enzymatic mechanism. During catalysis, free cysteine is converted into alanine with the concomitant formation of a persulfide bond with the catalytic cysteine residue, thus forming a covalent enzyme intermediate. Cysteine desulfurases in their persulfurated forms serve as donors at the intersection of various cellular sulfur-requiring pathways. Most Gram-positive bacteria, including Bacillus subtilis, contain a cysteine desulfurase gene sufS located adjacent to the gene encoding the proposed Fe-S cluster scaffold SufU. In this work, we identified the participation of SufU as a substrate in the SufS catalytic mechanism. Development of a sensitive method for detection of alanine formed in the SufS reaction enabled the identification of its associated mechanistic features. Steady-state kinetic analysis of alanine formation provided evidence of a double-displacement mechanism (ping-pong) of the cysteine:SufU sulfurtransferase reaction catalyzed by SufS. Results from site-directed mutagenesis of the catalytic cysteine (SufS(C361A)) and iodoacetamide alkylation of SufU support the occurrence of persulfide sulfur transfer steps in the mechanism of SufS.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

IscS Functions as a Primary Sulfur-donating Enzyme by Interacting Specifically with MoeB and MoaD in the Biosynthesis of Molybdopterin in Escherichia coli

The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from l-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in DeltacsdA and DeltasufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.

Mechanistic Insights into Sulfur Relay by Multiple Sulfur Mediators Involved in Thiouridine Biosynthesis at tRNA Wobble Positions

The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking.

Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: kinetic analysis of cleavage of the persulfide intermediate by chemical reductants.

Cysteine desulfurases (CDs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that cleave sulfur from cysteine via an enzyme cysteinyl persulfide intermediate. In vitro studies of these enzymes have generally employed dithiothreitol as a cosubstrate to reductively cleave the persulfide intermediate, and it has been suggested that persulfide cleavage is the rate-limiting step for catalysis. In this study, the kinetics and mechanisms of cleavage of the persulfide intermediate in Slr0387 (CD-0387), a sequence group I (NifS/IscS-like) cysteine desulfurase from Synechocystis sp. PCC 6803, by physiological and nonphysiological reductants have been examined, and the extent to which this step is rate-limiting for catalysis has been determined. The observations that dithiols such as dithiothreitol (DTT) cleave the persulfide with approximately 100-fold greater efficiency than structurally similar monothiols such as 2-mercaptoethanol (2-ME), that cleavage by DTT exhibits saturation kinetics, and that the dependence of the observed first-order rate constant for persulfide cleavage by DTT on the concentration of the dithiol corresponds precisely with that for formation of a complex between DTT and the PLP cofactor of the resting enzyme suggest that persulfide cleavage by dithiols occurs by prior formation of a complex, in which addition of one thiol to the cofactor positions the second thiol for attack. This conclusion and the observation that a second molecule of L-cysteine can bind to the cofactor in the persulfide form of CD-0387 explain why several CDs are subject to potent inhibition by L-cysteine during turnover with DTT: binding of L-cysteine prevents formation of the PLP-DTT adduct and renders the dithiol no better than a monothiol, which must react with the persulfide in bimolecular fashion. Consistent with this rationale, catalysis by CD-0387 with 2-ME as cosubstrate, while less efficient, is not subject to potent inhibition by L-cysteine. The similarity of the maximum rate constant for persulfide cleavage by DTT to k(cat) suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that k(cat) is approximately 10-fold greater when tris-(2-carboxyethyl)phosphine (TCEP), the most efficient persulfide cleaver identified, is used as the reducing cosubstrate. The faster turnover with TCEP provides a chemical model for activation of CD-0387 and other CDs by the presence of accessory factors that serve as efficient acceptors of the persulfide sulfur.

Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

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.

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.

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 assavable 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 · S 2O 3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.

Oxidation increases the proteolytic susceptibility of a localized region in rhodanese.

For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.