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.     

New crystalline derivatives of bovine liver rhodanese

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

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.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

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.     

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects on the sulfur-free and sulfur-containing catalytic intermediates

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave Km (Ks) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reversed by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.     

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects or the sulfur-free and sulfur-containing catalytic intermediates

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave K_m (K₅) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate-similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reserved by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.     

Kinetic evidence for the existence of an unstable intermediate in the trinitrophenylation-induced rhodanese inactivation reaction.

1. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.     

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.     

alpha-Crystallin facilitates the reactivation of hydrogen peroxide-inactivated rhodanese

It was previously shown that rhodanese, inactivated with hydrogen peroxide, could only be reactivated in the presence of a reductant or the substrate thiosulfate if these reagents were added soon after inactivation and if the oxidant was removed. Here, we report on the facilitated reactivation (75%) of hydrogen peroxide-inactivated rhodanese by the chaperone alpha-crystallin. Reactivation by the chaperone still required a reductant and thiosulfate. Without alpha-crystallin, but in the presence of the reductant and thiosulfate, the inactivated enzyme regained about 39% of its original activity. The alpha-crystallin-assisted reactivation of hydrogen peroxide-inactivated rhodanese was independent of ATP. Further, we found, that alpha-crystallin interacted transiently, but could not form a stable complex with hydrogen peroxide-inactivated rhodanese. Unlike in prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that alpha-crystallin can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein.     

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.     

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.     

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

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

Chemical Modification of Rhodanese with Sulphite

The essential sulphydryl group of bovine liver rhodanese (thiosulphate: cyanide sulphurtrasferase, E.C. 2.8.1.1.) is modified by sulphite produced during the enzymatic reaction or added to the fully active enzyme. The enzyme treated with labelled reagent incorporates 1 equivalent of SO²₃^- and loses one -SH group with the formation of a S-sulphonate group at the active site. Mercaptoethanol is effective in both restoring enzyme activity and removing bound sulphite from protein. The inactivation process is dependent on the presence of oxygen and is antagonized by chelation of metal ions, that catalyze sulphite autoxidation, or by scavenging free radicals with mannitol or benzoate. Since the presence of superoxide dismutase and/or catalase protects the enzyme only to a small extent, the inactivation process should be attributed to sulphite radicals rather than intermediates of oxygen reduction.     

Thermally perturbed rhodanese can be protected from inactivation by self-association

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30–40°C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 µg/ml is inactivated at 40°C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 µg/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30–40°C. Light scattering kinetics at 40°C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions: at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Gluteraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40°C but not at 23°;C. Precipitates formed at 40°C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.     

A study of dithiothreitol inactivation of the enzyme rhodanese.

When air oxidized, partially inactivated rhodanese (EC 2.8.1.1) is treated with dithiothreitol (DTT) to regenerate the reduced essential sulfhydryl group there is an initial reactivation followed by an anomalous slower inactivation. Fully active enzyme shows only inactivation. The inactivated enzyme may be completely reactivated on long incubation with the substrate thiosulfate ion. None of the normal potentialities of DTT appear to be responsible for the inactivation. The results are interpreted in terms of disulfide formation between DTT and an essential enzymic sulfhydryl group with the resulting complex being stabilized by secondary interactions which are particularly favorable due to similarities between DTT and lipoic acid--a normal sulfur acceptor substrate.     

The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase

The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.     

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.     

Differential binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid to rhodanese catalytic intermediates

Studies have been performed to quantitate the binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid (2,8-ANS) to catalytic intermediates of the enzyme rhodanese: the sulfur-substituted form (ES) and the sulfur-free form (E). The molecular 2,8-ANS has not been extensively used for protein studies, and some characterization is presented to demonstrate its usefulness as a probe for apolar binding sites. The molecule 2,8-ANS binds to at least two classes of sites on rhodanese. One class (class 1) is present in the ES form and has a Kd of 1.7 mM. The E form of rhodanese appears to have a second class of sites (class 2) in addition to the class 1 sites. Two independent fluorometric methods of analyzing the class 2 binding of 2,8-ANS to the E form gave an average value for Kd congruent to 179 microM. These fluorometric titrations, together with a Job plot, clearly indicate that 2,8-ANS binds to more than one site on the E form of rhodanese. The apparent apolarity is slightly higher for class 2 sites than for the class 1 sites, but both give Z factors of greater than 85. The substrate thiosulfate is able to displace the probe that is bound to the class 2 sites on the E form of the enzyme. Further, 2,8-ANS is found to be a competitive inhibitor of the catalyzed reaction with an apparent Kd of 170 microM. Circular dichroism measurements detect no significant changes in the average conformation of rhodanese that can be ascribed to the presence of 2,8-ANS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.