Observations on the Bisulfite Reductase (P582) Isolated from Desulfotomaculum nigrificans

The bisulfite reductase (P582) from Desulfotomaculum nigrificans was purified to homogeneity as judged by polyacrylamide gel electrophoresis. By colorimetric methods of analysis, the products of bisulfite reduction by this enzyme were determined to be trithionate, thiosulfate, and sulfide. Of these, trithionate was consistently found to be the major product, whereas the latter two were formed in lesser quantities. When [(35)S]bisulfite was incorporated as substrate, no labeled sulfide was detected. Furthermore, when trithionate and thiosulfate were isolated from reaction mixtures and chemically degraded, (35)S was found in all three sulfur atoms of trithionate; however, only the inner sulfur atom of thiosulfate was radioactive. From these data we conclude that the bisulfite reductase of D. nigrificans reduces bisulfite to trithionate and that thiosulfate and sulfide are endogenous side products of the reaction.     

Product analysis of bisulfite reductase activity isolated from Desulfovibrio vulgaris.

Bisulfite reductase was purified from extracts of Desulfovibrio vulgaris. By colorimetric analyses trithionate was found to be the major product, being formed in quantities 5 to 10 times more than two other detectable products, thiosulfate and sulfide. When [35S]bisulfite was used as the substrate, all three products were radioactively labeled. Degradation of [35S]trithionate showed that all of its sulfur atoms were equally labeled. In contrast, [35S]thiosulfate contained virtually all of the radioactivity in the sulfonate atom while the sulfane atom was unlabeled. These results, in conjunction with the funding that the sulfide was radioactive, led to the conclusion that bisulfite reductase reduced bisulfite to trithionate as the major product and sulfide as the minor product; the reason for the unusual labeling pattern found in the thiosulfate molecule was not apparent at this time. When bisulfite reductase was incubated with [35S]bisulfite in the presence of another protein fraction, FII, the thiosulfate formed from this reaction contained both sulfur atoms having equal radioactivity. This discovery, plus the fact that trithionate was not reduced to thiosulfate under identical conditions, led to the speculation that bisulfite could be reduced to thiosulfate by another pathway not involving trithionate.     

Purification of a unique bisulfite-reducing enzyme from Desulfovibrio vulgaris

An enzyme which catalyzes the reduction of bisulfite to sulfide and thiosulfate was purified from extracts of the sulfate-reducing bacterium, $\text{Desulfovibrio vulgaris}$. Trithionate was not a product of this reaction nor was it or thiosulfate reduced by the enzyme. High substrate concentrations inhibited sulfide but not thiosulfate formation. The enzyme was named bisulfite reductase II to distinguish it from bisulfite reductase which reduces bisulfite to trithionate.     

Reduction of bisulfite by the trithionate pathway by cell extracts from Desulfotomaculum nigrificans

Bisulfite was reduced to sulfide by cell extracts of Desulfotomaculum nigrificans. When trithionate was added to reaction mixtures reducing bisulfite, sulfide formation was inhibited with accumulation of thiosulfate. The thiosulfate reductase activity of cell extracts was found to be inhibited by trithionate. Trithionate alone was reduced to thiosulfate and purified bisulfite reductase (P582) was not affected by trithionate. It is concluded that the pathway for bisulfite reduction in Dt. nigrificans includes trithionate and thiosulfate as intermediate compounds.     

Dissimilatory reduction of bisulfite by Desulfovibrio vulgaris.

The reduction of bisulfite by Desulfovibrio vulgaris was investigated. Crude extracts reduced bisulfite to sulfide without the formation (detection) of any intermediates such as trithionate or thiosulfate. When the particulate fractions was removed from crude extracts by high-speed centrifugation, the soluble supernatant fraction reduced bisulfite sequentially to trithionate, thiosulfate, and sulfide. Addition of particles or purified membranes to the soluble fraction restored the original activity demonstrated by crude extracts, i.e., reduction of bisulfite to sulfide without the formation of trithionate and/or thiosulfate. By using antiserum directed against bisulfite reductase, the reduction of bisulfite by crude extracts was inhibited. This finding, in addition to several recycling studies of thiosulfate reduction, provided evidence that bisulfite reduction by D. vulgaris operated through the pathway involving trithionate and thiosulfate as intermediates. The role of membranes in this process is discussed.     

Characterization of a trithionate reductase system from Desulfovibrio vulgaris.

A trithionate reductase system was isolated and purified from extracts of Desulfovibrio vulgaris. This system reduced trithionate to thiosulfate and consisted of two proteins. One was bisulfite reductase, an enzyme that reduces bisulfite to trithionate, and the second component was designated TR-1. Both enzymes were required to reduce trithionate to thiosulfate. Flavodoxin and cytochrome c3 from D. vulgaris were tested for their ability to function as electron carriers during trithionate reduction. When molecular hydrogen was the source of electrons for the reduction, both flavodoxin and cytochrome c3 were required. In contrast, when the pyruvate phosphoroclastic system was the reductant, flavodoxin alone participated as the electron carrier. The results indicate that flavodoxin, but not cytochrome c3, interacted with the trithionate reductase system. The cytochrome in the hydrogenase-linked assay functioned as an electron carrier between hydrogenase and flavodoxin.     

Bisulfite reductase of Desulfovibrio vulgaris: explanation for product formation.

Bisulfite reductase, purified from Desulfovibrio vulgaris, was coupled with the pyruvate phosphoroclastic reaction. Moderate to low reducing conditions resulted in the formation of trithionate; however, when the concentration of reductant was high, a mixture of trithionate and thiosulfate was formed. Sulfide was also a detectable product, but only when the concentration of bisulfite was low. Flavodoxin mediated native coupling between bisulfite reductase and the phosphoroclastic reaction. A model for bisulfite reductase activity is proposed.     

Isolation of a bisulfite reductase activity from Desulfotomaculum nigrificans and its identification as the carbon monoxide-binding pigment P582

Crude preparations of Desulfotomaculum nigrificans were found to reduce bisulfite to trithionate, thiosulfate, and sulfide. The bisulfite reductase of this organism was partially purified and observed to reduce bisulfite to trithionate as the major product and with thiosulfate and sulfide as minor products. The enzyme exhibited spectral properties identical to the carbon monoxide-reacting pigment (P582) isolated from this organism. It is concluded that the bisulfite reductase of D. nigrificans is P582 and that this organism utilizes a pathway which involves trithionate during the reduction of bisulfite to sulfide.     

Formation of Thiosulfate from Sulfite by Desulfovibrio vulgaris

Crude extracts of Desulfovibrio vulgaris reduced sulfite to sulfide. Ammonium sulfate fractionation of crude extracts separated a thiosulfate-forming system from sulfite- and thiosulfate-reductase activities. Further purification by sucrose density centrifugation separated the thiosulfate-forming system into two components, both of which were required for the reaction. In addition to these two components, cytochrome c₃, ferredoxin, and hydrogenase were required to form thiosulfate from sulfite. By absorption spectra and from the effect of pH and substrate concentration, the ionic species acting as the substrate for thiosulfate-formation was concluded to be bisulfite.     

Formation of thiosulfate and trithionate during sulfite reduction by washed cells of Desulfovibrio desulfuricans

Deenergized cells of Desulfovibrio desulfuricans strain Essex 6 formed trithionate and thiosulfate during reduction of sulfite with H₂ or formate. The required conditions were pretreatment with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), low concentration of the electron donor H₂ or formate (25–200 M) and the presence of sulfite in excess (>250 M). The cells formed up to 20 M thiosulfate, and variable amounts of trithionate (0–9 M) and sulfide (0–62 M). Tetrathionate was not produced. Sulfate could not replace sulfite in these experiments, as deenergized cells cannot activate sulfate. However, up to 5 M thiosulfate was produced by cells growing with H₂ and excess sulfate in a chemostat. Micromolar concentrations of trithionate were incompletely reduced to thiosulfate and sulfide by washed cells in the presence of CCCP. Millimolar trithionate concentrations blocked the formation of sulfide, even in the absence of CCCP, and caused thiosulfate accumulation; sulfide formation from sulfate, sulfite or thiosulfate was stopped, too. Trithionate reduction with H₂ in the presence of thiocyanate was coupled to respiration-driven proton translocation (extrapolated H⁺/H₂ ratios of 1.5±0.6). Up to 150 M trithionate was formed by washed cells during oxidation of sulfite plus thiosulfate with ferricyanide as electron acceptor (reversed trithionate reductase activity). Cell breakage resulted in drastic decrease of sulfide formation. Cell-free extract reduced sulfite incompletely to trithionate, thiosulfate, and sulfide. Thiosulfate was reduced stoichiometrically to sulfite and sulfide (thiosulfate reductase activity). The formation of sulfide from sulfite, thiosulfate or trithionate by cell-free extract was blocked by methyl viologen, leading to increased production of thiosulfate plus trithionate from sulfite, or increased thiosulfate formation from trithionate. Our study demonstrates for the first time the formation of intermediates during sulfite reduction with whole cells of a sulfate-reducing bacterium oxidizing physiological electron donors. All results are in accordance with the trithionate pathway of sulfite reduction.With gratitude dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

Formation of thionates by freshwater and marine strains of sulfate-reducing bacteria

The formation of thionates (thiosulfate, trithionate and tetrahionate) during the reduction of sulfate or sulfite was studied with four marine and four freshwater strains of sulfate-reducing bacteria. Growing cultures of two strains of the freshwater species Desulfovibrio desulfuricans formed up to 400 M thiosulfate and 100 M trithionate under conditions of electron donor limitation. Tetrathionate was observed in lower concentrations of up to 30 M. Uncoupler-treated washed cells of the four freshwater strains formed thiosulfate and trithionate at low electron donor concentrations with sulfite in excess. In contrast, only one of four marine strains formed thionates. The freshwater strain Desulfobulbus propionicus transformed sulfite almost completely to thiosulfate and trithionate. The amounts produced increased with time, concentration of added sulfite and cell density. Tetrathionate was detected only occasionally and in low concentrations, and was probably formed by chemical oxidation of thiosulfate. The results confirm the diversity of the sulfite reduction pathways in sulfate-reducing bacteria, and suggest that thiosulfate and trithionate are normal by-products of sulfate reduction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone     

Studies on Biochemistry of the Thiobacilli

In the oxidation of thiosulfate at pH 4.5 tetrathionate was formed as an intermediate, and the thiosulfate-oxidizing enzyme was active in acidic pH range in contrast to the enzyme of T. thioparus and Thiobacillus X.

Phosphate did not seem to affect the oxidation of thiosulfate but rather affect the conversion of tetrathionate. In the absence of phosphate, tetrathionate, which was produced from thiosulfate oxidation, seemed to accumulate without undergoing further conversion.

Quantitative oxidation of tetrathionate to sulfate was achieved with freshly harvested cells of T. thiooxidans; pH optimum for the oxidation of tetrathionate by the washed cells was 2~3, and the activity fell markedly at pH above 3.5.

Tetrathionate might be enzymatically dismuted to pentathionate and trithionate under anaerobic conditions with crude extracts of T. thiooxidans; pH optimum for the reaction was about 2.7 and the activity fell strikingly at pH 4.7. The formed trithionate might be further hydrolyzed to thiosulfate and sulfate.     

Thiosulfate reduction in Salmonella enterica is driven by the proton motive force

Thiosulfate respiration in Salmonella enterica serovar Typhimurium is catalyzed by the membrane-bound enzyme thiosulfate reductase. Experiments with quinone biosynthesis mutants show that menaquinol is the sole electron donor to thiosulfate reductase. However, the reduction of thiosulfate by menaquinol is highly endergonic under standard conditions (ΔE°' = -328 mV). Thiosulfate reductase activity was found to depend on the proton motive force (PMF) across the cytoplasmic membrane. A structural model for thiosulfate reductase suggests that the PMF drives endergonic electron flow within the enzyme by a reverse loop mechanism. Thiosulfate reductase was able to catalyze the combined oxidation of sulfide and sulfite to thiosulfate in a reverse of the physiological reaction. In contrast to the forward reaction the exergonic thiosulfate-forming reaction was PMF independent. Electron transfer from formate to thiosulfate in whole cells occurs predominantly by intraspecies hydrogen transfer.     

Purification and properties of thiosulfate reductase from Desulfovibrio vulgaris, Miyazaki F

Thiosulfate reductase was purified to an almost homogeneous state from Desulfovibrio vulgaris, strain Miyazaki F, by ammonium sulfate precipitation, chromatography on DEAE-Toyopearl, Ultrogel AcA 34, and hydroxylapatite, and disc electrophoresis. The specific activity was increased 580-fold over the crude extract. The molecular weight was determined by gel filtration to be 85,000-89,000, differing from those reported for thiosulfate reductases from other Desulfovibrio strains. The enzyme had no subunit structure. When coupled with hydrogenase and methyl viologen, it stoichiometrically reduced thiosulfate to sulfite and sulfide with consumption of hydrogen. It did not reduce sulfite or trithionate. Cytochrome c3 was active as an electron donor. More than 0.75 mM thiosulfate inhibited the enzyme activity. o-Phenanthroline and 2,2'-bipyridine inhibited the enzyme and ferrous ion stimulated the reaction.     

Studies on Biochemistry of the Thiobacilli

The evidence for the reductive cleavage of trithionate to thiosulfate and sulfite in the presence of some reduced agents is presented with washed cells of T. thiooxidans.

The optimal pH upon the reaction was about 7.0.

α-Glycerophosphate was effective as an electron donor for the reduction, and it was suggested that reduced glutathione might be required as a co-factor for the reduction.     

Metabolism of sulfate-reducing prokaryotes

Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 °C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO₂. The most detailed knowledge is available on the metabolism ofDesulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.     

Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction?

The fate of 35-S during anaerobic metabolism of [35-S]sulfate, [35-S]thiosulfate, and [35-S]sulfate plus unlabeled thiosulfate by washed cell suspensions of Desulfovibrio spp, and of [35-S]thiosulfate by growing D. desulfuricans was examined. The results appear to be inconsistent with the hypothesis that thiosulfate is an intermediate in sulfate reduction. Since thiosulfate was produced from trithionate, the latter is also unlikely to be an intermediate in the reduction pathway. Extracts of D. desulfuricans catalysed exchange between sulfite and the sulfonate group of thiosulfate.     

Deletion of flavoredoxin gene in Desulfovibrio gigas reveals its participation in thiosulfate reduction

The gene encoding Desulfovibrio gigas flavoredoxin was deleted to elucidate its physiological role in the sulfate metabolism. Disruption of flr gene strongly inhibited the reduction of thiosulfate and exhibited a reduced growth in the presence of sulfite with lactate as electron donor. The growth with sulfate was not however affected by the lack of this protein. Additionally, flr mutant cells revealed a decrease of about 50% in the H2 consumption rate using thiosulfate as electron acceptor. Altogether, our results show in vivo that during sulfite respiration, trithionate and thiosulfate are produced and that flavoredoxin is specific for thiosulfate reduction.     

Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.     

Studies of sulfate utilization by algae. 5. Identification of thiosulfate as a major Acid-volatile product formed by a cell-free sulfate-reducing system from chlorella

Separation of the products formed from sulfate-³⁵S by cell-free extracts of Chlorella pyrenoidosa (Emerson Strain 3) has permitted the identification of thiosulfate as a major product which yields acid-volatile radioactivity. The products formed, as separated by Dowex-1-nitrate chromatography, are qualitatively the same whether extracts at pH 7.0 (using TPNH as the reductant) or extracts at pH 9 [using 2,3-dimercaptopropan-1-ol, (BAL) as reductant] are employed. While thiosulfate can be separated without the addition of carrier, the inclusion of carrier improves the recovery. High concentrations of ATP which have been shown previously to inhibit the formation of acid-volatile radioactivity from radioactive sulfate, inhibit the formation of thiosulfate almost completely. Degradation of the thiosulfate formed at normal ATP concentrations reveals that most of the radioactivity is in the SO₃-sulfur of the molecule suggesting that the SH-sulfur is derived from the enzyme extracts. If carrier sulfite is present during thiosulfate formation from sulfate-³⁵S, radioactive sulfite is recovered at the expense of radioactive thiosulfate. Reconstruction experiments utilizing specifically-labeled thiosulfates indicate that radioactive sulfite formation is probably not the result of trapping a normal intermediate, but can be attributed to non-enzymatic exchange between labeled thiosulfate formed from sulfate and the non-radioactive sulfite added, suggesting that free sulfite is not an intermediate in thiosulfate formation from sulfate.