Are Thiosulfate and Trithionate Intermediates in Dissimilatory

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1 JOURNAL OF BACTERIOLOGY, JUIY 1975, P Copyright American Society for Microbiology Vol. 123, No. 1 Printed in U.SA. Are Thiosulfate and Trithionate Intermediates in Dissimilatory Sulfate Reduction? L. A. CHAMBERS* MD P. A. TRUDINGER Baas Becking Geobiological Laboratory, P. 0. Box 378, Canberra City, A. C. T., 2601, Australia Received for publication 7 March 1975 The fate of 35S during anaerobic metabolism of [35S]sulfate, [35S Ithiosulfate, and [35S ]sulfate plus unlabeled thiosulfate by washed cell suspensions of Desulfovibrio spp. and of [35SIthiosulfate 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. The reduction of sulfite to sulfide is an intermediary stage in dissimilatory sulfate reduction by Desulfovibrio spp. (19). The mechanism of sulfite reduction, however, is not clear. Thiosulfate has been reported to be an intermediate product during sulfite reduction by cellfree extracts of Desulfovibrio vulgaris (4, 5, 14, 22) and Desulfotomaculum nigrificans (1), and the separation of a thiosulfate-forming enzyme from extracts of D. vulgaris has been described (22). Thiosulfate reductases have been demonstrated in D. desulfuricans (7), D. nigrificans (20), and D. vulgaris (6). Kobayashi et al. (14) prepared a fraction from extracts of D. vulgaris which reduced sulfite sequentially to trithionate and thiosulfate. A second fraction, containing trithionate and thiosulfate reductases, was required for complete reduction of sulfite to sulfide. They proposed the following sequence of reactions and suggested that three separate enzymes were involved. 3SO32- - S362- S2032-5s2- This proposal gained support from reports that trithionate was the major or sole product of sulfite reduction by purified bisulfite reductases from D. gigas (18) and D. nigrificans (1, 2). On the other hand, Kobayashi et al. (13) and Jones and Skyring (10; H. E. Jones and G. W. Skyring, Biochim. Biophys. Acta, in press) reported that sulfide, as well as trithionate and thiosulfate, S032- S032- was produced by preparations of sulfite reductases from D. vulgaris and D. gigas, respectively, even though the enzyme preparations had no trithionate or thiosulfate reductase activity (9). The relative proportions of the products varied according to the concentrations of sulfite and electron donor employed. It was suggested (13) that thiosulfate and trithionate are formed by chemical combination of sulfite with labile intermediates between sulfite and sulfide. Akagi et al. (2) challenged this suggestion. They reported that the enzyme preparation, rather than sulfite, was apparently the source of the sulfide and the sulfane atoms of thiosulfate which were produced in small amounts during [3"S ]sulfide reduction by bisulfite reductase from D. nigrificans. However, this would appear not to apply in the experiments of Kobayashi et al. (13) where yields of sulfide plus sulfane sulfur (in thiosulfate) were up to 44% (wt/wt) of the amounts of enzyme used. On the basis of relative rates of thiosulfate and sulfate reduction by washed cells of D. vulgaris (D. desulfuricans strain Hildenborough), Postgate (21) concluded that thiosulfate was not an intermediate between sulfate and sulfide. This paper reports results with D. desulfuricans and D. gigas which appear to support the view that neither thiosulfate nor trithionate is a normal intermediate in the reduction pathway. MATERIALS AND METHODS Organism and growth conditions. D. desulfuricans NCIB 8380 and D. gigas NCIB 9332 were grown for 48 h from 10% (vol/vol) inocula in 5-liter conical flasks completely filled with Medium B as described by Jones (8). Culture purity was checked by microscope examination and by plating on growth medium supplemented with 2% agar, 0.05% ferrous ammonium sulfate, and 0.01% 2-mercaptoethanol; these were incubated aerobically and anaerobically at 35 C. For experiments with growth on 35S-labeled thiosul- 36

2 VOi. 123, 1975 THIOSULFATE AND TRITHIONATE INTERMEDIATES 37 fate, the medium was modified by substituting MgCl,.6H20 for MgSO4.7H20 and by omitting Na2SO4; [35SNa2S20 and lactate were added as specified in Table 2. Inocula for these experiments were grown in sulfate-free thiosulfate media. Preparation of washed cells. Organisms were harvested by centrifugation, washed twice with 0.1 M potassium phosphate buffer (ph 6.8), and resuspended in the same buffer to 10 ml. Dry weight was estimated on 1 ml of the suspension after drying overnight at 105 C. Preparation of cell-free extract. Cells of D. desulfuricans were suspended in 0.1 M potassium phosphate buffer (ph 6.8) and passed twice through a French pressure cell at 20,000 lb/in2. The extract was centrifuged at 10,000 x g for 30 min and the supernate was dialyzed overnight against the same buffer. Incubations. Anaerobic incubations with washed and growing organisms were performed in Thunberg tubes. Substrates were placed in the side arm and the tube was evacuated and refilled with purified oxygenfree nitrogen (25). This was done six times before mixing and incubation under partial vacuum at 36 C. Sulfide was collected by evacuation through 5,5'- dithiobis(2-nitrobenzoic acid) in 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, ph 8, and its concentration was determined by the absorption at 412 nm. One molecule of sulfide and one molecule of 5,5'-dithiobis(2-nitrobenzoic) react to give two molecules of thionitrobenzoate with a molar absorption coefficient at 412 nm of 13,600 (3, 16). With washed-cell experiments the reaction was stopped by addition of ethanol to approximately 50% (vol/vol) before collection of sulfide. Chromatography. Sulfate and thiosulfate were separated by using ion-exchange resin Bio-Rad AG 1 x 2 as described previously (23). Analysis of thiosulfate and trithionate. Thiosulfate and trithionate were estimated as described previously (11). Degradation of radioactive thiosulfate. The sulfane and sulfonate atoms were separated as described by Kelly and Syrett (12). The precipitated Ag2S (from sulfane-s) was subsequently oxidized by Br2 and HNO3. Radioactive counting. Counting was done in a Packard, model 2003, Tri-Carb scintillation spectrometer with an overall efficiency of 56%. The scintillation fluid contained: xylene, 385 ml; dioxane, 385 ml; ethanol, 230 ml; 2,5-diphenyloxazole, 5 g; dimethyl 1,4-bis-(5-phenyloxazolyl)benzene, 50 mg; naphthalene, 80 g. Reagents. Radiochemicals were obtained from the Radiochemical Centre, Amersham, England. K,S,O0 was prepared as described by Trudinger (24). RESULTS Products of sulfate reduction. Washed-cell suspensions of D. desulfuricans were incubated anaerobically with Na235SO, in the presence of lactate. At various stages of the reaction sulfide was removed by evacuation and the reaction mixtures were analyzed by paper chromatography. In no instance were radioactive products other than sulfide detected, suggesting that there were no major pools of intermediates. Reduction of 35S-labeled thiosulfate. The formation of sulfide from sulfate- and sulfonate-labeled Na235S 203 by washed suspensions of D. desulfuricans and D. gigas is shown in Table 1. In each instance, the specific activity of sulfide was close to half that of the original thiosulfate. Similar results were obtained with growing D. desulfuricans (Table 2). A small amount of doubly labeled thiosulfate was formed from sulfonate-labeled thiosulfate. For example, in the experiment with D. desulfuricans (Table 1) the specific activity of the sulfane atom of the residual thiosulfate was about 2% that of sulfide. Incorporation of 35S from [35S]sulfate into thiosulfate. Labeled sulfur was incorporated into thiosulfate when washed cells of D. desulfuricans were incubated with Na235SO, and Na2S2O3 (Table 3). Over 90% of the incorporated 35S was in the sulfonate group, and the ratio of specific activity of the sulfide to that of the sulfane group was 34. Isotopic exchange between sulfite and thiosulfate. A dialyzed cell-free extract of D. desulfuricans (10 mg of protein/ml) was incubated anaerobically at 35 C with 33 mm sulfonate-labeled Na235S203 and 33 mm Na2SO3 in the absence of electron donors. The reaction was stopped by the addition of excess I,, and the sulfate and tetrathionate, formed by oxidation of sulfite and thiosulfate, respectively, were separated by ion exchange. The presence of 35S in sulfate indicated exchange between sulfite and the sulfonate group of thiosulfate. At 1.25 h the extent of exchange was 33% of the theoretical maximum. Reduction of trithionate. Trithionate was reduced by washed-cell suspensions of D. desulfuricans. A major product, apart from sulfide, was thiosulfate (Table 4). DISCUSSION The complete transformation of thiosulfate to sulfide is believed to proceed by two stages: (i) reductive scission of thiosulfate to sulfide and sulfite S,Os2- + 2e- _ S2- + SO,2- (1) and (ii) reduction of sulfite (see references 19, 20) Ḃy the use of sulfane- and sulfonate-labeled [35S ]thiosulfate, Findley and Akagi (5) observed that, in extracts of D. vulgaris, sulfide was produced at a greater rate from the sulfane

3 38 CHAMBERS AND TRUDINGER J. BACTERIOL. TABLE 1. -Specific activity of sulfide produced during anaerobic incubation of 38S-labeled thiosulfate by sulfate-reducing bacteriaa Initial S,O,2- Incubation S2- S2- sp act Organism Atom labeled sp act (counts/min time produced (counts/min (i)m,ml Reduction prjml per,umol)pe m) D. desulfuricans Sulfonatec 1.1 x x 105 (37 mgb) D. desulfuricans Sulfane 1.1 x x 104 (41 mgb) x x 104 D. gigas (28 mgb) Sulfonate 1.0 x x 105 Sulfane 1.0 x 10' x 10' athe incubation system contained: organisms; 250 umol of potassium phosphate (ph 6.8); 200 MAmol of sodium lactate; and 100,umol of sodium thiosulfate in a final volume of 4 ml. bdry weight. c The specific activity of the sulfane atom of residual thiosulfate was 1.07 x 103 (counts/min per umol). T TABLE 2. Specific activity of sulfide during growth ofd desulfuricans on "S-labeled thiosulfatea I Initial thiosulfate Sulfide produced Atom Growth Growth labeled period (h) (A/g[dry /ml) wt (Mmol) Amt (counts/min Sp act (Mmol) Amt % Sp act Reduction (counts/min per ;Lmol) per gmol) Sulfane x x 104 Sulfonate x x 104 athe media, containing M lactate and 3'S-labeled Na,S,O,, were inoculated with 39 'gg of D. desulfuricans per ml. TABLE 3. Distribution of 35S during incubation of D. desulfuricans with 35SO2- and unlabeled thiosulfatea Determination Amt (counts/m in per,mmol) S 2- produced x 103 Remaining S20' x 10' Sulfane 0.25 x 103 Sulfonate 3.1 x 103 athe incubation mixture contained 38 mg (dry weight) of organisms, 100 umol of Na2 35SOs (specific activity 36.3 x 103 counts/min per Mmol), and 50 smol of Na2S20. Incubation, 30 min. Other conditions as for Table 1. Overall recovery of 35S was 89%. atom than from the sulfonate group, and that doubly labeled thiosulfate was formed from sulfonate-labeled thiosulfate. They concluded that this was evidence for the intermediate formation of thiosulfate in the sulfite reduction pathway. By contrast, in the present experiments the TABLE 4. Reduction of trithionate by D. desulfuricansa Incubation S2- S,0,2- S,02- Recovery time produced produced utilized of S (min) (pmol) ('*mol) (jlmol) (%) athe incubation system contained: 200 jamol of potassium phosphate (ph 6.8); 33 umol of potassium trithionate; 200,umol of sodium lactate; and 75 mg (dry weight) of organisms in a final volume of 4 ml. sulfane and sulfonate groups of thiosulfate were reduced to sulfide by both washed and growing cells at approximately equal rates (Tables 1, 2). If thiosulfate were an intermediate in sulfite reduction this would imply that there was no exchange between intracellular and extracellular thiosulfate. The experiment described in Table 3, however, demonstrates that intact cells readily catalysed the incorporation of 35S into extracellular thiosulfate when incubated with

4 VOL. 123, 1975 THIOSULFATE AND TRITHIONATE INTERMEDIATES 39 35SO2- and unlabeled thiosulfate. Over 90% of the incorporated 85S was located in the sulfonate group, suggesting that incorporation was the result of exchange between this group and sulfite (or an intermediate at the equivalent oxidation state). In the reaction sequence produced by Kobayashi et al. (14) the immediate precursor of sulfide is the sulfane atom of thiosulfate. During reduction of sulfonate-labeled thiosulfate, or of [I3S ]sulfate in the presence of unlabeled thiosulfate, the specific activity of accumulated sulfide should be less than 50% that of the sulfane atom of residual thiosulfate. In the experiments described in Tables 1 and 3, however, the ratios of specific activities of sulfide and sulfane atom of residual thiosulfate were 51 and 34, respectively. We conclude, therefore, that the present results are not consistent with the hypothesis that thiosulfate is a normal intermediate in the dissimilatory sulfate reduction pathway. The small incorporation of HS from 35SO2- or from the sulfonate group of thiosulfate into the sulfane atom of thiosulfate (Table 3) might be due to exchange with an intermediate at the oxidation level of elemental sulfur. Transient formation of the latter in sulfite reduction has been suggested (13). Because thiosulfate is a product of trithionate reduction (Table 4), it would seem that trithionate is probably not an intermediate between sulfite and sulfide. The results reported in this paper could be accounted for if sulfite reduction in washed and growing cells were largely due to the assimilatory sulfite reductase reported to be present in a number of Desulfovibrio species (18). This enzyme catalyses the reduction of sulfite to sulfide without the formation of detectable intermediates. Should this explanation be correct, however, it would raise an important question as to the function of the trithionate-forming bisulfite reductase which, at least in D. vulgaris, accounts for more than 90% of the total sulfite reductase activity in crude extracts (18). An alternative explanation is that bisulfite reductase is the major sulfite reducing system in the cell and that, as suggested by Kobayashi et al. (13), trithionate and thiosulfate are by-products of the cell-free system. ACKNOWLEDGMENTS We gratefully acknowledge the skilled technical support of A. J. Rutter. The Baas Becking Geobiological Laboratory is supported by the Bureau of Mineral Resources, the Commonwealth Scientific and Industrial Research Organization, and the Australian Mineral Industries Research Association Limited. LITERATURE CITED 1. Akagi, J. M., and V. Adams Isolation of bisulfite reductase activity from Desulfotomaculum nigrificans and its identification as the carbon monoxide-binding pigment P582. J. Bacteriol. 116: Akagi, J. M., M. Chan, and V. Adams Observations on the bisulfite reductase (P582) isolated from Desulfotomaculum nigrificans. J. Bacteriol. 120: Ellman, G. L Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: Findley, J. E., and J. M. Akagi Evidence for thiosulfate formation during sulfite reduction by Desulfovibrio vulgaris. Biochem. Biophys. Res. Commun. 36: Findley, J. E., and J. M. Akagi Role of thiosulfate in bisulfite reduction as catalyzed by Desulfovibrio vulgaris. J. Bacteriol. 103: Haschke, R. H., and L. L. Campbell Thiosulfate reductase of Desulfovibrio vulgaris. J. Bacteriol. 106: Ishimoto, M., and J. Koyama Biochemical studies on sulfate-reducing bacteria. VI. Separation of hydrogenase and thiosulfate reductase and partial purification of cytochrome and green pigment. J. Biochem. 44: Jones, H. E Sulfate-reducing bacterium with unusual morphology and pigment content. J. Bacteriol. 106: Jones, H. E., and G. W. Skyring Reduction of sulphite to sulphide catalysed by desulfoviridin from Desulfovibrio gigas. Aust. J. Biol. Sci. 27: Jones, H. E., and G. W. Skyring Assay conditions and sulfite reduction catalysed by desulfoviridin from Desulfovibrio gigas. Proc. Aust. Biochem. Soc. 7: Kelly, D. P., L. A. Chambers, and P. A. Trudinger Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate. Anal. Chem. 41: Kelly, D. P., and P. J. Syrett [3S ]thiosulphate oxidation by Thiobacillus Strain C. Biochem. J. 98: Kobayashi, K., Y. Seki, and M. Ishimoto Biochemical studies on sulfate-reducing bacteria. XIII. Sulfite reductase from Desulfovibrio vulgaris-mechanism of trithionate, thiosulfate, and sulfite formation and enzymatic properties. J. Biochem. 75: Kobayashi, K., S. Tachibana, and M. Ishimoto Intermediary formation of trithionate in sulfite reduction by a sulfate-reducing bacterium. J. Biochem. 65: Kobayashi, K., E. Takahashi, and M. Ishimoto Biochemical studies on sulfate-reducing bacteria. XI. Purification and some properties of sulfite reductase, desulfoviridin. J. Biochem. 72: Kredich, N. M., and G. M. Tomkins The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J. Biol. Chem. 241: Lee, J-P., J. LeGall, and H. D. Peck, Jr Isolation of assimilatory- and dissimilatory-type sulfite reductases from Desulfovibrio vulgaris. J. Bacteriol. 115: Lee, J-P., and H. D. Peck, Jr Purification of the enzyme reducing bisulfite to trithionate from Desulfovibrio gigas and its identification as desulfoviridin. Biochem. Biophys. Res. Commun. 45: LeGall, J., and J. R. Postgate The physiology of sulphate-reducing bacteria. Adv. Microbiol. Physiol.

5 40 CHAMBERS AND TRUDINGER 10: Nakatsukasa, W., and J. M. Akagi Thiosulfate reductase isolated from Desulfotomaculum nigrificans. J. Bacteriol. 98: Postgate, J. R The reduction on sulphur compounds by Desulfovibrio desulphuricans. J. Gen. Microbiol. 5: Suh, B., and J. M. Akagi Formation of thiosulfate from sulfite by Desulfovibrio vulgaris. J. Bacteriol. J. BACTERIOL. 99: Trudinger, P. A Products of anaerobic metabolism of tetrathionate by Thiobacillus X. Aust. J. Biol. Sci. 17: Trudinger, P. A The metabolism of trithionate by Thiobacillu X. Aust. J. Biol. Sci. 17: Trudinger, P. A On the absorbancy of reduced methyl viologen. Anal. Biochem. 36: