J. Biochem., 75, 519-529 (1974) Biochemical Studies on Sulfate-reducing Bacteria . Sulfite Reductase from Desulfovibrio vulgaris-Mechanism of Trithionate, Thiosulfate, and Sulfide Formation and Enzymatic Properties Kunihiko KOBAYASHI, Yasuhide SEKI, and Makoto ISHIMOTO Department of Chemical Microbiology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo Received for publication, September 10, 1973 The reaction of sulfite reductase [EC 1. 8. 99. 1] from Desulfovibrio vulgaris was investigated using a purified enzyme preparation in a system coupled with methyl viologen and hydrogenase [EC 1.12.2.1]. Trithionate, thiosulfate, and sulfide were detected even in the early phase of sulfite reduction and the amount of each compound did not decrease during the reaction or after hydrogen uptake ceased. The specific radioactivity of sulfide formed from 35S-labelled sulfite was scarcely reduced on adding cold trithionate and only slightly on adding cold thiosulfate. These results, in addition to the fact that trithionate and thiosulfate are not reduced by the enzyme, indicate that these three compounds are produced by sulfite reductase. At high concentrations of sulfite and low concentrations of methyl viologen or hydrogenase, trithionate was the dominant product. Under the opposite conditions, the formation of relatively large amounts of sulfide or thiosulfate was observed. On the basis of these findings, a mechanism was proposed for the reaction, including labile intermediates, presumably sulfoxylate and elemental sulfur, which accept electrons from reduced methyl viologen to form sulfur and sulfide or react with sulfite to produce trithionate and thiosulfate, respectively. Some enzymatic properties were examined. K,,, was 3.6•~10-3M for sulfite. The optimum pH was 5.5 to 6.0. The enzyme was partially inhibited by arsenite at concentrations of 10-2 to 10-4 M. It could reduce hydroxylamine, nitrite, and trimethylamine N-oxide, but not nitrate, chlorate, cyanide, azide, adenosine N-oxide, or taurine. Disc electrophoresis and enzyme staining using oxidation of reduced methyl viologen with acceptors in polyacrylamide gel revealed that desulfoviridin had reducing activities not only for sulfite but also for hydroxylamine and nitrite. It was previously observed by Kobayashi et al. mediary formation of trithionate and thiosul (1) that sulfite reduction in the extract of fate. A stepwise reduction of sulfite involving Desulfovibrio vulgaris proceeds with inter- sulfite reductase, trithionate reductase (Eq. 1), Vol. 75, No. 3, 1974 519 XIII 520 K. KOBAYASHI, Y. SEKI, and M. ISHIMOTO and thiosulfate reductase (Eq. 2) was proposed. (6) was also used, where indicated. Sodium trithionate was synthesized ac- cording to Willstatter (7). Methyl viologen was a product of British Drug Houses. The sulfite reductase reaction was carried out in Warburg vessels under a hydrogen Sulfite reductase was recently purified atmosphere in the presence of hydrogenase from D. gigas (2) and D. vulgaris (3) and and methyl viologen (3). The standard mix was identified as desulfoviridin. Lee and Peck ture contained 0.5 unit of hydrogenase, 1 ƒÊmole (2) reported that the enzyme from D. gigas of methyl viologen, 10 ƒÊmoles of sodium bi produces only trithionate from sulfite, while sulfite, and enzyme in 1.0ml of 0.08M phos Skyring and Trudinger (4) showed, by en phate buffer, pH 6.0. The center well con zyme location in polyacrylamide gel after tained 0.1ml of 20% KOH absorbed in fluted disc electrophoresis of D. gigas extract, that filter paper. The reaction was started by the desulfoviridin band forms sulfide in sulfite tipping sulfite from the side arm into the main reduction. Kobayashi et al. (3) detected not compartment. Hydrogen uptake was followed only trithionate but also thiosulfate and sulfide manometrically at 30•Ž. as the products of sulfite reduction with Assays of sulfur compounds were per- purified D. vulgaris sulfite reductase, which formed as follows. Hydrogen sulfide absorbed had no ability to reduce trithionate or thio in the alkaline filter paper in the center well sulfate. of the Warburg vessel was determined accord- Further investigation of the sulfite reduc ing to St. Lorant (8). Trithionate and thio tase reaction was intended to ascertain that sulfate were determined by a cyanolysis a single enzyme produced the three com method, as described previously (3). Tetra pounds in sulfite reduction and to clarify the thionate was also determined, but the amount mechanism of formation of these compounds. was always negligible within the limit of ex We have found that the distribution of sulfur perimental error. Sulfite was determined by into these products varies according to the reac the p-rosaniline method (9) with a slight tion conditions, and a branched-chain scheme modification ; an aliquot (0.1 ml) was trans including two labile intermediates (3) is pro ferred to 0.9 ml of 0.1M Na2HgCl4 and, after posed. Enzyme activity for nitrogenous sub 30-fold dilution, subjected to color develop strates and the effects of inhibitors are also ment. shown. The theoretical amount of hydrogen uptake required for the formation of the three MATERIALS AND METHODS products from sulfite was calculated using the formula, Sulfite reductase [EC 1.8.99.1] was purified from Desulfovibrio vulgaris as previously de scribed (3). Preparations obtained by hydro where parentheses represent the amounts in xylapatite chromatography or repeated DEAE- moles of the compounds produced, according cellulose chromatography were used as purified to the following equations, enzyme. These preparations contained small amounts of a minor green protein (see Fig. 9A), but were not able to reduce trithionate or thiosulfate. Hydrogenase [EC 1.12.2.1] was solubilized from particulate fractions of D. vulgaris and was partially purified accord The distribution of sulfur in products was ing to Yagi (5). The preparation was not expressed as a percentage for each product able to reduce sulfite, thiosulfate, and trithio (g atoms of sulfur) of the total amount of nate. Particulate hydrogenase preparation sulfur in all the products. J. Biochem. SULFATE-REDUCING BACTERIA. XIII 521 Measurement of the specific activity of uptake in the presence of purified hydrogenase sulfide formed from 35S-labelled sulfite was (5) and methyl viologen under hydrogen. carried out using sulfide absorbed by the Trithionate and thiosulfate in the reaction alkaline filter paper in the center well of the mixture were determined, as well as hydrogen vessel (10). [35S]-Sodium bisulfite was pre sulfide absorbed in the alkali in the center pared from [35S]-sulfuric acid by reduction well of the vessels. with copper. The concentration of sulfite was As shown in Fig. 1, trithionate, thiosul adjusted after iodimetric titration of an aliquot. fate, and sulfide were deteced even at the After the enzymatic reduction, sulfide was beginning of the reaction ; trithionate was the converted to methylene blue with dimethyl-p- major product among the three under these phenylenediamine (8), followed by colorimetric conditions. None of the three products de determination. Methylene blue was then creased during the reaction or changed in adsorbed on activated charcoal (8mg for 11ml amount after hydrogen uptake ceased (Fig. of the colored solution). After washing with 1A) ; trithionate and thiosulfate formed were water on a filter paper, radioactivity on the not further reduced to produce thiosulfate charcoal was measured with a GM counter. and sulfide. These results are in agreement In experiments for acceptor specificity, with the fact that trithionate and thiosulfate hydrogen uptake was followed under the same did not serve as substrates of the enzyme conditions as for sulfite reduction except for (3). the acceptor. These results indicate that trithionate, To locate reductase activities on polyacryl thiosulfate, and sulfide are the terminal, not amide gels after disc electrophoresis, substrate- intermediary, products of sulfite reductase, dependent oxidation of reduced methyl vio and that neither trithionate reductase nor thio logen (11, 12) was applied. A partially sulfate reductase takes part in thiosulfate and sulfide formation. purified preparation of sulfite reductase The decrease in the relative amount of (DEAE-cellulose fraction) was subjected to electrophoresis on polyacrylamide gel (5•~50 trithionate among the products during hy mM) at pH 9 (13 ). After electrophoresis for drogen uptake (Fig. 1B) would be due to the 90 min, gels were transferred to a test tube decrease in sulfite concentration (see below). (7mM in diameter) which contained 5ml of 0.08M Tris-HC1 buffer, pH 7.0, containing 2 mM methyl viologen and 10 mg of sodium dithionite. Approximately 10 mg of either NH2OH • HCl, NaNO2, or NaHSO3 was added to the solution. After several minutes incu bation at room temperature (about 30•Ž), the gels were placed on a glass plate under air and colorless bands in the blue gels appeared. Protein was determined by the method of Lowry et al. (14) or by measuring absorb ance at 280 mp. The factor 0.6 mg protein/ ml for 1 unit of absorbance was used. Fig. 1. Time course of product formation in sulfite RESULTS reduction. Reaction was carried out under the standard conditions except for the amount of sulfite 1. Formation of Trithionate, Thiosulfate (5 ƒÊmoles). Hydroxylapatite fraction (1mg of pro and Sulfide by Sulfite Reductase-Time course tein) was used as the enzyme preparation. A) Hy of product formation in sulfite reduction