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 : drogen uptake and product formation. B) Distribu
Sulfite reduction catalyzed by purified sulfite tion of S in products.„Ÿ, hydrogen uptake ; • , reductase was followed by measuring hydrogen trithionate ;•¢, thiosulfate ; •›, sulfide.
Vol. 75, No. 3, 1974 522 K. KOBAYASHI, Y. SEKI, and M. ISHIMOTO
Sulfite was recovered as trithonat, thio presence and absence of trithionate or thio sulfate, and sulfide (Table I). The theoretical sulfate and the amount and the radioactivity value for the total amount of hydrogen re of hydrogen sulfide formed were determined quired for the formation of the three products (Table II). from sulfite was practically equal to the ob The specific radioactivity of sulfide was served value in most cases. only slightly reduced by the addition of cold Effect of trithionate and thiosulfate on trithionate. If all the sulfite molecules were sulfide formation from 35S-labelled sulfite by reduced by sulfite reductase to produce trithio sulfite reductase: Sulfite labelled with 35S was nate which, in turn, were further reduced to reduced by purified sulfite reductase in the produce sulfide, [35S]-trithionate formed from
TABLE I. Stoichiometry of sulfite reduction. The reaction mixture contained sulfite reductase (hydroxyl apatite fraction, 0.2 mg protein), solubilized hydrogenase (1.2 unit), 2 ƒÊmoles of methyl viologen, 80 ƒÊmoles of phosphate buffer, pH 6.2, and 10 ƒÊmoles of NaHSO3 in a total volume of 1.0 ml. Other conditions and assay methods are described in "MATERIALS AND METHODS."
Corrected for zero time value.
TABLE II. Effect of trithionate and thiosulfate on the specific radioactivity of sulfide formed from 35S- labelled sulfite. Reactions were carried out under the standard conditions. Particulate hydrogenase was employed.
J. Biochem. SULFATE-REDUCING BACTERIA. XIII 523
[35S]-sulfite would be diluted at least 3- or 5- the same amount (5 ƒÊmoles) of sulfite was fold by the addition of 2- or 4-fold equivalent added in all vessels. (g atom sulfur) of cold trithionate, respectively, Trithionate was a dominant product in and this would result in a similar decrease in the presence of lower concentrations of methyl the specific activity of sulfide formed. This viologen (Fig. 2B). At higher concentrations was not the case. of methyl viologen, however, a larger propor In the case of thiosulfate, a partial dilu tion of the sulfur was found in both thiosul tion was observed, but the extent of dilution fate and sulfide. The recovery of sulfur was (below 2-fold) was not as large as would be over 80% in all cases and the total amount expected on the basis of an assumption similar of hydrogen uptake calculated from the de to the above. tected amounts of products accounted for the It is thus untenable to suggest that hy observed amount of hydrogen uptake. drogen sulfide is formed from sulfite through At higher concentrations of hydrogenase, trithionate and/or thiosulfate. The apparent hydrogen uptake increased (Fig. 3A) with the dilution by thiosulfate can be accounted for increase in the relative amount of thiosulfate by sulfur exchange between thiosulfate and and the decrease in that of trithionate (Fig. sulfite or between thiosulfate and sulfide (15 ). 3B). The observed hydrogen uptake corre 2. Alteration of Product Composition sponded to the amount calculated from the under Various Condions-Effect of concentra amounts of products. tion of methyl viologen and hydrogenase on These results indicate that composition sulfite reduction : Figure 2A shows the effect of the products is altered by the activity of of concentration of methyl viologen on hy the electron-donating system. At lower con drogen uptake. Changes in methyl viologen centrations of electron donor, trithionate is a concentration altered not only the rate of hy major product of sulfite reduction, and, at drogen uptake for sulfite reduction but also higher concentrations of donor, more sulfur the amount of hydrogen consumed. The is found in a more reduced state, i.e., sulfide higher the concentration of methyl viologen, or thiosulfate. the more hydrogen was consumed, although Effect of sulfite concentration on sulfite
Fig. 2. Effect of methyl viologen concentration on Fig. 3. Effect of hydrogenase concentration on
sulfite reduction. Reaction conditions were same sulfite reduction. The reaction was carried out as in the experiment in Fig. 1, except for the con under the standard conditions except for the amount centration of methyl viologen. A) Time course of of solubilized hydrogenase (9.5 units/mg protein).
hydrogen uptake for sulfite reduction in the pres DEAE-cellulose fraction (2.2 mg of protein) was ence of the indicated concentration of methyl used as the enzyme preparation. A) Time course viologen (mM). B) Product formation during 60 min of hydrogen uptake for sulfite reduction in the pres reaction in the presence of various concentrations ence of the indicated amount of hydrogenase (units).
of methyl viologen. „Ÿ, hydrogen uptake; • , tri B) Products formed during 60 min in the presence thionate; •¢, thiosulfate; •›, sulfide ; •~ , total of various amounts of hydrogenase. The symbols sulfur. are same as in Fig. 2.
Vol. 75, No. 3, 1974 524 K. KOBAYASHI, Y. SEKI, and M. ISHIMOTO
reduction : Figure 4 shows time courses of major product, but the relative amounts of hydrogen uptake for the reduction of varying sulfide and thiosulfate increased slightly when amounts of sulfite at constant concentrations the initial concentration of sulfite was lowered. of methyl viologen and hydrogenase. Reduc Sulfite concentration has an effect opposite to tion of sulfite was terminated before 240 min. those of methyl viologen and hydrogenase, Hydrogen uptake accelerated somewhat with though it is not distinct, as the products were time in each case. The average velocity assayed after the consumption of sulfite. Al during the overall reaction was higher at teration in product composition during the higher initial concentrations of sulfite. From reaction, as shown in Fig. 1B, may be a re- a double reciprocal plot of initial concentra flection of the change in sulfite concentration. tion of sulfite and average velocity of hydrogen 3. Some Enzymatic Properties of Sulfite uptake (Fig. 5), the K,, value was obtained Reductase- Effect of pH on activity: As as 3.6 •~ 10-1 M for sulfite. shown in Fig. 6, the optimum pH of the sul Table III shows the result of product anal fite reductase lies between 5.5 and 6.0, in ysis after the reaction. Trithionate was the contrast to thiosulfate reductase and trithio nate reductase, both of which have optimum pH's at about 7.5 to 8.0 and have only small activities at pH's below 6.0 (unpublished data). The ionic species of sulfite as a substrate of sulfite reductase is perhaps HS03- rather than 5032 or S2052- (16, 17).
Fig. 4. Time course of hydrogen uptake for reduc tion of various amounts of sulfite. The reaction mixture contained 2 ƒÊmoles of methyl viologen,
particulate hydrogenase (0.5 unit) and hydroxyl apatite fraction as the sulfite reductase preparation. Fig. 5. Double reciprocal plot for sulfite reduction. The amounts of sulfite added (ƒÊmoles) are indicated. Experiments were the same as in Fig. 4.
TABLE III. Effect of sulfite concentration on the product composition of sulfite reductase. The experi ment was the same as in Fig. 4. Reaction time was 240 min.
J. Biochem. SULFATE-REDUCING BACTERIA . XIII 525
TABLE V. Acceptor specificity of sulfite reductase. The assay method was the same as that for the manometric assay for sulfite reductase except for electron acceptor at the indicated concentration. Hydroxylapatite fraction (0.2 mg of protein) was used as the enzyme preparation.
Fig. 6. Effect of pH on sulfite reduction. Sulfite reduction was conducted under the standard condi tions except for pH and buffer. Sodium acetate buffer (•œ) and sodium phosphate buffer (•›) were used at 80mM. T Hydrogen uptake did not occur in the absence of the enzyme. TABLE ‡W. Effect of enzyme inhibitors on sulfite reductase activity. Sulfite reductase activity was assayed by hydrogen uptake under standard condi hydrogenase activity but to that of sulfite tions in the presence of inhibitors. Mean values of reductase. from 2 to 4 experiments were indicated. No marked inhibition was observed in the presence of other reagents, as in the crude enzyme (18 ). Cyanide, a potent inhibitor of assimilatory-type sulfite reductase [EC 1. 8. 1. 2] (19 ), slightly stimulated the activity, and, in the presence of this compound, almost same amounts of hydrogen sulfide, trithionate, and thiosulfate were formed during sulfite reduc tion as in its absence. Addition of a suspension of cadmium car bonate (4 ƒÊmoles in 1ml), prepared from cad mium sulfate and sodium carbonate, had little effect on hydrogen uptake or on the forma Inhibition of sulfite reduction : The effect tion of trithionate and thiosulfate. In this of various enzyme inhibitors on the activity case, no hydrogen sulfide was detected in the of sulfite reductase was studied by measuring alkali in the center well, but the formation the rate of hydrogen uptake (Table ‡W). of yellow cadmium sulfide was observed in Among the inhibitors tested, only sodium the reaction mixture. arsenite showed a partial inhibition of sulfite Acceptor specificity: Sulfite reductase does reduction at 10-2 to 10-4 M. In the presence not reduce trithionate or thiosulfate (3). of 10-2 M arsenite, methyl viologen was in the Other compounds were tested as substrates of reduced state during the reaction, showing a the enzyme in place of sulfite coupled with blue color, so the depression of sulfite reduc the hydrogenase-methyl viologen system tion by arsenite was not due to inhibition of (Table V).
Vol. 75, No. 3, 1974 526 K. KOBAYASHI, Y. SEKI, and M. ISHIMOTO
Hydroxylamine was reduced several times reduction catalyzed by the sulfite reductase more rapidly than sulfite by the purified en was about 7.5 (Fig. 8). Hydroxylamine reduc zyme. Slow reduction was observed when tion was also partially inhibited by arsenite trimethylamine N-oxide was added as a sub to much the same extent as sulfite reduction.. strate. Hydrogen uptake was not caused by Location of reductase activity on electro the following compounds ; sodium nitrate, phoresis gel: The ability of the sulfite reduc potassium chlorate, potassium cyanide, sodium tase to reduce hydroxylamine and nitrite was azide, adenosine N-oxide, and taurine. confirmed by location of these activities in The addition of sodium nitrite caused polyacrylamide gel after disc electrophoresis bleaching of reduced methyl viologen without (Fig. 9). Two green bands of desulfoviridin, hydrogen uptake, so the nitrite reduction ac one intense and the other faint, were visible tivity was not determined by this method. in gels after electrophoresis (Fig. 9B). Gels In the assay system with dithionite-reduced were incubated for several minutes in a blue methyl viologen as the electron donor (20), solution of dithionite-reduced methyl viologen decrease of nitrite was stimulated by the en containing hydroxylamine, nitrite, or sulfite, zyme. The sulfite reductase seems to reduce then taken from the solution and held in air nitrite (see below). at room temperature. Bleaching due to oxi Reduction of hydroxylamine: As shown dation. of the reduced methyl viologen was in Fig. 7, reduction of hydroxylamine by the observed in a few minutes on the blue gel purified sulfite reductase terminated with the containing hydroxylamine at a position iden consumption of one mole of hydrogen per tical to that of the major band of desulfovi mole of hydroxylamine, indicating that hydro ridin (Fig. 9C). The colorless band spread xylamine was reduced to ammonia. The wider over a period of several minutes and reduction was not observed in the absence of two green bands of desulfoviridin were ob the enzyme, nor in the presence of boiled served in the middle of the colorless zone enzyme. The optimum pH of hydroxylamine (Fig. 9D). Enzyme activity reducing hydro xylamine with methyl viologen seems to be present not only in the major but also in the minor desulfoviridin bands. Formation of a - colorless band at the position of desulfoviridin also occurred in the gel containing nitrite
Fig. 7. Hydroxylamine reduction by sulfite reduc tase. The reduction of hydroxylamine (5 ƒÊmoles) was carried out as described in Table V. Hydrogen uptake was measured manometrically. •›, complete system ; •~, sulfite reductase omitted ; •œ, sulfite Fig. 8. Effect of pH on hydroxylamine reduction. reductase boiled for 5 min. Hydroxylapatite fraction Hydrogen uptake was measured manometrically at
(0.21 mg of protein, 23.3 munits of sulfite reductase) the indicated pH in Macllvaine's citrate-phosphate was used as the enzyme preparation. buffer.
J. Biochem. SULFATE-REDUCING BACTERIA. XIII 527
DISCUSSION
In the previous paper of this series (3), it was shown that purified sulfite reductase from D. vulgaris forms three compounds, trithio nate, thiosulfate, and sulfide, from sulfite, though no ability to reduce trithionate with formation of thiosulfate or to reduce thiosulfate with formation of sulfide was found. The addi tion of trithionate or thiosulfate to the reac tion mixture had no effect on the rate of hydrogen uptake for sulfite reduction or on the amounts of products (3). The results pre sented in this communication indicate that the Fig. 9. Disc electrophoresis and location of hydro three compounds are formed from the begin xylamine, nitrite, and sulfite-reducing activities on ning of sulfite reduction, and that the amount polyacrylamide gel. Experimental details are de of each compound, once formed, does not de scribed in the text. A) Protein bands of purified crease during or after hydrogen consumption, sulfite reductase stained with Amido black 10B. in contrast to the observation on transient ac B) Native desulfoviridin visible on the gel without staining. C-F) Enzyme location on gels. Each gel cumulation of trithionate and thiosulfate with was soaked in a solution containing reduced methyl a crude enzyme preparation of the bacterium viologen and electron acceptor, and exposed in air (1). These results support the previous con for the indicated time. C) NH20H.HCl, 5min, D) clusion (3) that all three compounds are the NH2OH. HCl, 20 min, E) NaNO2, 5 min, and F) products of sulfite reductase. The possibility NaHSO3, 30 min. The arrows indicate the two of intermediary formation of trithionate or bands, major and minor, of desulfoviridin. The thiosulfate in the sulfide formation by sulfite position of bromophenol blue is also indicated (BPB). reductase was excluded by the finding that the radioactivity of sulfide from [35S]-sulfite was (Fig. 9E), slightly later than in the gel con not greatly diluted by cold trithionate or thio taining hydroxylamine. Slight fading of sulfate. reduced methyl viologen occurred throughout Increase in the relative amount of trithio the gel in the presence of nitrite in one hour, nate at higher concentrations of sulfite and while bleaching of the gels due to autoxida increase in those of sulfide or thiosulfate in tion took place from the periphery. Colorless the presence of higher concentrations of elec band formation was observed at the position tron donor lead to the hypothesis that a labile of the desulfoviridin band in the gel contain intermediate is formed and converted either ing sulfite after about 30 min exposure to air to trithionate or to sulfide and thiosulfate, (Fig. 9F), but was not observed in the ab depending on the sulfite concentration and sence of added acceptor after prolonged ex electron-donating power. As a mechanism posure for more than an hour. for the formation of these compounds by sul These results show that the sulfite reduc fite reductase, the following scheme is pro tase itself has catalytic activity for reducing posed, as suggested in the previous paper hydroxylamine and nitrite with reduced (3) without evidence. methyl viologen. It was not clear whether the minor desulfoviridin band had catalytic activity for reducing nitrite and sulfite in these experiments. The difference from hy droxylamine reduction may be due to the relatively low activities of the reductase for sulfite or nitrite in addition to the small amounts of material in the minor band.
Vol. 75, No. 3, 1974 528 K. KOBAYASHI, Y. SEKI, and M. ISHIMOTO
Sulfite is reduced by sulfite reductase to pro the ability to reduce hydroxylamine and nitrite duce an intermediate X, which then either (Table V) has been ascertained by location of combines with two molecules of sulfite to these activities in gels after disc electropho produce trithionate or is reduced to form an resis of the purified preparation (Fig. 9). other intermediate Y. Y is, in turn, reduced Coincidence of the band showing sulfide to form sulfide or combines with sulfite to forming activity from sulfite and that of de produce thiosulfate. Cytochrome c3 may func sulfoviridin was also demonstrated by Skyring tion as an electron donor for sulfite reductase and Trudinger with crude extract of D. gigas under physiological conditions (3). Inter- (4). mediates X and Y may be, though they can Activity for the catalysis of hydroxyl not be definitely identified, sulfoxylate (SO22-) amine reduction to ammonia with reduced and elemental sulfur, respectively. Sulfoxy methyl viologen was also reported for the late, which would be formed as a labile com- cytochrome c3 of the bacterium as well as for pound from sulfur dichloride or diethyl sulf some other heme compounds (23, 25). As oxylate in aqueous solution, combines with cytochrome , c$ is a basic protein and is elimi sulfite to form trithionate (21, 22). It is nated in the enzyme purification procedure, known that elemental sulfur combines with the hydroxylamine-reducing activity of sulfite sulfite to give thiosulfate. In addition, it was reductase is independent of that of cytochrome. found that colloidal sulfur is reduced readily C3. by reduced methyl viologen or cytochrome c2 Desulfovibrio sulfite reductase is distingu (23). ished from sulfite reductases from sulfate In crude extracts, trithionate and thio assimilating organisms (19). They reduce sulfate thus accumulated from sulfite by sulfite sulfite with 3 moles of NADH or 6 electrons reductase are reduced to sulfide in the pres to produce only sulfide. They have pH optima ence of both trithionate reductase and thio at 7 and are inhibited by cyanide. It is inter sulfate reductase and thus sulfite is stoichio esting that, in spite of the distinct properties metrically reduced with 6 electrons to sulfide mentioned above, the ability to reduce hydro (24). xylamine or nitrite and absorption at about There may be another possible pathway 600 mp are shared by sulfite reductases from to produce the three compounds by reduction various sulfate-reducers (this communication of sulfite. Sulfite might be reduced solely to and Ref. 2, 3, 26, 27) and sulfate-assimilating sulfide by sulfite reductase and this might organisms (19). react with remaining sulfite to produce poly Sulfite reduction with crude extract of thionates and thiosulfate. In fact, sulfide and Micrococcus lactilyticus was reported by Wool sulfite react each other at pH 6, forming folk (28) to produce thiosulfate under hy mainly thiosulfate in the absence of enzyme drogen at acidic pH. As the enzyme has not (unpublished data). This possibility, however, been purified, it is rather difficult to make seems to be unlikely under the conditions for any comparison with Desulfovibrio sulfite the enzymatic reaction, where sulfide evapo reductase. rated from the reaction mixture at pH 6 into alkali in the center well. The elimination of The authors are grateful to Mrs. M. Takahara, Mr. sulfide would be accomplished by the addition E. Takahashi, and Mr. K. Kubota for help in some of cadmium carbonate to the reaction mixture ; parts of this investigation. This work was partly in fact this addition had little effect on trithio supported by a Scientific Research Grant from the Ministry of Education of Japan. nate and thiosulfate formation or on hydrogen consumption. This finding indicates that the REFERENCES formation of these compounds is independent 1. K. Kobayashi, S. Tachibana, and M. Ishimoto, of sulfide and excludes the possibility of inter J. Biochem., 65, 155 (1969). mediary formation of sulfide. 2. J.P. Lee and H.D. Peck, Jr., Biochem. Biophys. The fact that sulfite reductase itself has Res. Commun., 45, 583 (1971).
J. Biochem. SULFATE-REDUCING BACTERIA. XIII 529
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