Mechanistm of Hydrogen Sulfide Formation Fromi Thiosulfate1 Akira Kaji2 and W

Home , Thiosulfate

MECHANISTM OF HYDROGEN SULFIDE FORMATION FROMI THIOSULFATE1 AKIRA KAJI2 AND W. D. McELROY McCollrnt-Pratt Institute and Department of Biology, Johns Hopkins University, Baltimore, Maryland Received for publication October 17, 1958 Sulfidle formation by the reduction of sulfuir temperature to remove sulfite and thiosulfate compounds has been observed in several micro- ions. A pale yellow supernatant fluid, obtained organisms (Butlin, 1956). The earlier observation after removal of elemental sulfur by centrifuga- by Neuberg and Welde (1914) that yeast can tion, did not contain sulfite or thiosulfate as produce hydrogen sulfide from thiosulfate has tested by fuchsin and iodometric titration, re- been studied in detail by Tanner (1918). Al- spectively. The amount of sulfur in this solution though the reduction of thiosulfate by extracts was measured gravimetrically after the water from Escherichia coli (Artman, 1956) an(d )e- had been evaporated ip vacuo at room tempera- sulphovibrio desulphuricans (Ishimoto and Ko- ture. yama, 1957; Postgate, 1956) has been reported, Preparation of enzyme. Fresh Fleishmann the enzymatic mechanism of hydrogen sulfide l)aker's yeast was dried in air for about two weeks formation has so far remained obscure. at room temperature. One hundred grams of the The present work has shown that a mechanism dry yeast were suspended in 250 ml of 1 M dibasic of hydrogen sulfide production by an enzyme sodium phosphate solution, and kept at room preparation from yeast is dependent up0on the temperature for 3 hr with occasional stirring. The interaction of thiosulfate with sulfhydryl com- suspension was then centrifuged, the pellet re- pounds. The products of this enzymatic process extracted with 100 ml of 1 M dibasic sodium are hycdrogen sulfide, sulfite, and oxidized suilf- phosphate and both extracts combined. hydryls. The significance of the reaction in the In order to eliminate soluble cofactors, the biological synthesis of cysteine is discussed. extract was saturated with ammonium sulfate, the pH was adjusted to 7.0 by 0.7 M ammonium MATERIALS AND METHODS hydroxide and the resulting precipitate, after centrifugation, was dissolved in 0.2 M phosphate Coenzyrnes and other materials. Glutatblione buffer (pH 7.0). The protein was precipitated a (GSH), sodium isocitrate, and glucose-6- plos- second time by saturated ammonium sulfate phate were purchased from Schwartz Company. and after dissolving in phosphate buffer was Baker reagent grade sodium thiosulfate was used. dialyzed against 0.05 M phosphate buffer, pH Homocysteine and eysteine were obtained from 7.0, at 0 C for 24 hr. This fraction I had a specific General Biochemical Company and reduced tri- activity of 12.5. The enzyme responsible for phosphopyridine inucleotide (TPNH), and para- catalyzing the direct reduction of thiosulfate was chloromercuribenzoate from the Sigma Chemical partially purified by ammonium sulfate fractiona- Company. tion of the crude preparation I. The preparation Colloidal sulfur was prepared by a mo(lifica- II obtained from the 30 to 60 per cent saturated tion of the method described by Chitanii (1951). ammonium sulfate fraction had a specific activity Seventy grams of concentrated sulfuric acid (spe- of 20.0. The specific activity of fraction I1 was cific gravity 1.84) was cooled to -10 C and 30 ml almost doubled by heating at 60 C for 5 min of a sodium thiosulfate solutioni containing 50 g (pH 7.0). After removing the precipitate by cen- of Na2S203 .5H20 was added. To this mixture, trifugation, the supernatant fraction III had a 30 ml of distilled water was added. After the specific activity of 37.0. reaction was completed, the mixture was dialyzed There was a 75 per cent loss of enzymatic against running tap water for 24 hr at room activitY after boiling preparation I for 5 min in 1 Supported in part by Office of Naval Research 2 M phosphate buffer, and complete loss of activ- and the Atomic Energy Commission. McColluin- ity after 30 min of boiling. Ashed residue of Pratt Institute Contribution No. 250. preparation I had no activity. 2 Daizian Foundation for Medical Research Hydrogen sulfide determination. Hydrogen sul- Fellow. fide was measured by the method described by 630 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 631

Delwiche (1951). Lead acetate dissolved in gum 1.4 arabic solution was added to reaction mixtures at various time intervals and the colloidal lead sulfide formed was meastured by the increase in 1.2 optical density at 490 m,, using a Beckman spee- trophotometer (model B). F 0 Sidlftte determination. Sulfite was determined by 0 decolorization of basic fuchsin solution, measured at 540 mnu. It was necessary to eliminate the 0.8 hydrogen sulfide formed coneurrently with the z sulfite, since it also decolorized the fuchsin. This LL 0.6 was accomplished by adding 4 ml of zinc carbonate slurry (Kurtenacker, 1927) to 6.5 ml of 04 the reaction mixture, followed by centrifugation. C-)o--J To 10 ml of distilled water were added 1 ml of 0~ the supernatant fluid, 1 ml of fuchsin solution 02 (10 mg of basic fuchsin in 100 ml H20), 0.5 ml of 1 M phosphate buffer (pH 7.2), and 1 ml of 02 parachloromercuribenzoate (10 umoles/ml). The final volume was adjusted to 25 ml with water. 30 60 90 The parachloromercuribenzoate was added to TIME (MIN) avoid slight fuchsin decolorization by gluta- Figure 1. Glutathione reductase and H2S forma- thione. A standard curve for fuchsin decolorization. The complete system contained 30 mg of tion by sulfite was made by adding known protein (fraction I), 5 MAmoles of oxidized gluta- amounts of sulfite to the enzyme reaction mixture thione, 0.2 mg of triphosphopyridine nucelotide lacking thiosulfate. Thiosulfate and sulfite solu- (TPN), 135 ,moles of thiosulfate, and TPN re- tions were standardized by iodometric titration ducing systems (30 ,moles of glucose-6-phosphate (Hawk et al., 1954). and 40,umoles of isocitrate) in 6.5 ml of 0.15 m Measurement of oxidized glutathione. Oxidized phosphate buffer (pH 7.1). 0 = Complete system; glutathione concentration was measured by * = systems where one of the above components is missing, and complete system with boiled reducing it with TPNH using the glutathione enzyme. Incubation was carried out at 37 C. An reductase present in fraction I. Utilization of optical density change of 0.5 corresponds to 2.00 TPNH was measured by the change in optical /Amoles of H2S produced in the reaction mixture. density at 340 m,u (Rall and Lehninger, 1952). The rate of reduced triphosphopyridine nucleo- sulfide from glutathione. The stopper was re- tide disappearance during the first 15 min is moved and 4 ml of lead acetate solution contain- linearly proportional to the amount of oxidized ing gum arabic were added. A unit of enzyme glutathione added to the reaction mixtures (be- activity is defined as the increase in optical tween 4 X 10-2 ,umoles and 20 X 10-2 ,moles of density of 0.01 at 490 mu after 30 min incubation. oxidized glutathione in 3 ml of reaction mixture). The specific activity is equal to optical density Assay of enzymatic activity. The following reac- change X 100 per mg protein. tion mixture was employed for thiosulfate reduction: To 5 ml of glutathione (100 ,umoles) were RESULTS added 0.5 ml of 2 M phosphate buffer (pH 7.0), Reaction between glutathione and thiosldfate. 1 ml of thiosulfate (270 ,umoles), and 0.2 ml of Preliminary experiments indicated that veast enzyme solution. The pH of the reaction mixture extract would catalyze a reaction betweein glu- was 6.9. The reaction was carried out aerobically tathione and thiosulfate to produce hydrogen at 35 C in a Thunberg tube. Hydrogen sulfide suilfide. This reaction may be analogous to the was absorbed by 2 ml of 2 M NaOH placed in the nonenzymatic reaction between cysteine and thio- upper chamber of the Thunberg tube. After sulfate to produce hydrogen sulfide (Steigmann, 30 min incubation, the NaOH solution in the 1945). Continuous hydrogen sulfide production upper chamber was tipped into the reaction inix- from thiosulfate will take place provided a sys- ture and the tube was placed in an ice-bath to tem is present which is capable of maintaining prevent nonenzymatic production of hydrogen glutathione. As shown in figurp 1, all of the com- 632 KAJI AND McELROY [VOL. 77

0.5

0.4 0

0 0~) >30.3 0 z LuJ 02

C) 0~~~~~~~~ 0.1

30 60 90 120 6 TIME (MIN) PROTEIN CONC.- MG Figure 2. Time course of hydrogen sulfide production by the reaction between thiosulfate and Figure S. Relationship between enzyme con- glutathione. The reaction mixture contained 100 centration and H2S production. The reaction mix- ,Amoles of glutathione, 180,umoles of thiosulfate, ture contained 100 ,umoles of glutathione, 270 of fraction in 6.7 ml of ,umoles of thiosulfate, and 12 mg of the enzyme and enzyme (12 mg II) in ml M M buffer Incubation was (fraction II) 6.7 of 0.15 phosphate buffer 0.15 phosphate (pH 6.8). was out at 35 C for carried out at 36 C. An optical density change of (pH 7.4). Incubation carried An of 0.5 corre- 0.5 corresponds to 2.00,umoles of H2S produced 30 min. optical density change to of in the in the reaction mixture. * = With enzyme; 0 = sponds 2.00 ,umoles H2S produced with partially inactivated enzyme (5 min 100 C); reaction mixture. * = with enzyme (fraction A\ = with completely inactivated enzyme (30 II); 0 = with partially inactivated enzyme min 100 C). (5 min, 100 C). ponents of the reaction mixture are necessary for The rate of hydrogen sulfide production was pro- hydrogen sulfide production. The complete reac- portional to the amount of protein added (figure tion mixture contained TPNH generating sys- 3). In addition, the effect of thiosulfate concen- tems, oxidized glutathione, thiosulfate, and yeast tration on the rate of hydrogen sulfide production enzyme. In any of the reaction tubes where one in the presence of 100 ,umoles of glutathione was of the above mentioned components was absent, studied, and the Michaelis-Menten constant there was no hydrogen sulfide production. (Kin) was calculated to be 6 X 103 M. The results in figure 2 demonstrate that the Identification of sulfite as a product. Ishimoto yeast enzyme enhances the rate of reaction and Koyama (1957) showed that during the pro- between glutathione and thiosulfate. The de- duction of hydrogen sulfide from thiosulfate, crease in hydrogen sulfide production after 90 min sulfite is concurrently produced. Since thiosul- incubation at 35 C can be attributed to the pres- fate at high temperature can react nonenzymati- ence of sulfide oxidase in the preparation cally with glutathione to produce hydrogen sul- (Ichihara and McElroy, unpublished data). The fide and sulfite, this reaction was studied in incubation of sulfide with the enzyme prepara- detail. The results in table 1 show that hydrogen tion resulted in the rapid disappearance of sulfide sulfide and sulfite are produced in almost equi- and the formation of thiosulfate. The inhibitory molar amounts. The results suggest that two action of sulfite which is produced in the reaction moles of glutathione react with one mole of thio- must also be considered as contributing to the sulfate to form one mole of sulfite, hydrogen sul- decrease in rate of hydrogen sulfide production. fide, and oxidized glutathione. 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 633

TABLE 1 Nonenzynmatic formation of sulfite and hydrogen sulfide from thiosulfate and glutathione at high temperatures 500

Temp S03- produced H2S Produced SO3-/H2S

C J,moles IAmoles 400 70 2.5 1.95 1.28 70 2.5 2.05 1.21 80 3.0 2.38 1.26 80 3.2 2.4 1.33 300 _ The reaction mixture contained 100 ,moles of glutathione, 270 jAmoles of S 03 in 6.7 ml of 0.15 M phosphate buffer (pH 6.9). 200 -

0.4

() 0 0.3 p- -4

D rC 2 3 4 5 6 7 8 9 10

I- m S . z =L 0.2

0 Figure 6. Competitive inhibition of thiosulfate (I) reduction by sulfite. Reaction mixture contained z (D 100,umoles of glutathione, 6 mg of enzyme (frac- 0 tion I), varied amount of thiosulfate ranging from 0.1 c-l (- 27,moles to 270,umoles, and sulfite in total volume 0. C) of 7.6 ml of 0.15 M phosphate buffer (pH 6.9). 1/S n 0 is 1/ml of the thiosulfate solution (250,umoles/ml) added to the reaction mixture. 1/V is 100 X 1/optical density change in 30 min. The incubation was carried out for 30 min at 35 C. 0 = 26.7 X 30 60 120 10-2 jumoles of S03= (3.5 X 105 M). 0 = 8.9 X TIME (MIN) 10-2 moles of SO3- (1.16 X 10- M). Figure 4. Time course of sulfite and H2S production by the enzymatic reaction between glu- fite produced during the reaction was larger tathione and thiosulfate. Reaction mixture con- than the apparent hydrogen sulfide production. tained 100 ,umoles of glutathione, 270 jumoles of This was due to the action of hydrogen sulfide thiosulfate, and 12 mg of protein (fraction II) in oxidase in the preparation as mentioned before. 6.7 ml of 0.15 M phosphate buffer. Final pH was 6.8. Incubation was carried out at 35 C. 0 = There was no disappearance of sulfite when it Increase of sulfite measured at 540 m, by fuchsin was incubated with the enzyme preparation. decolorization. An optical density change of 0.4 The incubation of the enzyme with either gluta- corresponds to 7.14,smoles of sulfite produced in thione or thiosulfate alone did not lead to the the reaction mixture. Under the conditions de- production of hydrogen sulfide. scribed in Methods the optical density of 5.8 was Site of enzyme action. Since hydrogen sulfide no sulfite is obtained for the blank where present. production can take place slowly without the O = Parallel increase of H2S. An optical density enzyme, the enhancement of its production from change of 0.5 corresponds to 2.00,moles of H2S the enzyme produced in the reaction mixture. glutathione and thiosulfate by could be a result of the catalytic removal of one of the Formation of sulfite was also demonstrated to products. This possibility was eliminated by the occur in the enzymatically catalyzed reaction. following experiments. Incubation of varied Figure 4 shows the time course of sulfite and amounts of oxidized glutathione (from 2 to hydrogen sulfide production. The amount of sul- 10 ,umoles) at 35 C for 30 min with 6 mg enzyme 634 KAJI AND McELROY [VOL. 77 in 7.6 ml of 0.15 M phosphate buffer (pH 6.9) did possibility remained that the enzyme catalyzes not result in a decrease of oxidized glutathione. a reaction between oxidized glutathione and Also, sulfite did not decrease on incubation with sulfite. Although the reaction between oxidized the enzyme. Moreover, it was found that sulfite glutathione and sulfite took place, the enzyme is not bound by the protein in a detectable did not increase the rate of the reaction. This amount (using the fuchsin decolorization nonenzymatic reaction is analogous to that be- method). From these results it can be concluded tween cystine and sulfite described by Clarke that the enzyme catalyzes the reaction between (1932). thiosulfate and glutathione directly. The further Studies of inhibitors. Artman (1956) showe(d that sulfite strongly inhibits hydrogen stulfide production from thiosulfate bv an extract of TABLE 2 E. co/i. Sulfite wvas also found to inhibit thie Effect of cysteine and homocysteine on H2S reaction catalyzed by the yeast extract. As showni production front thiosulfate by the Lineweaver and Burk plot (figure 5) the inhibitor competes with thiosulfate. Sulfite in Optical SH Compound S203 Enzyme DCehnsty concentrations up to 10-3 M did not have an at 490 mpA inhibitory effect on the nonenzymatic reaction between glutathione and thiosulfate at 75 C, ymoles mg pH 7.0, whereas 3.5 X 10-5 M S03- was inhibi- Homocysteine 6 0.02 tory to the enzymatic reaction. Homocysteine ...... 270 6 0.34 Parachloromercuribenzoate (1.4 X 10-3 M) was Homocysteine ...... 270 0 0.03 not inhibitory. Also, preincubation of the enzyme DL-Cysteine 0...... 6 0.01 with this concentration of parachloromercuri- DL-Cysteine ...... 270 6 0.30 DL-Cysteine ...... 270 0 0.15 benzoate for 15 min at room temperature, pH 7.0, followed by dialysis against phosphate buffer The rest of the reaction mixture is as given in (0.05 M, pH 7.0) for 24 hr at 0 to 5 C did not cause the text. An optical density change of 0.5 corre- loss of activity. Cyanide (1.4 X 10- M) at pll sponds to 2.00 ,umoles of H2S produced in the 6.8 caused only 17 per cent inhibition of the reaction mixture. reaction. Versene did not inhibit the reaction. 0.8-

0.7

O 0.6 -

0.5-

z 0.4 -

- 0.3 -

H0.2 0.0.1r

Figure 6. Effect of pH on the enzymatic and nonenzymatic production of H2S. The reaction mixture contained 100,umoles of glutathione, 270j,moles of thiosulfate, and 12 mg of protein (fraction I) in 6.7 ml of buffer solution. Acetate buffer (0.08 M), below pH 5.2; phosphate buffer (0.08 M), between pH 6.0 and 7.2; tris(hydroxymethyl)aminomethane buffer (0.08 M), between pH 7.2 and 9.0; and carbonate buffer (0.08 M), above pH 9.4, were used. The reaction was carried out at 35 C for 30 min. 0 = Enzy- matic reaction; * = nonenzymatic reaction. An optical density change of 0.5 corresponds to 2.00,moles of H2S produced in the reaction mixture. 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 635

Oxidized glutathione had a slight iiihibitory oxidase and a rapid nonenzymatic formation of effect on the nonenzymatic reaction occurriing at thiosulfate from hydrogen sulfide in the presence 80 C. At 10-3 M oxidized glutathione, a 40 per of sulfite, and oxidized glutathione, it was not cent depression in the rate of the reaction was possible to study quantitatively the effect of the observed. The effect of oxidized glutathione on enzyme on the reversibility of the reaction. the enzymatic reaction was of the same order of Sulfite ion competitively inhibits the enzy- magnitude, and therefore may be due to non- matic formation of sulfide. This is similar to its enzymatic reversal of the reaction. effect on rhodanese (Sorbo, 1951) and suggests En2ymatic specificity. In addition to gluita- the mechanism for the reduction of thiosulfate tione, homocysteine, and cysteine were foutnd to by an enzyme (ENZ) as shown in schema 1. react with thiosulfate to produce hydrogen sill- fide. The nonenzymatic reaction between cys- S S-S-SO3- teine and thiosulfate was much faster than that ENZ + S-SO3= -> ENZ between glutathione and thiosulfate (table 2 an(d figure 2). In this experiment each reactioni miiix- S ture containing 100 ,umoles of the sulfhydryl compound was incubated at 37 C, pH 6.9, for S-S-SO3- 30 min. Ascorbic acid was found to be unable to reduce thiosulfate even in the presence of thie ENZ + 2 gltitathione enzyme. Effect of hydrogen ion concentration. Enzymatic catalysis of thiosulfate reduction was maximum S at about pH 8.6 (figure 6). However, it is douibt- ful whether this is the true enzymatic pH opti- ENZ + oxidized glutathione mum because glutathione is oxidized spontaneously at high pH values, thus decreasing the S + H2S + S03=. amount of available substrate. Schema 1 Thiosulfate is spontaneously decomposed to sulfite and elemental sulfur at the lower pH. The strong inhibition by sulfite can be ex- Sulfur reacts spontaneously witlh glutathione to plained in a manner similar to that proposed by produce hydrogen sulfide. Because of this decom- Fridovich and Handler (1956) for sulfite oxida- position reaction at lower pH, the production of tion in which they propose that the initial step is hydrogein sulfide by glutathione and thiosulfate a binding of the sulfite to the disulfide group of is increased as shown in figure 6. lipoate. DISCUSSION In the nonenzymatic reaction between glutathione and thiosulfate two steps are suggested as Since glutathione is very abundant in yeast, shown in schema 2. it seems very likely that it serves as the redtietanit in the enzymatic formation of hydrogen sulfide S S03 -* S + S03= from thiosulfate. HowAever, other sulfhvdryl coIn- S + 2GSH - H2S + GSSG taining compounds such as cysteine and homocysteine will also function in the same capacity. Over-all: Systems capable of reducing oxidized gluta- 2GSH+S203 -> GSSG + S03= + H2S thione were found essential for the continuous reduction of thiosulfate to sulfide and sulfite. Schema 2 The coupling of yeast glutathione reductase to The increase in the rate of the nonenzymatic the TPNH-glucose-6-phosphate dehydrogenase formation of hydrogen sulfide at low pH implies and isocitrate dehydrogenase systems was found that the reaction is acid catalyzed. Assuming the to be satisfactory for this purpose. In the light of reduction of S203= does proceed in the manner this finding, the report of Artman (1956) that as shown in schema 2, a possibility that the en- pyruvate acts as the best hydrogen donor for zyme increases the rate of only one or the other sulfide production is understandable. of the steps was tested. On the assumption that Because of the presence of hydrogen sulfide colloidal sulfur could participate in the reaction 636 KAJI AND McELROY [VOL. 77 the effect of the enzyme on the spontaneous reac- ing hydrogen sulfide oxidase activity in the yeast tion between sulfur and glutathione was studied. extract. Varied amounts of colloidal sulfur were incubated with 100 j,moles of glutathione in the SUMMARY absence and the presence of fraction III at pH A crude enzyme preparation from yeast which 6.3. The enzyme did not increase the rate of catalyzes the formation of hydrogen sulfide from hydrogen sulfide production from colloidal sulfur thiosulfate and glutathione was studied. The and glutathione. protein fraction responsible for this catalysis If the reaction proceeds by these two steps, the has been purified nearly fourfold. Sulfite and sul- enzyme must catalyze the first reaction. However, fide are the equimolar products formed in the the incubation of thiosulfate and large amounts reaction. When the reaction is coupled to gluta- of enzyme under standard experimental condi- thione reductase, sulfide is produced continuously tions did not produce a detectable amount of from thiosulfate. Sulfite was found to be a com- free sulfite. petitive inhibitor of the enzymatic reaction. From these experimental data, it is suggested Thiosulfate could reverse this inhibition. A that yeast enzyme catalyzes hydrogen sulfide mechanism similar to that proposed for rhoda- production from thiosulfate and glutathione by nese has been postulated for this reaction. The enhancing the splitting of the S-S bond in physiological significance of this hydrogen sulfide thiosulfate only in the presence of SH-com- formation is discussed. pounds. It is possible that the SH-compound removes the elemental sulfur as it is formed on REFERENCES the surface of the enzyme. Ishimoto and Koyama (1957) and Ishimoto ARTMAN, M. 1956 The production of hiydro- et al. (1957) have suggested from studies on the gen sulfide from thiosulphate by E. coli. J. mechanism of thiosulfate reduction in bacteria, Gen. Microbiol., 14, 315-322. that molecular hydrogen, activated by hydro- BUTLIN, K. R. 1956 Formation enzymatiqiie for the de sulfure a partir de substrats mineraux par genase, was responsible reduction of cyto- les microorganismes. Colloque sur la bio- chrome C3 which in turn reduced thiosulfate by chemie du soufre. .ditions du Centre Na- means of an unknown intermediary carrier. It is tional de la Recherche Scientifique 13, Quai possible that some SH-compound like gluta- Anatole France, Paris. thione plays the role of this intermediary elec- CHITANI, T. 1951 Inorganic chemistry. Sang- tron carrier. However, from inhibitor studies no yo Tosho Co., Tokyo. evidence was obtained for participation of metal CLARKE, H. T. 1932 The action of sulfite ion in the reaction catalyzed by the yeast en- upon cystine. J. Biol. Chem., 97, 235-248. zyme. Concerning the biological significance of DELWICHE, E. A. 1951 Activators for the hydrogen sulfide production in yeast, the recent cysteine desulfhydrase system of an Escher- report by Schlossmann and Lynen (1957) that ichia coli. J. Bacteriol., 62, 717-722. FRIDOVICH, I. AND HANDLER, P. 1956 The cysteine is formed from hydrogen sulfide and initial step in enzymatic sulfite oxidation. L-serine in the presence of pyridoxal phosphate J. Biol. Chem., 223, 321-325. and an enzyme from yeast, is of considerable HAWK, P. B., OSER, B. L., AND SUMMERSON, interest. In view of these experiments, the split- W. H. 1954 Practical physiological chein- ting of thiosulfate, followed by reduction by istry, 13th ed. Blakiston Co., New York. some SH-compound in the cell, could play an ISHIMOTO, M. AND KOYAMA, J. 1957 Biochem- important role in over-all sulfur metabolism. ical studies on sulfate reducing bacteria. However, it is also possible, as suggested by VI. Separation of hydrogenase and thiosul- Shepherd (1956) that thiosulfate can be incor- fate reductase and partial purification of into sulfur before cytochrome and green pigment. J. Biochem. porated directly organic being (Tokyo), 44, 233. split, thus by-passing the stage of hydrogen ISHIMOTO, M., KOYAMA, J., YAGI, T., AND SHI- sulfide. RAKI, M. 1957 Biochemical studies on ACKNOWLEDGMENTS sulfate-reducing bacteria. VII. Purification of the cytochrome of sulfate reducing bac- The authors are greatly indebted to Dr. Ballen- teria and its physiological role. J. Biochem. tine for his interest and to Dr. Ichihara for assay- (Tokyo), 44, 413. 1959] HYDROGEN SULFIDE FORMATION FROM THIOSULFATE 637

KURTENACKER, Z. 1927 Zur iodometrischen SCHLOSSMANN, K. AND LYNEN, F. 1957 Bio- Analyse eines Gemenges von Sulfid, Sulfit, synthese des Cysteins aus Serin und Schwefel- und Thiosulfat. Z. anorg. u. allgem. Chem., wasserstoff. Bioch. Z., 438, 591-594. 161, 201-209. SHEPHERD, C. J. 1956 Pathways of cysteine NEUBERG, C. AND WELDE, E. 1914 Phyto- synthesis in Aspergillus nidulans. J. Gen. chemische Reduktionen IX Die Umwandelung Microbiol., 15, 29-38. von Thiosulfat in Schwefelwasserstoff und SORBO, B. 0. 1951 On the active group in Sulfit durch Hefen. Biochem. Z., 67, 111-118. rhodanese. Acta. Chem. Scand., 5, 1218- POSTGATE, J. R. 1956 Cytochrome C3 and 1219. desulphoviridin; pigments of the anaerobe STEIGMANN, A. 1945 Reactions of sulphur with Desulphovibrio desulphuricans. J. Gen. Mi- lipins, soaps, and cysteine. J. Soc. Chem. crobiol., 14, 545-572. Ind. (London), 64, 119-120. RALL, W. T. AND LEHNINGER, A. L. 1952 Glu- TANNER, F. W. 1918 Studies on the bacterial tathione reductase of animal tissues. J. metabolism of sulfur. J. Am. Chem. Soc., Biol. Chem., 194, 119-130. 40, 663-669.