Studies on Biochemistry of the Thiobacilli Part IX
Total Page：16
File Type：pdf, Size：1020Kb
[Agr. Biol. Chem., Vol. 30, No. 8, p. 713•`716, 1966] Studies on Biochemistry of the Thiobacilli Part IX. Reduction of Trithionate by Thiobacillus thiooxidans By Masayo OKUZUMI Tokyo University of Fisheries Received October 20, 1964 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. ƒ¿-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. INTRODUCTION hydrolysis of trithionate to thiosulfate and In previous paper,1) the author observed sulfate was present in T. thioparus (equa the fact that tetrathionate was converted to tion 2). pentathionate, trithionate and thiosulfate un 2S4O62-+S5O62-+S5O62- ... (1) der anaerobic conditions with crude extracts. S3O62-+H2O•¨S2O32-+SO42-+2H+ ...(2) The formation of thiosulfate was attributed to hydrolysis of the trithionate which was It was also suggested that the biological formed from dismutation of tetrathionate. hydrolysis of trithionate might take place in If the hydrolysis of trithionate did not take anaerobic reactions of tetrathionate at pH 3.0 place, the formation of trithionate would with crude extracts of T thiooxidans. On proceed at the same rate as the formation of the other hand, it has been observed by the pentathionate, and would be, therefore, equi author that trithionate was reduced to thio molar to pentathionate. On the other hand, sulfate and sulfite at neutral pH with washed in every manometric experiment on tetra cells of T. thiooxidans. In this paper, results thionate oxidation, 0.5 mole of oxygen was of some experiments are described on the consumed per mole of tetrathionate. The reductive cleavage of trithionate by T thio oxygen uptake observed in the tetrathionate oxidans. oxidation seemed not to be caused by oxida MATERIALS AND METHODS tion of tetrathionate itself. This suggested that tetrathionate was dismutated secondarily Organism and Crude Extract to consume oxygen. The organism used throughout these studies was Thiobacillus thiooxidans, which was isolated by our Tamiya et al.2) observed trithionate oxida laboratory. tion by T. thioparus, and Huzisige and Haga3) Cells were cultured and harvested by the same pro expressed that trithionate hydrolase catalyzing cedures as described in the previous paper.h) The 1) M. Okuzumi, This Journal, 29, 1069 (1965). washed cells were suspended in 0.1 as phosphate buffer 2) H. Tamiya, K. Haga and Huzisige, Acta Phytochim. (pH 7.0) at a final concentration of 14,16 mg dry (Japan), 12, 173 (1941). 3) H. Huzisige and K. Haga, ibid., (Japan), 14, 141 (1944). 4) M. Okuzumi and Y. Kita, This Journal, 29, 1063 (1965). 714 Masayo OKUZUMI weight per ml. pH of the cell suspension was adjusted thiosulfate and sulfate (equation 2). to requisite pH with 1N H2SO4 or 1N NaOH. Recently, when the investigations of tri The suspension was exposed to a Raytheon sonic thionate conversion under anaerobic condition oscillator (20kc/sec., 200 W) for fifteen minutes and were carried out in neutral pH range with centrifuged in the cold for thirty minutes at 12,000 r.p.m. to remove intact cells and debris. Solid ammo washed cells of T thiooxidans, formation of nium sulfate was added to the supernatant with both thiosulfate and sulfite other than sulfate stirring up to 80% saturation. The pH of solution as ultimate products of trithionate conversion was kept at 7.0 with ammonium hydroxide during the was confirmed by means of paper chromato ammonium sulfate fractionation. The preciptate was graphic analysis. In addition, the fact that collected and dissolved in 0.1 M phosphate buffer (pH the yield of sulfite was roughly equal with 7.0). that of thiosulfate was verified by the quanti Substrates tative analysis. These facts suggested that Sodium trithionate was synthesized according to trithionate might undergo a reductive cleavage Auerbach's methods) by oxidation of sodium thiosulfate to thiosulfate and sulfite (equation 4). with hydrogen peroxide. The product was recrystal lized three times from 50% ethanol. S3O62-+2e•¨S2O32-+S2O32-+S032- ...(4) Measurement of Trithionate Reduction Thus, effect of pH on the reaction was Experiments of trithionate reduction were conducted investigated with washed cells under anaero with washed cells under anaerobic condition by using usual Thunberg tubes. The washed cells and some bic condition. The result is shown in Fig . 1. other reagents were placed in the main compartment The usual assay was used except that the pH and trithionate as substrate was added from the side was changed as indicated in the figure . arm, The reactions were carried out at 29•`30•Ž , and, As shown in the figure, the velocity of after cessation of the reaction, the various sulfur com - trithionate reduction was affected by the pounds present in the reaction mixtures were deter- change in pH between 3 and 8, and the activ- mined. Determinations and Paper Chromatographies Determinations and paper chromatographies of in- organic sulfur compounds were carried out by the methods as described in the previous paper.4) RESULTS AND DISCUSSION Effect of pH on the Reductive Cleavage of Tri thionate Tamiya et al.2) reported that trithionate was directly oxidized to sulfate with washed cells of T. thioparus (equation 3) . FIG. 1. Effect of pH on Reduction of Trithionate by Washed Cells of S3O62-+2O2+2H2O•¨3SO42-+4H+ ...(3) T. thiooxidans Huzisige and Haga3) observed that sulfate Each reaction mixture contained 3 ml of washed cells (48 mg dry weight) suspended in was not the product obtained directly from 0.1 M phosphate buffer (pH 7.0) and 100 P moles of trithionate to total volume of the oxidation of trithionate , and that trithio 4.0 ml. pH was adjusted to desired value by nate was enzymatically hydrolyzed to form I N H2SO4 or 1 N NaOH . Incubation was continued for 360 minutes at 30°C under an . aerobic condition 5) F. Auerbach and I. Koppel, (editors) 1927. " Handbuch . der anorganischen Chemie," Vol. 4, pt. 1, Hirzel, Leipzig. Studies on Biochemistry of the Thiobacilli . Part IX 715 ity reached a maximum at pH 7.2 and fell considered that ƒ¿-glycerophosphate is effective steeply in the acid pH range. as electron donor for the reduction. More if trithionate was reduced by equation 4, over, thiosulfate was also employed as an the equimolar formation of thiosulfate and electron donor for the reduction, since the sulfite might be observed. However, the yield electron generated from oxidation of thio of sulfite was less than that of thiosulfate at sulfate to tetrathionate might be transferred each pH, and both curves were nearly parallel to trithionate through an intermediary elec .as shown in the figure. It is supposed that tron carrier. This experiment was carried the hydrolysis in addition to the reductive out with washed cells at pH 5.8. cleavage of trithionate takes place at each pH, In consequence, the reduction of trithio and that the velocity of trithionate hydrolysis nate by coupling with the oxidation of thio may be scarcely affected by the change in pH sulfate did not take place; it may be caused between 3 and 8, while the velocity of the by the effect of pH on oxidation of thiosulfate, reduction is greatly affected by its change. since thiosulfate is hardly oxidized to tetra thionate at pH above 5 by T. thiooxidans Reduction of Trithionate in Presence of ƒ¿-Gly cerophosphate . Anaerobic Reaction of Trithionate ƒ¿-Glycerophosphate was employed as an It was supposed that the formation of sulfite electron donor for the reduction of trithio might originate in the sulfate reduction. Ac nate. Provided that the used cells were fresh, cording to Peck's investigation,6) APS reduc trithionate would be reduced actively without tase and ADP sulfurylase are concerned with addition of ƒ¿-glycerophosphate. In such cases, the oxidation of sulfite to sulfate, and these ƒ¿-glycerophosphate was little effective as reactions seem to be reversible. Accordingly, electron donor on the reduction. Such fresh sulfate formed from the trithionate hydrolysis -cells probably contain a great deal of various might be then reduced to sulfite. Thus, to electron donors. Thus, the cells starved for investigate further whether the formation of twenty hours at 30°C were subjected to ex periments. The result is shown in Tabel I. TABLE 1. EFFECT OF ƒ¿-GLYCEROPHOSPHATE ON REDUCTION OF TRITHIONATE BY WASHED CELLS Each reaction mixture contained 3 ml of washed cells (48 FIG. 2. Time Course of Anaerobic mg dry weight) suspended in 0.1 M phosphate buffer (pH Reaction of Trithionate by Washed 7.3), and 108 p moles of trithionate to total volume of 6.0 ml. Cells. All reaction mixtures were under anaerobic condition. * Heated cells (at 100•Ž for 10 minutes) Reaction mixture contained 4 ml of washed cells (56 mg dry weight) suspended in 0.1M phosphate buffer (pH 7.0) and 100 p moles The yield of both thiosulfate and sulfite of trithionate to total volume of 6.0 ml. in the case of addition of ƒ¿-glycerophosphate Reaction was carried out at 30°C under an- aerobic condition. was roughly 1.5 times as great as the yield obtained in the case of no addition of it. It is 6) H.D. Peck, Jr., Proc. Natl. Acad. Sci. U.S., 45, 239, (1959). 716 Masayo OKUZUMI sulfite was attributed to reduction of trithio The sum of equation a to d (equation e) nate itself, or to secondary reduction of sulfate indicates that net productions of elemental which was formed from hydrolysis of tri sulfur and sulfate should occur during the thionate, the relationship between sulfite and oxidation of tetrathionate.