Desulfotomaculum nigrificans

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1969, p Vol. 98, No American Society for Microbiology Printed In U.S.A. Thiosulfate Reductase Isolated from Desulfotomaculum nigrificans WALTER NAKATSUKASA AND J. M. AKAGI Department of Microbiology, University of Kansas, Lawrence, Kansas Received for publication 18 February 1969 A thiosulfate reductase from Desulfotomaculum nigrificans has been partially purified by ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, and sucrose density gradient centrifugation. With inner- and outer-labeled 35S-thiosulfate, the enzyme reduced only the outer sulfur atom to hydrogen sulfide. The enzyme was inhibited by sulfite and also by several sulfhydryl inhibitors. The Km value for this enzyme was calculated to be 1.3 X 10-' M. Other inorganic sulfur compounds, such as sulfate, sulfite, tetrathionate, and dithionate, were not reduced by this enzyme. The reduction of inorganic sulfate to hydrogen sulfide through a dissimilatory pathway, by members of the genera Desulfovibrio and Desulfotomaculum (5), is a well known phenomenon. Although the reduction to the sulfite level, involving adenosine-5'-phosphosulfate (APS), was characterized (7, 14), subsequent steps leading to the formation of H2S are not clearly understood. A thiosulfate reductase from Desulfovibrio desulfuricans was partially purified (9), and it was demonstrated that, in this organism, cytochrome C3 (8, 9, 10, 16) functioned as a link between hydrogenase and thiosulfate reductase. Whole cells and extracts of D. desulfuricans were reported to reduce tetrathionate and thiosulfate to H2S. Tetrathionate was cleaved by an enzyme tetrathionate reductase to two thiosulfate molecules, and the thiosulfate was reduced to H2S by thiosulfate reductase (16, 17). The latter enzyme was also demonstrated in yeast (11), and this enzyme cleaved thiosulfate to H2S and sulfite with reduced glutathione, cysteine, or homocysteine participating as a source of electrons. Recently, a direct reduction of thiosulfate by molecular hydrogen was observed with cell-free extracts of Desulfovibrio gigas (L. J. Guarrala, Bacteriol. Proc., p. 133, 1968). This system required ferredoxin or flavodoxin which functioned as the electron carrier in the reduction of thiosulfate. This report describes some of the properties of a thiosulfate reductase isolated from the thermophile, Desulfotomaculum nigrificans (4, 5). Evidence is presented that only the outer sulfur atom of thiosulfate is reduced to H2S, and the inner sulfur atom accumulates as sulfite. MATERIALS AND METHODS Cultivation of organisms. D. nigrificans strain 8351 was grown at 55 C in the medium previously described (2), with the modification that the yeast extract concentration was reduced to 4 g per liter. Desulfovibrio vulgaris (18) was cultivated as previously reported (2). The methods for large-scale cultivation and harvesting of these organisms have been described (1, 2). The cells were stored as a wet paste under molecular hydrogen at -20 C. Preparation of cell-free extracts. Cell-free extracts were prepared by passing a 25 or 50% cell suspension, in 0.05 M potassium phosphate buffer (ph 7.0), through a French pressure cell at 20,000 psi. The disrupted cell mixture was centrifuged at 27,000 X g for 20 min to remove cellular debris; the supernatant liquid represented the crude extract. Preparation of D. vulgaris hydrogenase. The crude extract of D. vulgaris was centrifuged at 105,000 X g for 2 hr, and the supernatant liquid was discarded. The pellet was washed twice with water, homogenized in 0.05 M tris(hydroxymethyl)aminoethane (Tris)- hydrochloride buffer (ph 7.0), and centrifuged at 27,000 X g for 30 min. The light yellow supernatant liquid contained hydrogenase activity and was stored in l-ml portions at -20 C. Preparation of the pyruvic phosphoroclastic extract of D. nigrificans. The phosphoroclastic system used in this study was prepared essentially as reported for obtaining the ferredoxin-free clastic system from Clostridium pasteurianum (13). Crude extracts of D. nigrificans were passed through a small diethylaminoethyl (DEAE) cellulose column (phosphate form), and the unabsorbed fraction, containing clastic activity, was collected, immediately lyophilized, and stored at -20 C. All of the ferredoxin and most of the thiosulfate reductase activity remained adsorbed on the resin. Purification of D. nigrificans thiosufate reductase. 429

2 430 NAKATSUKASA AND AKAGI J. BACTMROL. Crude extracts of D. nigrificans, usually 200 to 400 ml, were employed for purification purposes. All fractionations were conducted at 0 to 5 C. To the crude extract, we added solid (NH)2SO4, with stirring, to 40% saturation. After standing for 30 min, the mixture was centrifuged at 27,000 X g for 20 min, and the black precipitate was discarded. The supernatant fraction was further saturated with (NH4)2SO4 to 70%, and the precipitate which formed was collected by centrifugation and dissolved in a minimum amount of cold distilled water. It was dialyzed against 0.01 M potassium phosphate buffer (ph 7.0; five changes of 4-liter amounts), and the salt-free fraction was applied to a DEAE cellulose collumn (4 by 10 cm). The column was washed with 0.1 M phosphate buffer (ph 7.0) until the absorbancy at 260 nm (A28. M) dropped below 0.1. The thiosulfate reductase remaining on the column was eluted with 0.5 M phosphate buffer (ph 7.0) and was collected by centrifugation after the addition of solid (NH4)2SO4 to 70% saturation. The precipitate was dissolved in a minimum amount of water and subjected to sucrose density gradient centrifugation. The gradient was prepared in a stepwise fashion with 4.5 ml each of 10, 20, 30, and 40% sucrose in 0.05 M phosphate buffer, ph 7.5. The sucrose gradient was allowed to equilibrate for 5 hr before sample application (2 ml). Polycarbonate screw-cap tubes, manufactured for use with a no. 30 rotor (Beckman Instruments, Inc.), were employed for these experiments. The sucrose gradient centrifugation was conducted at 78,000 X g for 15 hr at 0 C. The sedimentation pattern of the sample showed a clear area, approximately 2 cm in height, at the top of the liquid and below this was a light yellow fraction (fraction 2) which layered just above an amber-colored substance (fraction 3). The light yellow fraction contained most of the thiosulfate reductase activity, and it was collected with a hypodermic syringe and needle. After removing the sucrose from this fraction by dialysis, it was assayed for activity. The purification steps are summarized in Table 1. When the thiosulfate reductase activity in the crude extract was low, increases in purification as high as 70-fold were observed. The reason for using outer-labeled "8Sthiosulfate for following the purification of this enzyme is discussed in a later section. TABLE 1. Enzyme assays. Thiosulfate reductase activity was determined by measuring the amount of H2S formed from thiosulfate with methyl viologen as the electron carrier. The electron donor for this reduction was pyruvate when using the phosphoroclastic system, or molecular hydrogen when the hydrogenase from D. vulgaris was employed. All assays were conducted at 50 C in a conventional Warburg apparatus. Unless otherwise indicated, the incubation time for these experiments was 20 min. When the phosphoroclastic system was used, the reaction mixture contained, in micromoles: potassium phosphate buffer (ph 7.0), 100; sodium pyruvate, 50; coenzyme A, 0.13; sodium thiosulfate, 10; methyl viologen, 1.0; clastic system, 2 mg; thiosulfate reductase fraction; and water. In the center well, 0.1 ml of 20% CdCl2 was added. One side arm contained 0.1 ml of 20 N H,PO4, whereas the second side arm contained a mixture of the clastic system and the thiosulfate reductase fraction. Total volume was 1.2 ml. The flasks were gassed with nitrogen and, after equilibration, the reaction was initiated by tipping the mixture of the clastic-thiosulfate reductase fraction into the main compartment. The second assay, employing the hydrogenase from D. vulgaris, contained the following, in micromoles: potassium phosphate buffer (ph 7.0), 100; methyl viologen, 1.0; sodium thiosulfate, 10; hydrogenase, 1 mg; thiosulfate reductase fraction; and water. The center well contained 0.1 ml of 20% CdClI. One side arm contained 0.1 ml of 20 N H,P04, and the other side arm contained the thiosulfate reductase fraction, Flasks were gassed with hydrogen for 10 min, and after equilibration, the reaction was initiated by tipping the thiosulfate reductase fractiou into the main compartment. The reactions of both assays were terminated by tipping H3PO4 into the reaction mixture; after shaking the flasks for an additional S min, the CdCl2-impregnated filter papers were removed and analyzed for H2S. Degradation of inner- and outer-labeled "S-thiosulfate. The authenticity of the inner- and outer-labeled 85S-thiosulfate was confirmed by degrading both compounds by a modification of the method of Ames and Willard (3). A 50-ml, pear-shaped flask containing Purification scheme for thiosulfate reductase from D. nigrificans Fraction Vol (ml) Protein (mg) Specific activitya Total unitsa -urecovey (%) per min per mag)moeofh85)rcer(% Crude extract , % (NH4) 2S , DEAE cellulose column. 0.1 M POj3- eluate M PO4,3-eluate Sucrose density gradient. Fraction Fraction a Converted to micromoles of H23,S from observed counts/min. Activities of all fractions were measured with the clastic assay. Outer-labeled 5S-thiosulfate (0.5,gc per flask) was employed.

3 VOL. 98, 1969 THIOSULFATE REDUCTASE FROM D. NIGRIFICANS 3 ml of 0.1 M sodium thiosulfate was placed in a freezer (-20 C) until the liquid was frozen. To the surface of the frozen liquid, we added 1.5 ml of concentrated HCI and 1.0 pliter of the respective labeled compound, containing 0.1 JAc of "5S. The flask was connected to an absorption train of two test tubes, each containing 10 ml of hydroxide of hyamine. Through an inlet tube, a continous stream of nitrogen was passed, and the flask was placed into a water bath at 90 C and allowed to remain at this temperature for 30 min. During this time, sulfur dioxide, originating from the inner sulfur atom of the thiosulfate, was evolved and trapped in the hyamine solution. The outer sulfur atom remained in the reaction flask in the form of elemental sulfur. After 30 min of incubation, 1 ml of the hyamine solution was removed and its radioactivity was measured. The elemental sulfur remaining in the reaction flask (outer sulfur atom) was extracted with 10 ml of toluene, and a sample of this was counted in a Packard liquid scintillation spectrometer. By employing the above procedure, we verified that the inner- and outer-labeled 35S-thiosulfate (commercial source) preparations were authentic. Recoveries of 92 and 100% (based on radioactivity) were obtained for the inner and outer labels, respectively. Other assays. Hydrogen sulfide was estimated by the method of Fogo and Popowski (6). Protein was measured according to Lowry et al. (12). The 35Shydrogen sulfide was trapped as the cadmium salt on CdCl2-impregnated filter paper in the center well of the Warburg flasks. After terminating the reaction, the paper was carefully removed, placed in 10 ml of counting fluid, and counted with a Packard liquid scintillation spectrometer (model 3375). All counts were corrected for quenching. Coenzyme A was purchased from P-L Biochemicals, Inc. Methyl viologen was obtained from Mann Research Laboratories. All radioactive compounds were purchased from New England Nuclear Corp. RESULTS Effect of enzyme concentration, ph, and buffers. The amount of H2S produced from thiosulfate was a linear function of thiosulfate reductase concentration (Fig. 1). This enzyme operated equally well in the ph range of 6.0 to 8.0. Its activity above or below these ph values was not tested because of the possibility that the D. nigrificans clastic system or the D. vulgaris hydrogenase would be affected. The buffer in which thiosulfate reduction functioned most effectively was potassium phosphate. In this buffer system, the amount of H2S was two times greater than in Tris, borate, or triethanolamine buffers under identical conditions. Because the clastic system assay depends on the presence of phosphate, the hydrogenase assay was used for this determination as the various buffers do not affect the hydrogenase activity. Stability of thiosulfate reductase. The enzyme showed no loss in activity after lyophilization and storage for at least 1 month at -20 C. In the frozen state, the activity decreased 33 % after 2 weeks, and heating the enzyme to 70 C in 0.01 M thiosulfate solution resulted in a 30% loss of activity. Lowering the ph to 5.0 and adjusting back to 7.0 decreased its activity by 40 %. Reduction of inner and outer-labeled 35S-thiosulfate. When the various fractions were tested for thiosulfate reductase activity during purification, we observed that the H2 to H2S ratio was frequently 2. Since, theoretically, the reduction of thiosulfate to H2S and sulfite requires 1 mole of hydrogen per mole of thiosulfate, the ratio of 2 suggested that both inner and outer sulfur atoms of thiosulfate were reduced to H2S. When innerlabeled '5S-thiosulfate and outer-labeled 35Sthiosulfate were tested, both sulfur atoms were reduced to H2S (Fig. 2) by the fraction eluted from DEAE by 0.5 M phosphate. When the same enzyme preparation was tested for its ability to reduce sulfite, we observed that, in addition to thiosulfate, sulfite was reduced to H2S (Table 2). Furthermore, the concentration of sulfite which was reduced most effectively was 10-3 M, whereas higher concentrations were inhibitory. The contaminating sulfite reductase was removed by sucrose density centrifugation; the thiosulfate reductase preparation obtained (sucrose density gradient, fraction 2) gave an H2 to H2S ratio of 1.0 and reduced only the outer "5S-thiosulfate (Table 3). These observations indicated that thiosulfate reductase preparations, contaminated with a sulfite reductase, may show higher specific activities if methods other than outer-labeled 35S-thiosulfate reductions are used for assay. Inhibitors of thiosulfate reductase. Sulfite was reported to be a competitive inhibitor of thiosulfate reductase from yeast, and the Km of this 9.0-1'0 2x S C) to to, ALIGRAMS PRTON 431 FIG. 1. Effect of protein concentration on thiosulfate reduction. Enzyme protein (sucrose density gradient, fraction 2) as indicated; 0.5 uac of "S-thiosulfate was added to each flask. The units of the ordinate are 103 counts/min.

4 432 NAKATSUKASA AND AKAGI J. BACTERIOL. 10 x 12 w OUTER LABEL S~~~203 z 6.0 :E ~ *A ILi 3.0 INRLABEL. 0 $ TIME, MINUTES FIG. 2. Reduction of outer- and inner-labeled 35- thiosulfate by the fraction eluted from DEAE by 0.5 M phosphate. Reaction was conducted as described for the hydrogenase assay. Enzyme protein, 2.2 mg; 0.5,uc oj the respective isotopes was employed. The units of the ordinate are 103 counts/min. TABLE 2. Effect of sulfite concentration on sulfite reductase activity of a DEAE cellulose column, 0.5 M phosphate eluatea Sulfite concn (umoles) H2S formed ,pmoles of thiosulfate 0.80 a Reaction mixtures were as described for the hydrogenase assay. Enzyme protein, 3.4 mg. enzyme was calculated to be 6 X 10O3 M for thiosulfate. The thiosulfate reductase from D. nigrificans was also inhibited by sulfite; however, the mechanism of this inhibition was not determined. In addition, the thiosulfate reductase was inhibited by N-ethylmaleimide, iodoacetamide, and p-hydroxymercuribenzoate. Restoration of p- hydroxymercuribenzoate-inhibited activity by cysteine suggested that the enzyme contains one or more sulfhydryl groups on the enzyme surface which participate in the catalysis. From the data analyzed by a Lineweaver-Burk plot, the Km of the thiosulfate reductase from D. nigrificans was calculated to be 1.3 X 10- M. Effect of other sulfur compounds. Several other sulfur compounds were tested for their ability to be reduced by thiosulfate reductase. These were sulfate, sulfite, tetrathionate, dithionate, and colloidal sulfur. With the exception of colloidal sulfur, none of the other inorganic compounds TABLE 3. Effect of outer- and inner-labeled 35Sthiosulfate and "3S-sulfite on a partially purified thiosulfate reductase from D. nigrificansa Isotope added Amt C(umoles) fmoles) 85S-SO1> S_3'SO "5SO SO32... a Reaction mixtures were as described for the hydrogenase assay. Sucrose density gradient fraction 2 was used as the enzyme source (1.1 mg); 0.5,uc of the respective isotopes was used; reaction time, 30 min. b Converted to micromoles of HI 3"S from measured counts/min. was affected. Because colloidal sulfur was reduced nonenzymatically by reduced methyl viologen, it was not possible to determine whether the enzyme could directly reduce elemental sulfur. Other electron donors. In addition to pyruvate and molecular hydrogen, other electron donors were tested. No H2S was produced when the electron donors were reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, reduced glutathione, or cysteine. In addition, no stimulatory effects were noted when reduced glutathione, cysteine, or 2- mercaptoethanol was added to the standard assays. DISCUSSION Sulfate-reducing bacteria belonging to the genera Desulfovibrio and Desulfotomaculum reduce inorganic sulfate to H2S by a dissimilatory pathway involving APS as the activated form of sulfate (7, 14). The subsequent reduction of APS to adenosine monophosphate and sulfite has been described (15), and it has been generally accepted that a sulfite reductase system reduces the sulfite to H2S by a mechanism still not clear at the present time. In addition to a sulfite-reducing system, these organisms contain a thiosulfate reductase which reduces thiosulfate to H2S (8, 9, 10, 17). In this study, the thiosulfate-reductase from D. nigrificans was partially purified and was shown to be relatively free from sulfite reductase activity. The enzyme catalyzed the reduction of the outer sulfur atom of thiosulfate, whereas the inner sulfur atom presumably remained as sulfite. This was concluded from experiments in which a sulfite reductase was present as a contaminant in thiosulfate reductase preparations and, in these cases,

5 VOL. 98, 1969 THIOSULFATE REDUCTASE FROM D. NIGRIFICANS both the inner- and outer-labeled 35S-thiosulfate appeared as H23"S. Although thiosulfate reductase has been shown to occur in sulfate-reducing bacteria, the physiological role it plays in sulfate reduction has not been described. Woolfolk (19) presented evidence that cell-free extracts of Micrococcus lactilyticus reduced metabisulfite (pyrosulfite), in equilibrium with sulfite, to thiosulfate, which was subsequently reduced to H2S by a thiosulfate reductase; dithionite was reported to be the intermediate compound between metabisulfite and thiosulfate. He suggested that the pathway of sulfite reduction in sulfate-reducing bacteria may be similar to that found for M. lactilyticus. Another possible explanation for the role of thiosulfate reductase in sulfate reducers may be related to the "thiosulfate-forming system" reported to be present in D. vulgaris and D. nigrificans (B. Suh et al., Bacteriol. Proc., p. 133, 1968). If sulfite were transformed reductively to thiosulfate, then a mechanism for reducing thiosulfate to H2S is necessary for the organism to continue its metabolic processes. Since sulfite inhibited thiosulfate reductase activity and at higher concentrations inhibited sulfite reductase activity (Table 2), it is tempting to speculate that, when the concentration of sulfite in these organisms attains a certain level, the "thiosulfateforming system" serves as a mechanism to regulate the intracellular concentration of sulfite. The relatively high Km value observed for the thiosulfate reductase insures that thiosulfate will not be effectively utilized until a relatively high concentration is established. When the level of sulfite becomes sufficiently low, the sulfite and thiosulfate reductases can operate and effectively reduce their respective substrates to H2S. Preliminary data indicated that the system which forms thiosulfate from sulfite has a relatively high Km value. The purification of the sulfite reductase and the "thiosulfate-forming system" is necessary to elucidate this hypothesis. ACKNOWLEDGMENTS We thank H. D. Peck for his advise and constructive criticisms during the preparation of this manuscript. This study was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases, Public Health Career Development Award K3-GM- 30,262, and Public Health Training Grant GM-703 to the University of Kansas. LITERATURE CITED Akagi, J. M The participation of a ferredoxin of Clostridium nigrificans in sulfite reduction. Biochem. Biophys. Res. Commun. 21: Akagi, J. M., and L. L. Campbell Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84: Ames, D. P., and J. E. Willard The kinetics of the exchange of sulfur between thiosulfate and sulfite. J. Amer. Chem. Soc. 73: Campbell, L. L., Jr., H. A. Frank, and E. R. Hall Studies on thermophilic sulfate reducing bacteria. I. The identification of Sporovibrlo desulfuricans as Clostridium nigrificans. J. Bacteriol. 73: Campbell, L. L., and J. R. Postgate Classification of the spore-forming sulfate-reducing bacteria. Bacteriol. Rev. 29: Fogo, J. K., and M. Popowski Spectrophotometric determination of hydrogen sulfide. Methylene blue method. Anal. Chem. 21: Ishimoto, M., and D. Fujimoto Adenosine-5'-phosphosulfate as an intermediate in the reduction of sulfate by a sulfate reducing bacterium. Proc. Jap. Acad. 35: Ishimoto, M., and J. Koyama On the role of a cytochrome in thiosulfate reduction by a sulfate reducing bacterium. Bull. Chem. Soc. Jap. 28: 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. (Tokyo) 44: Ishimoto, M., J. Koyama, and Y. Nagal Biochemical studies on sulfate reducing bacteria. IV. Reduction of thiosulfate by cell-free extracts. J. Biochem. (Tokyo) 42: Kaji, A., and W. D. McElroy Mechanism of hydrogen sulfide formation from thiosulfate. J. Bacteriol. 77: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Mortenson, L. E., R. C. Valentine. and J. E. Carnahan An electron transport factor from Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 7: Peck, H. D The ATP dependent reduction of sulfate with hydrogen in extracts of Desulfovibrio desulfuricans. Proc. Nat. Acad. Sci. U.S.A. 45: Peck, H. D Evidence for the reversibility of the reaction catalyzed by adenosine-5'-phosphosulfate reductase. Biochim. Biophys. Acta 49: Postgate, J. R Cytochrome C3 and desulphoviridin; pigments of the anaerobe Desulphovlbrio desulphuricans. J. Gen. Microbiol. 14: Postgate, J. R The economic activities of sulfate reducing bacteria. Progr. Ind. Microbiol. 2: Postgate, J. R., and L. L. Campbell Classification ot Desulfovibrio species, the nonsporulating sulfate-reducing bacteria. Bacteriol. Rev. 30: Woolfolk, C. E Reduction of inorganic compounds with molecular hydrogen by Micrococcus lactilyticus. II. Stoichiometry with inorganic sulfur compounds. J. Bacteriol. 84: