Vol. 132, No. 1 Printed in U.S.A.

JOURNAL OF BAcTRjoLOGY, Oct. 1977, p. 139-143

Copyright C 1977 American Society for Microbiology

Bisulfite Reductase ofDesulfovibrio vulgaris: Explanation for Product Formation H. L. DRAKE AND J. M. AKAGI* Department of Microbiology, University ofKansas, Lawrence, Kansas 66045 Received for publication 25 April 1977

Bisulfite reductase, purified from Desulfovibrio vulgaris, was coupled with the pyruvate phosphoroclastic reaction. Moderate to low reducing conditions resulted in the formation of trithionate; however, when the concentration of reductant was high, a mixture oftrithionate and thiosulfate was formed. Sulfide was also a detectable product, but only when the concentration of bisulfite was low. Flavodoxin mediated native coupling between bisulfite reductase and the phosphoroclastic reaction. A model for bisulfite reductase activity is proposed.

Bisulfite reductase is thought to play an important role in the dissimilatory pathway for the reduction of bisulfite. Lee and Peck (15) reported that the bisulfite reductase purified from Desulfovibrio gigas reduced bisulfite solely to trithionate. Subsequently, Kobayashi et al. (14) showed that, in addition to the formation of trithionate, bisulfite reductase isolated from Desulfovibrio vulgaris also formed thiosulfate and sulfide. Kobayashi et al. (13) and Jones and Skyring (11) later demonstrated that enzymatic assay conditions played an important role in determining the pattern of product formation. Akagi et al. (4) proposed that trithionate was the only true enzymatic product of bisulfite reductase isolated from Desulfotomaculum nigrificans. In addition, Drake and Akagi (7, 8) showed that both trithionate and sulfide were formed by bisulfite reductase purified from D. vulgaris, whereas thiosulfate formation, if present (8), was not considered a true enzymatic product (7). To date, a hydrogenase-methyl viologen system has been utilized to generate the source of electrons for the study of bisulfite reductase. However, the most common source of carbon and energy for growing sulfate-reducing bacteria is lactate. Lactate is oxidized to acetate and C02 via lactate dehydrogenase, the pyruvate phosphoroclastic reaction, and acetokinase (1, 2, 5, 16). It is likely that the electrons abstracted during this oxidation are channeled into the dissimilatory sulfate-reducing pathway. This study was initiated to determine whether bisulfite reductase could be coupled with the oxidation of pyruvate via the phosphoroclastic reaction. In addition, we sought to investigate and resolve the apparent discrepan-

cies that exist in the literature concerning product formation by bisulfite reductase. MATERIALS AND METHODS Organism. D. vulgaris NCIB 8303 was grown and harvested as previously described (3). Enzyme assay conditions. By use of standard manometric techniques, all enzyme assays were performed in Warburg flasks of an 8-ml capacity. The particulate hydrogenase of D. vulgaris was prepared as described previously (20). Unless otherwise indicated, the standard reaction mixture contained (in micromoles): coenzyme A, 0.12; sodium pyru-

vate, 50; potassium phosphate buffer (pH 6.0), 100; electron carriers, enzymes, and substrate in a total volume of 1.0 ml. The center well contained 0.1 ml of either 20% KOH for trapping CO2 (thus allowing the measurement of H, evolution via hydrogenase activity) or 20% CdCl, for trapping sulfide. Both the KOH and CdCl2 were absorbed on fluted filter paper. The gas phase was N, when the phosphoroclastic reaction was coupled with hydrogenase. When bisulfite reductase activity was studied, CO was utilized as the gas phase to inhibit any hydrogenase activity present in the phosphoroclastic extract. All assays were performed at 30°C. Purification of the enzymes. The purification and general properties of bisulfite reductase (desulfoviridin) have been described (7, 8). The phosphoroclastic system was partially purified from cell extracts of D. vulgaris. One hundred milliliters of cell extract, prepared as described earlier (3), was fractionated by ammonium sulfate salt precipitation. The fraction precipitating between 0.33 and 0.45 saturation was dissolved in a small amount of distilled water and dialyzed against 1,000 volumes of 0.01 M potassium phosphate buffer, pH 7.0, for 15 h. The dialyzed material was passed through two consecutive Amberlite CG-50 columns (2 by 1 cm) to remove cytochrome c3 and two consecutive diethylaminoethylcellulose columns (2 by 1 cm) (chloride) to remove ferredoxin and flavodoxin. Due to the instability of pyruvate dehydrogenase activity, the unabsorbed 139

140

DRAKE AND AKAGI

J. BACTERIOL.

TABLE 1. Electron carriers for the phosphoroclastic reaction a

were required for optimal coupling between the

phosphoroclastic reaction and hydrogenase. The optimum pH for the phosphoroclastic reaction was 6.0. Electron carrier phosphate (jumol Effect of bisulfite on the clastic system. Before attempting to couple bisulfite reductase 6.60 Methyl viologen 6.20 with the phosphoroclastic reaction, we sought 0.94 Flavodoxin 0.45 0.10 0 Cytochrome C3 to determine whether any of the inorganic sul2.80 Completeb 2.40 fur anions involved in the dissimilatory patha Each flask contained 1.8 mg of phosphoroclastic way would affect the clastic system. Table 2 extract, 0.1 mg of hydrogenase, and 50 ,umol of shows an inhibitory effect of bisulfite on the pyruvate. Electron carrier concentrations were: formation of acetyl phosphate by the phosmethyl viologen, 1.0 ,umol; flavodoxin, 4.9 mg; cyto- phoroclastic system. The inhibition occurred chrome C3, 4.3 mg. Incubation time was 30 min. when either methyl viologen or native electron Standard assay conditions. carriers were utilized, although the extent of b Flavodoxin plus cytochrome C3. inhibition was reduced somewhat with the native carrier system. Raising the pH of the reacmaterial was immediately stored in 1.0-ml portions tion to 7 or 8 did not alleviate this inhibition. In at -20°C and thawed once for each experiment. At contrast, tetrathionate caused a complete supthe concentrations utilized, this carrier-free phos- pression of the phosphoroclastic reaction, phoroclastic extract was found to be essentially de- whereas thiosulfate, trithionate, and sulfate void of all enzymatic activities associated with the had no appreciable effects. The nature of the reduction of inorganic sulfur compounds. The purifi- partial inhibition by bisulfite and the total incation of flavodoxin has been described (8). Cyto- hibition by tetrathionate has not been deterchrome C3 was obtained by modification of the methods of Horio and Kamen (10) as previously described mined at this time. However, we have observed in native carrier studies that the inhibitory (2). All purifications were performed at 4°C. Analytical determinations. Acetyl phosphate was effect of bisulfite can be minimized by optimizdetermined by the method of Lipmann and Tuttle ing the concentration of flavodoxin. What ef(17). Analyses of thiosulfate and trithionate were fect, if any, these inorganic sulfur anions may performed by the method of Kelly et al. (12). Sulfide have on the phosphoroclastic system within the was determined by the method of Fogo and Po- cell is not currently known. powski (9). Protein was estimated by the method of Coupling of the phosphoroclastic reaction Lowry et al. (18), using bovine serum albumin as a standard. Sodium bisulfite solutions were prepared with bisulfite reductase. Table 3 outlines the fresh in 0.001 M disodium ethylenediaminetetraace- pattern of product formation by bisulfite reductase when coupled with the phosphoroclastic tate prior to each experiment. system. By varying the amount of pyruvate, we RESULTS found that we could control the nature of the General properties of the pyruvate phosphoroclastic reaction. The pyruvate phosphoroclastic reaction of the sulfate-reducing bacte- TABLE 2. Effect of bisulfite on the phosphoroclastic reactiona ria is well documented (1, 2, 16, 19). The oxidation of pyruvate to acetyl phosphate and CO2 NaHSO3 Acetyl phosrequires coenzyme A, orthophosphate, thiamine Electron carrier phate added diphosphate (present in the phosphoroclastic (AO) formed (Mmol) (jszmol) extracts utilized in this study), and electron Methyl viologen 0 11.89 carrier. Table 1 outlines the phosphoroclastic Methyl viologen 10 4.89 reaction of D. vulgaris. When the phosphoroMethyl viologen 5 7.04 clastic system was coupled with hydrogenase Methyl viologen 1 9.15 via the artificial electron carrier methyl violoNC' 0 5.95 gen, the formation of acetyl phosphate was NC 10 3.73 coincident with the evolution of an equimolar a Each flask contained 3.7 mg of phosphoroclastic amount of molecular hydrogen. Cytochrome C3 0.1 mg of hydrogenase, and 50 Amol of extract, was not observed to couple with the phosphoroElectron carrier concentrations were: clastic system, although it did couple with the pyruvate. methyl viologen, 1.0 ,umol; flavodoxin, 4.51 mg; cyhydrogenase used in this study (8). Flavodoxin tochrome C3, 2.94 mg. Incubation time was 30 min. alone coupled with the phosphoroclastic reac- Standard assay conditions. tion, but did not readily couple with hydrogenb NC, Native carriers, flavodoxin plus cytoase. Thus, both cytochrome C3 and flavodoxin chrome C3. Acetyl

H, evolved

VOL. 132, 1977

141

BISULFITE REDUCTASE OF D. VULGARIS

TABLE 3. Coupling bisulfite reductase with the phosphoroclastic reaction a Product formed Pyruvate added

(gmol)

HS03-

(Mmol)

S30 62-

S2032-

S2-

50 25 10 8 5 50c 50, TCd 10, TC

0.66

1.81

Trace

1.51

1.12

0

4.9

2.50 2.70 1.28 0.23 1.25 2.60

0.29 0 0 0 2.27 0.52

0 0 0 0 0.28 0.53

4.6 3.2 1.3 0.5 8.7 6.5

Acetyl phosphate 5.0

c

sumed b

(Simol) 5.60 6.77 8.08 8.10

3.84 0.69 8.57 9.37

a Each flask contained: phosphoroclastic extract, 1.8 mg; bisulfite reductase, 2.0 mg; NaHSO3, 10 AmoI; methyl viologen, 0.5 ,umol. Incubation time was 60 min. Standard assay conditions. b Calculated by the summation of the micromoles of sulfur atoms accounted for in the products. c Bisulfite reductase heated in a boiling water bath for 10 min. d TC, Reaction was allowed to go to completion, as determined by the loss of CO2 evolution; reaction time was 150 min.

products formed. With a 50 mM pyruvate concentration, a mixture of trithionate and thiosulfate was formed by bisulfite reductase. However, as the pyruvate concentration in the system was lowered, only trithionate was formed. Regardless of the initial pyruvate concentration, sulfide was only detected when the reaction was allowed to proceed to completion (Table 3). Assuming that the numbers of electrons required for the reduction of bisulfite to trithionate, thiosulfate, and sulfide are two, four, and six, respectively, the amount of acetyl phosphate formed (two electrons abstracted per pyruvate oxidized) was in good agreement with the amount of sulfur products detected. Effect of time on product formation. Since sulfide was formed only when the reaction was allowed to go to completion, the effect of time on the product profile was investigated. As shown in Fig. 1, trithionate was initially the only product detected when 10 mM pyruvate was 0

o 25

E

0

r

20

Q

0 15

-I~~~~~

LL

U 0

>/ 0//-

10

,/.

0

o 05

It

0

20

40

60

80

100 120

140

TIME (min.)

FIG. 1. Effect of time on product formation. Each flask contained: phosphoroclastic extract, 1.8 mg; bisulfite reductase, 1.6 mg; sodium pyruvate, 10 iLmol; sodium bisulfite, 10 umol; and methyl viologen, 0.5 ,umol. Standard assay conditions. Symbols: trithionate; 0, thiosulfate; A, sulfide; broken line, acetyl phosphate. 0,

used. As the reaction proceeded, trithionate formation reached a plateau and thiosulfate formation ensued. Lastly, sulfide was formed, but only when the concentration of bisulfite was low. When the reaction had reached completion (140 min), the number of micromoles of sulfur atoms in the three products accounted for all of the bisulfite added (10 ,umol). From this profile, it can be seen that trithionate was the predominant product. Thiosulfate and sulfide were formed only after the concentration of bisulfite was lowered, sulfide being formed last. Trithionate was interpreted to be the preferred product of bisulfite reductase. Coupling with flavodoxin. We have shown that flavodoxin can assume the role of native electron carrier for bisulfite reductase (8). Since flavodoxin was also shown to couple with the phosphoroclastic reaction (Table 1), we sought to determine whether a similar product pattern could be obtained when methyl viologen was replaced with flavodoxin in the phosphoroclastic extract-bisulfite reductase system. As shown in Table 4, a high pyruvate concentration (50 mM) again resulted in the formation of a mixture of both trithionate and thiosulfate. When the pyruvate concentration was lowered, only trithionate was formed. No sulfide was formed when the concentration of bisulfite was high. However, when the concentration of bisulfite was lowered to 1 gmol, a small amount of sulfide was detected. It should be noted that sulfide formation by bisulfite reductase was almost negligible when the native electron carrier flavodoxin was utilized. The relatively large amount of sulfide formed when methyl viologen was used may not be indicative of the in vivo reaction catalyzed by bisulfite reductase.

142

J. BACTERIOL.

DRAKE AND AKAGI TABLE 4. Coupling with tlavodoxina Substrate added

Product formed (Atmol)

(Amol)

Carrier

NaHSO,

Pyruvate

S,306-

S,O,'"

Si-

Acetyl phosphate

Fvd 10 0.99 50 1.08 0 2.8 0 Fvd 8 10 2.07 0 1.7 Fvd 1 50 0.32 0 0.09 1.0 1 0.10 Fvd 8 0.32 0 1.0 MeV 10 8 2.70 0 0 3.2 0.54 MeV 1 8 0.16 2.3 Trace a The flavodoxin (Fvd) concentration was 2.0 mg, and the methyl viologen (MeV) concentration was 0.5 ,umol. Each flask contained 1.8 mg of phosphoroclastic extract and 2.0 mg of bisulfite reductase. Incubation time was 60 min. Standard assay conditions.

thetical; as such, it is not meant to necessarily DISCUSSION depict the actual detailed enzymatic mechaThe results obtained in this study show that nism. The active site is visualized to have three with the bisulfite reductase can be coupled pyruvate phosphoroclastic reaction. Bisulfite is adjacent sites, A, B, and C, available for bindreduced to trithionate, thiosulfate, and sulfide, ing bisulfite. It is possible that site C must be the pattern of product formation being depend- occupied before site A or B is in the proper ent upon the pyruvate concentration and the configuration for binding additional bisulfite. concentration of bisulfite, i.e., the ratio of pyru- Once bound, the bisulfite at site C is reduced by vate to bisulfite. A moderate to low pyruvate two electrons to sulfoxylate (Fig. 3). If availaanother bisulfite is bound (site B), forming concentration and high concentrations of bisul- ble, a two-sulfur intermediate; no reduction is refite favor the formation of trithionate, whereas an increase in the pyruvate concentration re- quired to form this compound. A third bisulfite sults in the formation of a mixture of tri- (site A) could then react with the two-sulfur thionate and thiosulfate. Sulfide formation is intermediate, forming trithionate. However, if concentration ofthe electron donor (reduced dependent upon a low bisulfite concentration. the Lee and Peck (15) first demonstrated that methyl viologen) is high enough, the two-sulfur may undergo a reduction to thiobisulfite reductase from D. gigas formed only intermediate trithionate. In contrast, Kobayashi et al. (13, sulfate. If the available reductant concentra14) and Jones and Skyring (11) found that thio- tion is not high, trithionate is the initial prodsulfate and sulfide were also formed. These in- uct. As the reaction proceeds and the bisulfite is vestigators utilized a hydrogenase-methyl viol- depleted, a critical point is reached (Fig. 1) at ogen system. In the present study, it was read- which site A does not readily become occupied ily apparent that trithionate is the only product by bisulfite. Instead, the reduction of the twoformed by bisulfite reductase when the bisulfite sulfur intermediate predominates and thiosulconcentration is high and the amount of availa- fate is formed. A third critical point is reached (Fig. 1) when the bisulfite concentration is alble reductant is moderate to low. Kobayashi et al. (13) and Jones and Skyring most entirely depleted. The sulfoxylate inter(11) found that high reductant (methyl violo- mediate is now reduced to sulfide. It should be gen) concentrations and low bisulfite concen- noted that both the sulfoxylate and the twotrations favored the formation of thiosulfate sulfur intermediate would be labile compounds, and sulfide. The information obtained in our thus making their isolation unlikely. Several investigators have speculated that a phosphoroclastic extract-coupled experiments dissimilatory pathway might not be operating support their findings. To account for the observations obtained in in the sulfate-reducing bacteria (6, 13). Kobaythis study, a model is proposed for bisulfite ashi et al. (13, 14) have proposed a reaction reductase (Fig. 2). The proposed model is hypo- sequence for product formation by bisulfite reductase in which sulfide is seen to be the true product, trithionate and thiosulfate being (A) H8HSQ_SO formed by the nonenzymatic reaction of sulfite 'AT(E 2IT with the labile intermediates sulfoxylate and elemental sulfur, respectively. It has been imFIG. 2. Model for the active site of bisulfite reduc- plied that assimilatory and dissimilatory sultase. fite reduction may closely parallel one another

BISULFrrE REDUCTASE OF D. VULGARIS

VOL. 132, 1977 HS03

H. HS£ 3. o0S0] .O o03_SH20

H20

H20 12e2H 2 H20 H20*.2-1 ssoj s2~ FIG. 3. Proposed pathway for the formation of products by bisulfite reductase. 1e14H

(6, 13). We have recently characterized a novel thiosulfate-forming enzyme that facilitated the removal of the trithionate formed by bisulfite reductase (8). The thiosulfate-forming enzyme is not trithionate reductase, but does constitute the first reported case in which a purified protein has been shown to utilize trithionate in the formation of thiosulfate. One cannot say with certainty what role bisulfite reductase might play in the cell. One possibility is that this enzyme effectively depletes the bisulfite pool, which is inhibitory to certain enzyme systems (Table 2), by storing this sulfur species in the form of trithionate. In our experiments with the purified enzyme, trithionate formation is seen to be favored. Once formed, trithionate can be reduced to thiosulfate via the thiosulfate-forming enzyme. Thiosulfate reductase would complete the pathway leading to the formation of sulfide from bisulfite. We believe that dissimilatory reduction does occur in the sulfate-reducing bacteria and that bisulfite reductase plays an integral role in this process. ACKNOWLEDGMENTS This study was supported by Public Health Service grant 04672 from the National Institute of Allergy and Infectious Diseases and by a grant from the University of Kansas General Research Fund. H.L.D. is the recipient of a Wakaman Fellowship from the ASM Foundation. LITERATURE CITED 1. Akagi, J. M. 1964. Phosphoroclastic reaction ofClostridium nigrificans. J. Bacteriol. 88:813-814. 2. Akagi, J. M. 1967. Electron carriers for the phosphoroclastic reaction of Desulfovibrio desulfuricans. J. Biol. Chem. 242:2478-2483. 3. Akagi, J. M., and L. L. Campbell. 1962. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194-1201.

143

4. Akagi, J. M., M. Chan, and V. Adams. 1974. Observations on the bisulfite reductase (P582) isolated from Desulfotomaculum nigrificans. J. Bacteriol. 120:240244. 5. Brown, M. S., and J. M. Akagi. 1966. Purification of acetokinase from Desulfovibrio desulfuricans. J. Bacteriol. 92:1273-1274. 6. Chambers, L. A., and P. A. Trudinger. 1975. Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction? J. Bacteriol. 123:36-40. 7. Drake, H. L., and J. M. Akagi. 1976. Product analysis of bisulfite reductase activity isolated from Desulfovibrio vulgaris. J. Bacteriol. 126:733-738. 8. Drake, H. L., and J. M. Akagi. 1977. Characterization of a novel thiosulfate-forming enzyme isolated from Desulfovibrio vulgaris. J. Bacteriol. 132:132-138. 9. Fogo, J. K., and M. Popowski. 1949. Spectrophotometric determination of hydrogen sulfide. Anal. Chem. 21:732-734. 10. Horio, T., and M. D. Kamen. 1961. Preparation and properties of three pure crystalline bacterial haem proteins. Biochim. Biophys. Acta 48:266-286. 11. Jones, H. E., and G. W. Skyring. 1975. Effect of enzymic assay conditions on sulfite reduction catalyzed by desulfoviridin from Desulfovibrion gigas. Biochim. Biophys. Acta 377:52-60. 12. Kelly, D. P., L. A. Chambers, and P. A. Trudinger. 1969. Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate. Annal. Chem. 41:898-901. 13. Kobayashi, K., Y. Seki, and M. Ishimoto. 1974. Biochemical studies on sulfate-reducing bacteria. XIII. Sulfite reductase fromDesulfovibrio vulgaris. Mechanism of trithionate, thiosulfate, and sulfide formation and enzymatic properties. J. Biochem. (Tokyo) 75:519-529. 14. Kobaysahi, K., E. Takahashi, and M. Ishimoto. 1972. Biochemical studies on sulfate-reducing bacteria. XI. Purification and some properties of sulfite reductase, desulfoviridin. J. Biochem. (Tokyo) 72:879-887. 15. Lee, J. P., and H. D. Peck. 1971. Purification of the enzyme reducing bisulfite to trithionate from Desulfovibrio gigas and its identification as desulfoviridin. Biochem. Biophys. Res. Commun. 45:583-589. 16. LeGall, J., and J. R. Postgate. 1973. The physiology of sulfate-reducing bacteria. Adv. Microb. Physiol. 10:81-133. 17. Lipmann, F., and L. C. Tuttle. 1945. A specific micromethod for the determination of acyl phosphates. J. Biol. Chem. 159:21-28. 18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 19. Suh, B., and J. M. Akagi. 1966. Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans. J. Bacteriol. 91:2281-2285. 20. Suh, B., and J. M. Akagi. 1969. Formation of thiosulfate from sulfite by Desulfovibrio vulgaris. J. Bacteriol. 99:210-215.

Bisulfite reductase of Desulfovibrio vulgaris: explanation for product formation.

Vol. 132, No. 1 Printed in U.S.A. JOURNAL OF BAcTRjoLOGY, Oct. 1977, p. 139-143 Copyright C 1977 American Society for Microbiology Bisulfite Reduct...
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