THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 237, No. 1, January 1962

Printed in U.S.A.

The Oxidation of Thiosulfate and Phosphorylation

in Extracts of Thiobacillus thioparus

H. D. PECK. JR., AND E. FISHER, JR.*

From the Biology Division, Oak Ridge National Laboratory,t Oak Ridge, Tennessee

(Received for publication, June 26, 1961)

Thiobmillus thioparus is an autotrophic microorganism that obtains the energy required for growth by the oxidation of re- duced inorganic sulfur compounds to sulfate. When it is grown on high concentrations of thiosulfate, the organism accumulates elemental sulfur and produces sulfate. The pathway of thio- sulfate oxidation has been essentially unknown; however, it has been postulated that tetrathionate and other polythionates are intermediates in this oxidation (1). Recent evidence indicates that inorganic orthophosphate is involved in the oxidation of thiosulfate by this organism since the presence of inorganic orthophosphate or arsenate (2) is required for the complete oxidation of thiosulfate to sulfate. Santer (3) showed that during the oxidation of thiosulfate in the presence of 01*-labeled inorganic orthophosphate, 0’8 is transferred to the sulfate that is produced in the oxidation. The specific activity of the 018 found in the sulfate was approximately one-fourth of that found in the inorganic orthophosphate.

Recently, Peck (4) postulated a reaction sequence for the oxidation of thiosulfate by T. thioparus that explains these ob- servations (Equations 1 to 6).’

thiosulfate

4H+ + 4e + 2S203= reductase

- 2SO3= + 2HzS (1)

2HzS + 02 sulfide oxidase , 2s” + 2HzO (2)

2SO3’ + 2AMP y APS-reductase

\ PAPS + 4e (3)

2APS + 2Pi , ADP-sulfurylase \ 2ADP +2SOa= (4)

2ADP , adenylic kinase

’ AMP + ATP (5)

ZSzOz- + 02 + AMP + 2Pi + 4H+ +

2S0 + 2SO4- + ATP + 2H20 (6)

This scheme was based on the observations that cell-free extracts of T. thiopmus metabolize thiosulfate in the presence of gluta- thione and that the enzymes that catalyze Reactions 1 to 5 were present in the cell-free preparations. The most interesting observation was the presence of APS-reductase (4, 5), which

* Research Participant. Present address, Department of Ophthalmology, Tulane University, New Orleans, Louisiana.

t Operated by Union Carbide Corporation for the United States Atomic Energy Commission.

1 The abbreviation used is: APS, adenosine 5’-phosphosulfate.

until this time was found only in the “sulfate-reducing bacteria.” The presence of this enzyme in high specific activity (equal to that found in the “sulfate-reducing bacteria”) and the absence of the enzyme that reduces 3’-phosphoadenosine-5’-phospho- sulfate (6) suggested that APS-reductase, functioning in reverse to form APS from AMP and sulfite, is an essential.-enzymatic component in the oxidation of thiosulfate by this organism. It has recently been shown by Peck (7) that extracts of T. thiopurus, other thiobacilli, and Desulfovibrio desulfuricans catalyze the formation of APS from AMP and sulfite in the presence of Fe(CN)6-;. This reaction seems to be identical to the APS- reducing activities previously reported in extracts of these organisms. It is presumably the enzymatic process whereby a high energy sulfate is formed by these extracts that can be ex- changed for Pi to form ADP and sulfate by the enzyme ADP- sulfurylase (8).

The scheme also offers an explanation for the observation made with whole cells concerning the role of Pi in this oxidation. The absence of Pi during the oxidation of thiosulfate in the cell should lead to an accumulation of APS that would result in a depletion of the AMP present in the cells and a consequent failure to oxidize sulfite to sulfate. The fact that arsenate can replace Pi in this oxidation is consistent with the observations of Robbins and Lipmann (8) that ADP-sulfurylase can catalyze the arsenolysis of APS. In addition, the reaction sequence indicates a possible mechanism for the transfer of 018 from Pi to sulfate (3) that is consistent with known mechanisms of 0’8 transfer. 0’8 would be introduced first into AMP by the combined action of ADP- sulfurylase and adenylate kinase (Equations 7 and 8).

0; 0 II 1

li; 2Ado-O-PTO- ,, -0- + 2POh’*’ ti I

(j-I ij

0 0'8 (7)

II 2Ado-O-P-018- ! -0’8- + SO,-

A- 0’”

ii

I 0’8 0 : II

2Ado-O- -O’8~P-O’8- + Ado-O- I-O’*- + ATP !J (8)

0- j !I* 0-

Since the amount of intracellular AMP is small when compared to the total amount of substrate dissimilated, the phosphate group of AMP will rapidly become randomly labeled with 018 upon continued recycling. This labeled AMP then forms APS

190

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January 1962 H. D. Peck, Jr., and E. Fisher, Jr. 191

that will now be labeled with 01* between the sulfur and phos- phorus atoms:

SOS- + Ado-O- as -0’” =

O-

Acl-O-~-018-~-O- + 2e

d- 8 Cleavage of APS by ADP-sulfurylase between the phosphate- oxygen bond will produce sulfate labeled with 0’8:

A,,-,-;;,Sw~-,- + PO*’ 5 ADP + Oj:()-

8 A- (10)

The atom % excess of 018 in the sulfate will be 25 y0 of that found in the Pi, and Santer (3) observed a value of 22 to 23%.

In the present communication, some properties of a cell-free system from T. thiopurus that oxidizes thiosulfate to sulfate and esterifies phosphate are described.

EXPERIMENTAL PROCEDURE

T. thiopurus (ATCC 8158) was grown on Starkey’s medium No. 2 (9) and aerated with a gas mixture containing 95% air and 5% CO*. After 3 to 4 days of incubation at room tempera- ture, the cells were harvested in a Sharples centrifuge. The cell paste was suspended in distilled water, and the elemental sulfur formed during growth was removed by low speed centrifugation. The cells were then washed twice with distilled water, and a 30 to 50% suspension of cells (based on wet weight) was prepared in 0.05 M Tris, pH 8. The suspension of cells was then passed once or twice, depending upon the degree of breakage, through a French pressure cell (American Instrument Company). I f the extract was too viscous for centrifugation, the extract was sup- plemented with 100 pg of DNase that served to liquefy the extract in 5 to 10 minutes at 30”. The extract was then cen- trifuged at 20,000 x g for 30 minutes. Active preparations could also be prepared by sonic oscillation; however, extracts prepared in this manner exhibited hydrolytic activities for adenosine nucleotides with the result that phosphorylative activ- ity was low. Extracts were either used directly or dialyzed for 18 hours against 0.025 M Tris, pH 8, plus 5 X 10-3 M EDTA. To show nucleotide requirements, the dialyzed extracts were treated with 100 mg of acid-washed charcoal per 5 ml.

Activity measurements were made in Warburg vessels (vol- ume, 20 ml) at 30” with the gas phase air and 0.2 ml of 20% NaOH in the center well. The reactions were initiated by the addition of enzyme from the side arm of the Warburg vessel and stopped either by the addition of 1.0 ml of 10% trichloroacetic acid or by removal of the contents of the Warburg vessel to a centrifuge tube, which was then placed in a boiling water bath for 3 minutes. Pi was determined by the method of Fiske and SubbaRow (lo), sulfate by the method of Gleen and Quastel (1 l), thiosulfate and polythionates by the method of Sarbo (12) (removing sulfide with a Cd++ precipitation), and esterified phosphate by adsorption on charcoal and hydrolysis of labile phosphate. Electrophoresis was carried out as described by Robbins and Lipmann (8) in 0.03 M citrate, pH 5.5. Protein

was determined by the method of Lowry et al. (13). Radio- activity was measured in a liquid scintillation spectrometer (Packard Instrument Company) in 10 ml of the following solvent: 2,5-diphenyloxazole, 4 g; 1,4-bis-2’-(5’-phenyloxazolyl)benzene, 100 mg; toluene, 700 ml; absolute ethanol, 300 ml. S3504- was isolated and prepared for counting by precipitation with an excess of acid BaClz and washed 4 or 5 times with 0.01 M Na2S203. Insoluble elemental sulfur was prepared for counting by washing 5 or 6 times with water containing 1: 1000 of Triton X-100.

Adenylic deaminase was prepared by the method of Nikiforuk and Colowick (14) and tetrathionate by the method described by Mellor (15). Adenylic kinase, hexokinase, DNase, and nucleotides were obtained from commercial sources. P320*’ was purchased from the Oak Ridge National Laboratory, NazSS3503 (inner label) from Volk Radiochemical Company, and Na&35S03 (outer label) from New England Nuclear Company.

RESULTS

Oxidation of Thiosulfate--The oxidation of thiosulfate by cell- free extracts of T. thioparus has been previously reported in brief (4). The reaction mixture contained, in addition to cell-free extract, substrate amounts of GSH and thiosulfate. The ra- tionale for the addition of substrate amounts of GSH with the substrate, thiosulfate, is based upon several observations. Skarzynski et al. (16) reported that T. thiopixus preferentially incorporates the outer sulfur atom of thiosulfate into cellular material and elemental sulfur. However, this observation has recently been questioned by Santer et al. (2). An enzyme has been isolated from yeast by Kaji and McElroy (17) that catalyzes t,he reductive cleavage of thiosulfate to sulfite and sulfide, and an apparently similar system has been described in extracts of D. desulfuricuns (18). The former employed GSH as electron donor, and the latter used molecular hydrogen. The presence of thiosulfate reductase in extracts of Thiobacillus thiooxidans was suggested by the observation of Suzuki and Werkman (19) that sulfide is produced from thiosulfate in the presence of GSH. The presence of this enzyme in extracts of T. thioparus was demonstrated, and it was proposed that the enzyme was involved in thiosulfate oxidation (4).

To detect phosphorylation coupled with thiosulfate oxidation, the reaction mixture was supplemented with fluoride and EDTA to minimize hydrolysis of any phosphate esters that might be produced. Neither ADP-sulfurylase (8), thiosulfate reductase (17), nor APS-reductase (5) have been shown to exhibit metal requirements. AMP and Pi were included as substrates for the postulated phosphorylation. The oxidation of thiosulfate was carried out in Warburg vessels. After 30 minutes, the reaction was stopped by the addition of 2 ml of 10% trichloroacetic acid, and analyses were made for phosphate and sulfate. Although insensitive, the barium method of Gleen and Quastel (11) was employed for the determination of sulfate since sulfite, thiosul- fate, and sulfide do not interfere with the determination. As shown in Table I, during the oxidation of thiosulfate, phosphate was esterified and sulfate was produced. Approximately 1 pmole of phosphate is esterified for each micromole of O2 utilized and sulfate produced. These data are in agreement with the reaction sequence postulated previously for this oxidation. Since the electrons for the reduction of thiosulfate are supplied by GSH, the oxidation will now require 2 atoms of oxygen for each sulfate formed. Oxygen uptake observed with GSH alone is nrohn.hlv

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192 Thiosulfate Oxidation and Phosphorylation in Thiobacillus Vol. 237, No. 1

TABLE I

Oxygen utilization, phosphate esterifkation, and sulfate production during thiosulfate oxidation

The complete reaction mixture contained, in micromoles: Tris, pH 8, 300; AMP, 10; Pi, 5; NaF, 100; EDTA, 10; GSH, 40; Nak&03, 10. T. thioparus extract, 17.7 mg in a total volume of 2.0 ml. Time, 40 minutes.

system

(1) Complete. 5.0 (2) Complete without &03=. 1.6 (3) Complete without GSH 0.6

Net change : (1) without [(2) and (3)l. 2.8

/mhs pm&s 2.25 3.35 0.55 0 0.00 1.67

1.70 1.68

TABLE II

Esterijkation of orthophosphate during oxidation of thiosulfate by cell-free extracts of T. thioparus

The complete system contained, in micromoles: Tris, pH 8, 300; EDTA, 10; NaF, 100; AMP, 10; Na&203, 40; GSH, 40; TPN, 0.67; Pi, 5 (containing 2.28 X 106c.p.m. perpmole). T. thioparus extract, 26 mg in a total volume of 2 ml. Time, 30 minutes.

Additions

/.lmozes pf?ZOleS

Complete.................... 2.53 3.47 Complete with 2,4-dinitro-

phenol, 2 X 1OW M. 3.06 4.66

@%OkS pnzoles

2.69 2.26

3.72 3.22

caused by a TPNH-oxidase and GSH-reductase that have been reported to be present in similar organisms (20). Usually, oxygen utilization was greater than sulfate production, and sulfate production was greater than phosphate esterification. In the absence of fluoride, it was not possible to observe phos- phate esterification although oxygen utilization and sulfate pro- duction could be readily observed. The extracts contained enzymes that catalyzed the release of Pi from ATP, ADP, and AMP and all these activities were stimulated by sulfide and inhibited by fluoride. In spite of the presence of hydrolytic enzymes in the extract, it seemed that there was an esterification of phosphate occurring rather than an inhibition of hydrolytic enzymes, since there was less phosphate at the termination of the reaction than was added initially to the reaction mixture.

Nature of Phosphorylation-It has been assumed that T.

thioparus obtains the energy required for growth from oxidative phosphorylation occurring during the oxidation of sulfur com- pounds (I, 21). Because the phosphorylation observed in these extracts could be a result of oxidative phosphorylation, the effect of 2,4-dinitrophenol on the phosphorylation was determined. The results are shown in Table II. Control values of the reac- tion mixture without thiosulfate and without GSH have been subtracted. Pi labeled with P32 was included in the reaction mixture to facilitate the identification of the product of the phosphorylation. After 30 minutes, the reaction was stopped by the addition of 2.0 ml of 10% trichloroacetic acid and Pi determined. Nucleotides were adsorbed on charcoal, the char- coal washed and divided into two portions. One portion was

hydrolyzed in 1 N HCl for 15 minutes, and the released Pi and radioactivity determined. The Pi lost from the reaction mix- ture, 15-minute hydrolyzable phosphate adsorbed on charcoal, and the esterified phosphate determined by radioactivity are all in good agreement. The addition of 2,4-dinitrophenol to the reaction mixture did not inhibit the phosphorylation but seemed to stimulate slightly. This result indicated that the phos- phorylation was probably not the consequence of oxidative phosphorylation. The radioactive compound was eluted from the other portion of the charcoal with ammoniacal ethanol and subjected to high voltage paper electrophoresis. Essentially all of the radioactivity was present in an area that corresponded to ADP. In the presence of adenylic kinase, glucose, and hexo- kinase, AMP (identified by adenylic deaminase), and glucose 6-phosphate were formed from this nucleotide. The appearance of the P32 exclusively in the ADP and the dependence of this incorporation on the presence of both GSH and thiosulfate are in accord with the postulated reaction sequence. The presence of fluoride would inhibit adenylic kinase and lead to the accumu- lation of ADP from the phosphorolysis of APS by ADP-sulfuryl- ase.

To eliminate the possibility that the ADP had been formed in some manner from ATP, the oxidation of thiosulfate in the pres- ence of Pi32 was carried out with and without a pool of unlabeled ATP. These results are presented in Fig. 1. In the “complete” and “complete with ATP,” there were 3 pmoles of phosphate esterified. The nucleotides were adsorbed on charcoal, eluted, and subjected to electrophoresis. The absence of label in the ATP indicated that ATP was not an intermediate in the forma- tion of ADP.

E$ect of AMP on Oxygen Utilization and Phosphate Esterijka- tion-Since AMP seemed to be the phosphate acceptor in the oxidation of thiosulfate, it should be possible to show a depend- ence on AMP for oxygen utilization and phosphorylation. ADP was not tested as an acceptor because of the hydrolytic enzymes present in the extracts. When extracts were treated with char- coal and used immediately, a partial dependency on AMP for oxygen utilization and phosphate esterification was obtained. If the values for Pi uptake and oxygen utilization obtained with the “complete” and “complete without ,4MP” are subtracted, as shown in Table III, good agreement is obtained between oxygen utilization and phosphate esterification. IMP, GMP, UMP, and CMP were also tested for activity as phosphate

ORIGIN

---I+ AMP ADP ATP

COMPLETE

COMPLETE WITHOUT S,03-- 0

%TMHpLAETTpE 0 @a0

COMPLETE WITHOUT S20,--

t 0 0

WITH ATP

FIG. 1. Incorporation of P32 into ADP during the oxidation of thiosulfate in the presence of ATP. The complete system con- tained, in micromoles: Tris, pH 8, 300; AMP, 10; P3204s, 5, con- taining 8.5 X lo5 c.p.m.; NaF, 100; EDTA, 10; GSH, 40; Na&03, 10; and where indicated ATP, 20. T. thioparus extract, 15 mg in a total volume of 2 ml; time, 40 minutes. Outlined areas, ultraviolet quenching; shaded areas, radioactivity, which was determined by radioautography.

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January 1962 H. D. Peck, Jr., and E. Fisher, Jr. 193

acceptors in this system. None of these nucleotides showed any significant activity. Sulfate was not determined in these reac- tion mixtures; however, the dependency of phosphorylation and sulfate production on the presence of Pi is shown in Table IV. The production of sulfate may occur without phosphorylation in other ways still consistent with this mechanism. Sulfite, produced by thiosulfate reductase, may be oxidized to sulfate by a nonphosphorylating sulfite oxidase (22). In the presence of AMP and absence of phosphate, APS may accumulate. Addi- tion of trichloroacetic acid or acid-BaClp for the determination of sulfate would hydrolyze the APS to AMP and sulfate. Both of these mechanisms for nonphosphorylative sulfate formation would make the oxygen utilized and sulfate produced greater than the phosphate esterified. In Table IV, the fact that the sulfate formed in the “complete” is equal to the amount of AMP added suggests that the sulfate arises here from the acid hy- drolysis of APS.

Effect of Arsenate-Santer el al. (2) showed that arsenate will replace phosphate during the oxidation of thiosulfate. The role of arsenate in replacing phosphate in enzymatic reactions is well known, and it seemed that arsenate was functioning in a similar manner in T. thiopurus. However, the effect of arsenate in this system might be direct or indirect. The suggestion for a direct effect of arsenate comes from the observations of Robbins and Lipmann (8), who showed that in the presence of arsenate, ADP- sulfurylase catalyzes an arsenolysis of APS. Arsenate could also liberate Pi or AMP by reactions other than ADP-sulfurylase and thereby indirectly stimulate thiosulfate oxidation by making Pi or AMP available td the system. Arsenate does not inhibit

TABLE III

E$ect of AMP on oxygen utilization and phosphate esterijication

The complet,e reaction mixture contained, in micromoles : Tris, pH 8,300; AMP, 10; Pi, 5; EDTA, 10; NaF, 100; GSH, 40; NazSz03, 10. T. thioparus extract, 17.7 mg in a total volume of 2 ml. Time, 40 minutes..

system 02 utilized

pmoles

(1) Complete.. 5.3 (2) Complete without Sz03=. 1.8 (3) Complete without AMP.. 3.4 (4) Complete without AMP,

szoa-. 1.5 Net change : (1) without (3)

TABLE IV

A

pntoles

3.5

1.9

1.6

Pi re- maining

pmoles

4.5 6.4 5.8

6.1

A

pzoles

1.9

0.3

1.6

Effect of arsenate and phosphate on sulfate formation

The complete system contained, in micromoles: Tris, pH 8, 300; EDTA, 10; NaF, 100; AMP, 1; Na&03, 10; GSH, 40; and where indicated Pi, 5, and NazAs04, 10. T. thioparus extract, 28.1 mg in a total volume of 2 ml. Time, 40 minutes.

system Pi esterified SOh= formed

pmles

Complete............................. 0 1.07 Complete with P04’-. 1.35 2.70 Complete with As04’. 0 3.06

TABLE V

Requirement of GSH for oxidation and phosphorylation

Each reaction mixture contained, in micromoles: Tris, pH 8, 300; EDTA, 10; NaF, 100; AMP, 10; Pi, 5; NazSz03, 10; and GSH, as indicated. T. thioparus extract, 20 mg in a total volume of 2 ml. Time, 30 minutes.

Additions SOa= produced Pi esterified

pmoles

None............................... 0 0

GSH,2pmoles...................... 0.8 0.7 GSH, 5 pmoles 2.0 1.2 GSH, 20 pmoles 3.5 1.9 GSH, 40 pmoles 4.5 1.8

sulfate production or phosphate esterification in extracts when arsenate and phosphate are present in equimolar quantities. In the absence of phosphate and presence of 10 pmoles of AMP, the formation of sulfate was observed. The sulfate in this nstance could have arisen from the acid hydrolysis of APS and not by the arsenolysis of APS. An experiment was performed in which the reaction mixtures contained only catalytic amounts of AMP. The results are shown in Table IV. As much sulfate was produced in the presence of arsenate as in the presence of phosphate, and 3 times as much sulfate was produced in the presence of arsenate as there was AMP added to the system. This result indicated that it was possible for arsenate to stimulate sulfate production by an arsenolysis type of reaction. In whole cells, the stimulation of thiosulfate oxidation can therefore be the result of the release of AMP by the arsenolysis of APS.

Role of GSH-The presence of thiosulfate reductase (equation 11) in extracts of T. thiopurus has been noted previously, and the formation of both sulfite and sulfide has been observed (4). This enzyme, which was described by Kaji and McElroy (17) from yeast, increases the rate of reaction between GSH and thiosulfate. The enzyme is remarkably stable to heat and is inhibited by sulfite, one of the products of the reaction. GSH can be replaced by homocysteine or cysteine, and the reaction with GSH can be summarized as follows:

2GSH + SzOJ= ---f GSSG + SO,= + H2S (11)

The main evidence for the involvement of this enzyme in the oxidation of thiosulfate by extracts of T. thioparus, besides the presence of the enzyme in extracts, is the observation that sub- strate amounts of GSH are required for the formation of sulfate and phosphorylation (Table V). GSSG was not active in the system, an observation indicating that the requirement was for GSH and not for GSSG. Cysteine is inactive for thiosulfate oxidation and phosphorylation but was itself very rapidly oxi- dized by the system.

In Table VI, the rates of oxygen utilization, phosphate esterifi- cation, and sulfate production at different hydrogen ion concen- trations are shown. The reaction proceeds most rapidly at pH values between 8 and 9. Below a pH value of 7, there is essen- tially no reaction. The pH optimum for the thiosulfate reduct- ase of T. thioparus as well as that of yeast (17) lies between the pH values of 8 and 9 and activity drops very rapidly between the pH values of 8 and 7. This behavior of the thiosulfate- oxidizing system is in agreement with data described in a later

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TABLE VI

E$ect of pH on oxidation of thiosulfate Each reaction mixture contained, in micromoles: Tris-maleate

buffer, 300; AMP, 10; TPN, 0.67; Pi, 5; NaF, 100; EDTA, 10; GSH, 40; Na&s03, 10. T. thioparus extract, 14 mg in a total volume of 2 ml. Time, 40 minutes. Control values for reaction mixtures without thiosulfate have been subtracted.

PH

6.1 7 7.4 8.6 9.2

Oa utilized Pi esteri6ed Sod= produced

@ales p?f%OleS /moles

0 0 0 0.51 0 0 1.16 0.32 0.8 3.2 1.6 2.62 2.6 0.65 1.56

TABLE VII

Formation of polythionate by extracts

The reaction mixture contained, in micromoles: Tris, pH 8, 300; AMP, 10; NaF, 100; EDTA, 10; Pi, 5; NazSz03, 9.0. 7’. thio- parus extract, 17.7 mg in a total volume of 2 ml.

Time SzOa’ Polythionate Total as SrOa’

nzin

0

10 20

$moles remaining pmoles tetrathionate

formed p?d‘3

8.6 0.2 9.0 6.5 1.25 9.0 5.7 1.65 9.0

section of this paper that implicate the reductive cleavage of thiosulfate by thiosulfate reductase as the limiting reaction in the oxidation of thiosulfate.

Thiosulfate can be oxidized directly to tetrathionate by tetra- thionase, and tetrathionate and other polythionates have been suggested as intermediates in the oxidation of thiosulfate (1). Tetrathionase exhibits maximal activity between the pH values of 5 and 6 (22). At a pH value of 8, the specific activities of tetrathionase and thiosulfate reductase are of the same magni- tude (0.3 to 0.6 pmole per hour per mg of protein). This value for thiosulfate reductase is considerably larger than that pre- viously reported (4) but the similar specific activities of these two enzymes indicated that the possible role of tetrathionate in this system should be investigated.

Oxidation of Tetrathionate, Suljite, Suljide, and Elemental Sulfur-The first experiments, in which the utilization of thio- sulfate was examined, indicated that the same amount of thio- sulfate had disappeared in the flasks containing thiosulfate alone as in those containing thiosulfate and GSH. As shown in Table VII, when the utilization of thiosulfate (in the absence of GSH) was followed with time, the disappearance of thiosulfate could be accounted for by the appearance of polythionates, presumably tetrathionate. In reaction mixtures containing both thiosulfate and GSH, there was disappearance of thiosulfate that could not be accounted for by the appearance of polythionate, but rather by the appearance of sulfate (see Table V). In terms of micro- moles, the amount of thiosulfate disappearing was usually less than the amount of sulfate formed. The reason for this is that sulfide itself can be oxidized to thiosulfate,2 in addition to ele- mental sulfur, by these extracts and the greater the proportion

2 H. D. Peck, Jr., unpublished experiments.

of sulfide oxidized to thiosulfate, the less will be the disappearance of thiosulfate. Whole cells of T. thioparus (1) and extracts of Thiobacillus X (23) oxidize thiosulfate to tetrathionate, and a cyclic mechanism involving polythionate has been proposed to account for the oxidation of thiosulfate to sulfate (1). Since tetrathionate was formed by the extract at a rate comparable with the rate of sulfate formation, tetrathionate, recrystallized several times to remove impurities, was utilized as substrate for phosphate esterification and sulfate production. As shown in Table VIII, tetrathionate alone was not metabolized; however, in the presence of GSH, phosphate esterification and sulfate produc- tion were identical to that observed in the presence of thiosulfate and GSH. The fact that the rates of sulfate production and phosphate esterification were the same with tetrathionate and thiosulfate suggested that tetrathionate was being hydrolyzed to thiosulfate and metabolized as described previously. This interpretation was substantiated by the observation that tetra- thionate was nonenzymatically and quantitatively cleaved to thiosulfate by an excess of GSH. These observations do not completely rule out the possibility that tetrathionate is an inter- mediate in the oxidation of thiosulfate by whole cells; however, it does seem that in this cell-free system of thiosulfate oxidation, tetrathionate is not an intermediate.

According to the reaction sequence for the oxidation of thio- sulfate, it should be possible for this extract to utilize sulfite as substrate for phosphate esterification and sulfate production. In addition, with sulfite as substrate, GSH should not be required for these activities. When sulfite is employed as sub- strate (Table IX), oxygen is utilized, sulfate produced, and phos- phate esterified. In accord with the postulated reaction se- quence, in other experiments, it was shown that GSH is neither required nor does it enhance these reactions. The fact that sulfate production and phosphate esterification are greater than observed with thiosulfate and GSH indicates that, in this cell-free system, the reductive cleavage of thiosulfate is the limiting re- action.

The possible involvement of sulfide in the oxidation of elemental sulfur was suggested by Starkey (24), who observed the forma- tion of small amounts of HzS from elemental sulfur. This idea was reiterated by Parker and Prisk (25), who observed the oxida- tion of sulfide to sulfate by whole cells of T. thiooxidans as well as of T. thioparus. Possible intermediates in this oxidation are indicated by the observation of Vishniac and Santer (1) that, during the oxidation of S35-labeled sulfide, the transitory forma- tion of labeled thiosulfate and polythionate can be observed. Suzuki’and Werkman (19), employing extracts of T. thiooxidans,

TABLE VIII Oxidation of thiosulfate and tetrathionate by extracts

Each reaction mixture contained, in micromoles: Tris, pH 8, 300; AMP, 10; EDTA, 10; NaF, 100; Pi, 5; and where indicated Na.&03, 10; K&06, 10; GSH, 40. T. thioparus extract, 40 mg in a total volume of 2 ml. Time, 40 minutes.

Additions Pi ester&d SO,= produced

pmoles

s,oa- . . 0 0

GSH . . . . . 0 0 SzOa- + GSH 1.7 2.0 S,O6’. . 0 0 Sa06- + GSH. . 1.9 / 2.1

-

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demonstrated the oxidation of sulfide to thiosulfate. However, they did not observe either the formation of sulfate and the esterification of phosphate or the formation of polythionates. When the starting substrates were elemental sulfur and GSH (that react to form sulfide), polythionates were produced by a nonenzymatic reaction. The oxidation of sulfide in this organism seems to be similar to the sulfide-oxidizing system present in rat liver (26). The significance of this reaction may be questioned, however, since ferritin (27) and modified heme compounds (28) also catalyze the formation of thiosulfate from sulfide.

When sulfide was added to a reaction mixture designed to permit phosphorylation, the formation of sulfate and phosphate esterfication was observed (Table IX). This observation in- dicated that the whole system for the oxidation of sulfide to sulfate was present in these extracts of T. thioparus.

Oxidation of S3%03- and &Y503- by Whole Cells-Whole cells of T. thioparus deposit large amounts of elemental sulfur when grown under certain cultural conditions (see Vishniac and Santer (1)). The first indication that the sulfur atoms of thiosulfate were metabolized by different pathways was presented by Skar- zynksi et al. (16). They showed that during growth the culture medium (after cells and elemental sulfur were removed) lost radioactivity when the cells were grown on S35SOsB (outer label) but did not lose radioactivity when grown on SL!Y603- (inner label). They further demonstrated that only the outer sulfur atom was incorporated into cellular material. These observa- tions are not necessarily opposed to the scheme of Vishniac and Santer (l), in which elemental sulfur arises by the breakdown of pentathionate. In fact, it seems essential that whether thio- sulfate is metabolized by an initial reductive cleavage to sulfide and sulfite or metabolized via a cycle involving polythionates, elemental sulfur should arise mainly from the outer sulfur atom of thiosulfate.

Recently, however, Santer et al. (2) reported equal utilization of both sulfur atoms for sulfate production. Since the mecha- nism of thiosulfate oxidation involving an initial reductive cleavage of thiosulfate requires that there be at least a preferen- tial utilization of the inner sulfur atom for sulfate production, the problem was reinvestigated. Whole cells of T. thioparus were allowed to oxidize thiosulfate labeled either in the outer (S35S03-) or inner (SS3503-) sulfur atom. An unlabeled pool of

TABLE IX . .

Oxzdatzon of SZO~~, SOP, and S3 Each reaction mixture contained, in micromoles: Tris, pH 8,

300; AMP, 10; EDTA, 10; NaF, 100; Pi, 5; and where indicated Na&&03, 10; GSH, 40; N&03, 10; Na& 10; and T. thioparus extract, 29.5 mg in a total volume of 2 ml. Time, 30 minutes. NaOH was not added to the center wells of flasks containing sulfite or sulfide.

system . . SO&’ 02 uthzed Pi esterified produced

pnoles pmoles pmoles

Extract . . 0 0 0 Extract + SzOz-.................. 0 0 0 Extract + GSH.. . 0 0 0 Extract + S203- + GSH. . . 2.7 2.0 4.2 Extract + SOa--. 2.5 4.5 7.8 Extract + S-. . . 3.3 1.8 4.0 so3’. . . . . 0 0 0 s- . 0 0 0

TABLE X

Distribution of radioactivity in sulfate and elemental sulfur formed during oxidation of S%!303- and SSs608-

Each flask contained: phosphate buffer, pH 7, 100 pmoles; So, 100 mg; and where indicated 0.5 ml of a 10% suspension of whole cells in 0.01 M Tris, pH 8; Sa5S03-, 40 pmoles containing 2.5 X 107 c.p.m., and SS3603-, 40 pmoles containing 3.5 X 107 c.p.m. Reac- tion mixtures (total volume, 2 ml) were incubated for 60 minutes.

Additions SO4” Total radioactivity

produced Recovery

BaSO4 SO

p?noles c.p.m. %

SW0 a- 0 4.2 X lo3 SEF503- + cells. . . 24.0 2.07 X 107 1.5 X lo6 60 s35so3-. 0 3.2 X lo4 S35SOd + cells. 28.4 1.23 X lo7 1.03 X 10’ 90

elemental sulfur was included in each reaction mixture to pre- vent the reutilization of any labeled elemental sulfur that might be formed. Control mixtures that were only lacking whole cells also were analyzed. As shown in Table X, when SS3503- was oxidized, significant label was found only in the sulfate formed. Oxidation of LY5S03” under identical conditions resulted in equal labeling in both the sulfate and elemental sulfur. These results indicate that there is preferential utilization of the sulfur atoms of thiosulfate. It is of interest that in the absence of the pool of elemental sulfur, i.e. the conditions used by Santer et al. (2), there was only a small indication of the preferential utilization of the sulfur atoms of thiosulfate.

DISCUSSION

The first step in the oxidation of thiosulfate to sulfate seems to be the reductive cleavage of thiosulfate to sulfite and sulfide. The oxidation of sulfite and formation of sulfate in extracts of T. thioparus are postulated to require the presence of Pi, AMP, and two enzymes, APS-reductase (Equation 3) and ADP-sul- furylase (Equation 4). Pi is esterified and appears in the ter- minal phosphate of ADP. The direct AMP-dependent oxidation of sulfite to APS in the presence of Fe(CN)6Z has also been ob- served (7), and presumably the same enzyme catalyzes this reaction that catalyzes the reduction of APS to AMP and sulfite. Although extracts can form 3’-phosphoadenosine-5’-phosphosul- fate as well as APS,2 they are unable to reduce this sulfur-contain- ing nucleotide to sulfite; i.e. these preparations lack a yeast-type sulfate reductase (4).

Another pathway of sulfite oxidation that does not involve a phosphorylation has been reported to occur in many tissues (22). The nonphosphorylative pathway does not seem to have any resemblance to the phosphorylative pathway since the former is inhibited by arsenite and the latter is not affected by this inhibitor. A stimulatory effect of AMP on sulfite oxidation has previously been described (29) ; however, neither the esterifi- cation of phosphate nor the formation of APS was observed. The nonphosphorylative pathway of sulfite oxidation does not seem to be operative in T. thioparus since the 018 studies of Santer (3) can be interpreted to indicate that essentially all of the sulfate formed during the oxidation of thiosulfate was at some time in a covalent bond through oxygen with phosphate. The presence of APS-reductase in extracts of T. thiooxidan,+ and Thiobacillus denitrijkans2 as well as in extracts of T. thio-

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196 Thiosulfate Oxidation and Phosphorylation in Thiobacillus Vol. 237, No. 1

parus, and the observations on the system described here, indi- cate that Equations 3 and 4 correctly represent both the pathway of sulfite oxidation in T. thioparus and a general mechanism for the oxidation of sulfite to sulfate in the thiobacilli.

The origin of sulfite from tetrathionate, sulfide, and elemental sulfur is still in doubt and poorly understood, particularly with regard to the role of polythionates in the oxidation of these sulfur compounds. The enzymatic activity, described in ex- tracts of yeast (17) and D. desulfuricans (18), that catalyzes the reductive cleavage of thiosulfate to sulfite and sulfide is present in extracts of T. thioparus and seems to be the only way that sulfite is formed from thiosulfate by these extracts.

It has been postulated repeatedly (see Tamiya et al. (30), Vishniac and Santer (l), and Lees (31)) that sulfite or sulfate is formed from more reduced sulfur compounds by a cyclic mecha- nism involving polythionates. The possible role of tetrathionate in this pathway has been most intensively investigated, and many strains of thiobacilli produce tetrathionate from thiosulfate and are able to utilize it as a substrate for growth (1). However, this does not seem to be a universal observation (25). Other evidence for this pathway was obtained with whole cells by the isolation of polythionates formed during the oxidation of labeled sulfur compounds (1, 32). Enzymological evidence for the in- volvement of tetrathionate is scanty and consist mainly of the observation that extracts of Thiobacillus X osidize thiosulfate to tetrathionate (23). This enzyme seems to be similar to tetra- thionase previously described from Proteus vulgaris (33).

Extracts of T. thioparus, although able to form tetrathionate from thiosulfate, do not form sulfate unless GSH is added to the reaction mixture. When GSH is added, tetrathionate seems to be hydrolyzed to thiosulfate and is further metabolized. Since the oxidation of sulfide to thiosulfate does not seem to involve the formation of polythionates (19), a pathway for the oxidation of elemental sulfur and sulfide can be proposed that utilizes known reactions and does not involve polythionates:

So + 2GSH --f H& + GSSG (12) 2SH- + 202 ---t SzO3- + Hz0 (13)

Thiosulfate is then oxidized to sulfate, as described in Equations 1, 3, and 4. The reaction described by Equation 12 has been reported by Suzuki and Werkman (19) and may be nonenzy- matic. The second reaction (Equation 13) has been shown in rat tissue (26) as well as extracts of T. thiooxidans. Tetra- thionate does not have a role in this scheme and its occurrence is regarded as incidental to the over-all scheme. With regard to the elemental sulfur formed by whole cells, sulfide originating from thiosulfate may be oxidized to elemental sulfur to supply electrons for the reduction of GSSG to GSH that is required for the reductive cleavage of thiosulfate.

Since T. thioparus can generate a high energy sulfate during the oxidation of sulfite to sulfate, the possible importance of this reaction in the over-all energy economy of the cell is of interest. It had been assumed previously that the thiobacilli obtained all the energy required for growth from oxidative phosphorylation (1). The evidence for oxidative phosphorylation consists of the presence of cytochromes (23) and the stimulation of oxidative metabolism by 2,4-dinitrophenol (21). However, it has been claimed that T. thiooxidans possesses no cytochromes (34). Oxidative phosphorylation has not been demonstrated in ex- tracts of these organisms, and in the absence of this data, the question can be asked whether the high energy phosphate

formed by the substrate sulfurylation is sufficient to account for the growth of the organism.

The proposed reaction sequence is a mechanism whereby the chemical energy liberated by the oxidation of reduced sulfur compounds can be transformed into biologically utilizable energy in the form of adenosine phosphates. However, it is possible that in some energy-requiring reactions of the cell, the energy of the nucleotide-bound sulfate is directly utilized. The production of a high-energy sulfate by oxidation of sulfite seems to be uniquely characteristic of the Thiobacilli and possibly some photolithotropic microorganisms.2

SUMMARY

We prepared cell-free extracts of Thiobacillus thioparus that oxidize thiosulfate to sulfate. The oxidation requires substrate amounts of reduced glutathione, and orthophosphate is esterified with the formation of adenosine diphosphate in an amount equal to the sulfate produced in the oxidation. The initial reaction is the reductive cleavage of thiosulfate to sulfite and sulfide with- out the intermediate formation of polythionates. The sulfite is subsequently oxidized in the presence of adenosine monophos- phate to adenosine 5’-phosphosulfate. Phosphorolysis of adeno- sine 5’-phosphosulfate by adenosine diphosphate sulfurylase yields sulfate and adenosine diphosphate. These extracts also oxidize sulfite and sulfide to sulfate with the concomitant esteri- fication of phosphate.

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H. D. Peck, Jr. and E. Fisher, Jr.thioparus

ThiobacillusThe Oxidation of Thiosulfate and Phosphorylation in Extracts of

1962, 237:190-197.J. Biol. Chem. 

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