JouRNAL OF BACTERIOLOGY, Dec. 1978, p. 916-9230021-9193/78/0136-0916$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 136, No. 3

Printed in U.S.A.

Dissimilatory Reduction of Bisulfite by Desulfovibrio vulgarisH. L. DRAKEt AND J. M. AKAGI*

Department ofMicrobiology, University ofKansas, Lawrence, Kansas 66045

Received for publication 6 September 1978

The reduction of bisulfite by Desulfovibrio vulgaris was investigated. Crudeextracts reduced bisulfite to sulfide without the formation (detection) of anyintermediates such as trithionate or thiosulfate. When the particulate fractionwas removed from crude extracts by high-speed centrifugation, the solublesupernatant fraction reduced bisulfite sequentially to trithionate, thiosulfate, andsulfide. Addition of particles or purified membranes to the soluble fractionrestored the original activity demonstrated by crude extracts, i.e., reduction ofbisulfite to sulfide without the formation of trithionate and/or thiosulfate. Byusing antiserum directed against bisulfite reductase, the reduction of bisulfite bycrude extracts was inhibited. This finding, in addition to several recycling studiesof thiosulfate reduction, provided evidence that bisulfite reduction by D. vulgarisoperated through the pathway involving trithionate and thiosulfate as interme-diates. The role of membranes in this process is discussed.

Sulfate-reducing bacteria, belonging to thegenera Desulfovibrio and Desulfotomaculum,are unique because they can utilize inorganicsulfate as a terminal electron acceptor and formcopious amounts of hydrogen sulfide as an endproduct. This dissimilatory process is in contrastto the assimilatory reduction of sulfate wheresmall amounts of sulfate are reduced and sub-sequently assimilated into cellular material. Thereductive processes for the reduction of sulfatecan be separated into two phases: the reductionof sulfate to (bi)sulfite and the reduction of(bi)sulfite to sulfide.

In the first phase sulfate is activated via ATPsulfurylase (EC 2.7.7.4, ATP:sulfate adenylyl-transferase) activity (1, 14, 21, 24), forming ad-enylylsulfate, which is subsequently reduced to(bi)sulfite plus AMP by adenylylsulfate reduc-tase (15, 29, 31). The formation of adenylylsul-fate and pyrophosphate, from ATP and sulfate,is a reversible reaction in favor of ATP andsulfate; however, the reaction is driven to theright by the hydrolysis of pyrophosphate byinorganic pyrophosphatase (2, 24, 39, 40). Thesecond phase of the dissimilatory reduction, in-volving the reduction of bisulfite to sulfide, hasnot been clearly established. The main issue hasbeen whether or not bisulfite is directly reducedto sulfide without any detectable intermediates,or whether bisulfite is reduced through a path-way consisting of trithionate and thiosulfate asinternediates as predicted by the earlier works

t Present Address: Department of Biochemistry, School ofMedicine, Case Western Reserve University, Cleveland, OH44106.

of Kobayashi et al. (21) and Suh and Akagi (38).We recently reported the in vitro reconstitutionof a thiosulfate-forming pathway of Desulfovi-brio vulgaris which consisted of bisulfite reduc-tase, a thiosulfate-forming enzyme (TF), hydro-genase, and the native electron carriers cyto-chrome C3 and flavodoxin (8). This, togetherwith the purification ofthiosulfate reductase (12,13), suggests that one pathway for the reductionof bisulfite to sulfide involves the intermediatestrithionate and thiosulfate. Although trithionatereductase activity has not been purified to date,it is possible that this enzyme may also be in-volved in the dissimilatory pathway. Figure 1illustrates the possible pathways for the dissi-milatory reduction of bisulfite to sulfide.This study was initiated to further probe the

route(s) of bisulfite reduction by extracts of D.vulgaris. We present evidence which suggeststhat trithionate and thiosulfate are intermedi-ates in the in vivo reduction of bisulfite to sulfideand that membranes play a paramount role inthis process.

MATERLALS AND METHODSOrganism. D. vulgaris NCIB 8303 was grown and

harvested as previously described (1).Assay conditions. Standard manometric tech-

niques were used throughout this study, employingWarburg flasks of approximately 8-ml capacity. Unlessotherwise indicated, the standard assay mixture con-tained: potassium phosphate buffer, pH 7.0, 50 pmol;extract and substrate(s) in a total volume of 1.1 ml.The center well contained 0.1 ml of a 20% CdC12solution absorbed on fluted filter paper. The gas phasewas H2, and the incubation temperature was 30°C.

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DISSIMILATORY REDUCTION OF BISULFITE 917

ASSIMILATORY REDUCTION, 6 E

I HO IHS03 2

2 E I

2 32

HSO~- 2E 002C s2QE3s2BISULFITE S3O6 S 03 THIOSULFATEREDUCTASE REDUCTASE so 2-

TRITHIONATE A,NREDUCTASE So32-

FIG. 1. Pathways for the reduction of bisulfite to sulfide.

Since the pH optima for bisulfite reductase, TF, tri-thionate reductase, and thiosulfate reductase activitiesare 6, 6, 7, and 8, respectively (6-8, 12, 13, 23; this lab,unpublished data), a compromising pH of 7.0 was used.

Preparation of crude extract, soluble fraction,and membranes. Unless otherwise indicated, all pro-cedures were performed at 0 to 40C. A 50% cell sus-

pension of a wet cell paste in 0.05 M potassium phos-phate buffer (PB), pH 7.0, containing 0.1 mg of deox-yribonuclease per 100 ml was passed through a Frenchpressure cell at 7,500 lb/in2. The lysate was centrifugedat 8,000 x g for 20 min. The supernatant fraction,designated crude extract (CE), was stored under an H2atmosphere at room temperature for 60 min to depletethe CE of endogenous inorganic sulfur compounds. ACdCl2 trap (absorbed on a filter paper and suspendedfrom the rubber stopper) was used to remove thesulfide formed during this time. The CE was centri-fuged at 100,000 x g for 90 min, and the supernatant,designated USS, was stored at -20°C under H2. Thisrepresented the soluble fraction. The precipitate wassuspended in 0.05 M PB, pH 7.0, and repelleted bycentrifugation (100,000 x g, 30 min). The precipitatewas suspended in the same buffer (35%, wt/vol), and2-ml aliquots were applied to centrifuge tubes contain-ing 23 ml of 20 to 60% linear sucrose gradients equili-brated with 0.01 M PB, pH 7.0. After centrifugation at78,000 x g for 90 min, the milky gray band whichformed three-fourths of the way down the gradientwas removed and dialyzed against 0.01 M PB, pH 7.0,until free of sucrose. The dialyzed material was con-centrated by Amicon ultrafiltration (PM-30 mem-brane) and subjected to a second similar sucrose gra-dient centrifugation. The membrane fraction, whichmigrated to the bottom of the tubes, was suspended in0.05 M PB, pH 7.0, dialyzed, and stored at -20°C.This fraction represented the partially purified mem-brane fraction. Other than hydrogenase activity, themembranes did not contain any of the reductasesinvolved in dissimilatory bisulfite reduction. Hydro-genase activity was measured by the reduction ofmethyl viologen by the enzyme in a hydrogen atmo-sphere.

Preparation of rabbit antiserum directedagainst bisulfite reductase. Bisulfite reductase waspurified as described previously (7, 8). One milligramof bisulfite reductase, in complete Freund adjuvant,was injected via three routes (subcutaneously, intra-muscularly, and intravenously) into a 10-week-old rab-bit. A secondary injection, identical to the first, was

made 3 weeks later. Three weeks later, the animal wasbled from the ear, and the antiserum fraction (a-BR)was stored in 2-ml aliquots at -20°C.Treatment of CE and USS with a-BR serum.

Standard titration techniques were used to determinethe optimum extract-a-BR serum ratio which yieldedmaximum precipitation. After the precipitation was

complete (20 to 30 min at room temperature), thegreen precipitate was removed by low-speed centrifu-gation. The supernatant fluid was used as a-BR-treated extract. Removal of the precipitate was notrequired for inhibition of bisulfite reductase activity.Bisulfite reductase fluoresces red when exposed to UVlight (365 nm) under alkaline conditions (33, 34). Sinceno fluorescence was observed with a-BR-treated ex-

tracts, we concluded that the precipitation procedureeffectively removed all of the bisulfite reductase activ-ity.

Analytical determinations. Thiosulfate and tri-thionate were determined according to Kelly et al. (18)as previously described (3). Sulfide was analyzed bythe method of Fogo and Popowski (11). [3S]sulfidewas determined as described previously (27). Proteinwas estimated according to Lowry et al. (25), usingbovine serum albumin as a standard. [3S]bisulfite wasvolatilized as 'SO2 by the addition of 0.1 ml of 20 NH3PO4 and quantitated as described previously (8).The isolation and degradation of [3S]thiosulfate

was performed by methods previously described (7,10). [35S]trithionate was isolated from reaction mix-tures by thin-layer chromatography, as described ear-

lier (3). An alternate and faster method of trithionatedegradation was developed; this method involves thedirect precipitation of the inner (sulfane) atom withsilver. To a suitable fraction of trithionate is added 1.5ml of 0.2 M AgNO3. The reaction is stoppered and

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918 DRAKE AND AKAGI

placed in a 50°C water bath for 5 min. The reactionoccurring is: -03S-S*-SO3- + 2 Ag++ 2 H20-+ Ag2S*+ 2 S042 + 4 HW. The silver sulfide precipitation iscollected by filtration, dried, and counted. The sulfo-nate atoms are quantitated by methods previouslydescribed for the sulfonate sulfur group of thiosulfate(7). The validity of this degradation scheme was con-firmed by utilizing [3S]sulfane- and [3S]sulfonate-la-beled trithionate. Furthermore, identical results were

obtained with previously described methods of tri-thionate degradation (7).

Trithionate was synthesized by modification of themethod of Roy and Trudinger (36) as described byAkagi et al. (3). 3S-inner-labeled trithionate (03S-'S-SO3 ) was synthesized by supplementing the sulfurdichloride-ether solution with 3SC12 (4 mCi). [3S]sul-fonate-labeled trithionate (O03'S-S-'SO3 ) was syn-thesized by supplementing the metabisulfite solutionwith Na2'SO3 (4 mCi). Na23SO3, [3S]sulfane-labeledsodium thiosulfate, and 'SC12 (special order) were

purchased from New England Nuclear Corp. [3S]sul-fonate-labeled sodium thiosulfate was purchased fromAmtrsham Corp. Sodium bisulfite, potassium trithion-ate, and sodium thiosulfate solutions were freshly pre-pared for each experiment in 0.001 M disodium eth-ylenediaminetetraacetate. Periodically, the Na2'SO3solutions were checked for any oxidation to Na235SO4by acid volatilization and trapping the volatile 'SO2in hyamine hydroxide.

RESULTS

Bisulfite reduction by cell extracts. Table1 shows the products formed from the substratesbisulfite, thiosulfate, and trithionate by CE andthe soluble fraction (USS) of the CE. Both ex-tracts reduced trithionate to thiosulfate plus sul-fide and thiosulfate to sulfide. The combinationof bisulfite and trithionate was reported to berequired for thiosulfate formation (8) by a thio-sulfate-forming enzyme (TF). When this com-

bination was tested as substrates for CE andUSS, a marked increase in hydrogen consump-tion and products formation was observed withCE. A significant difference in the productsformed from bisulfite by CE and USS was ob-served (Table 1). The CE reduced bisulfite tosulfide without the formation (detection) of tri-thionate or thiosulfate. In contrast, the solublefraction reduced bisulfite primarily to thiosul-fate. Since the difference between CE and USSwas the lack of particulate material in the latterfraction, membranes were suspected to be in-volved in the bisulfite-reducing process. Whenpartially purified membranes and USS were in-cubated with bisulfite, a restoration of CE activ-ity was observed; i.e., sulfide was essentially thesole product.The effect of time on bisulfite reduction by

USS is seen in Fig. 2. Most apparent is thatthiosulfate accumulated in the reaction mixture

J. BACTERIOL.

and subsequently disappeared. Concomitantwith decreasing thiosulfate concentration, sul-fide formation increased. This suggests that bi-sulfite was reduced to sulfide through the inter-mediate, thiosulfate. Whereas trithionate for-mation was not readily apparent (Fig. 2), it ispossible that bisulfite was reduced to trithionate,which rapidly converted to thiosulfate. We pre-viously reported (8) that a thiosulfate-formingsystem (bisulfite reductase plus TF) reducedbisulfite to thiosulfate without forming signifi-cant quantities of the intermediate, trithionate.Perhaps, under steady-state conditions of bisul-fite reduction, the level of trithionate remainslow and thiosulfate formation becomes appar-ent. This possibility might exist with USS sincewe noted (unpublished data) that bisulfite ionswere inhibitory to thiosulfate reductase activity.This inhibition was more apparent with USSthan with CE. If thiosulfate reductase is in-hibited by bisulfite ions, thiosulfate would notbe expected to be reduced until the bisulfiteconcentraton was reduced. This would explainwhy thiosulfate accumulated before sulfide for-mation in Fig. 2.Reduction of [35Sjbisulfite. When CE re-

duced H3SO3- to 3S2-, no decrease in the spe-cific activity of the isotope was noted (Table 2).When unlabeled trithionate or thiosulfate wasadded to the reaction mixture, the specific activ-ity of the 3S2- was considerably lower, suggest-ing that H3SO3- was reduced to 3S2- throughthe intermediates trithionate and thiosulfate. If,during H3SO3- reduction by CE, unlabeled tri-thionate was added and immediately isolatedfrom the reaction mixture, no radioactivity wasdetected in the trithionate molecule. When un-labeled thiosulfate was added, instead of tri-thionate, the thiosulfate molecule became radio-active. However, upon chemical degradation ofthe thiosulfate molecule, the sulfonate sulfuratom was the only species which contained thelabel. It was subsequently determined that arapid exchange reaction occurred betweenH SO3- and the sulfonate group of thiosulfate.This exchanged reaction also occurred in reac-tion mixtures incubated under nitrogen and ox-ygen but did not occur in nonenzymatic controlsor with CE after exposure to a boiling-waterbath for 10 mn.

In contrast, when the identical experimentswere conducted with USS, 35S was found to besignificantly distributed in the sulfur atoms mak-ing up the trithionate and thiosulfate molecules(Table 3). For the thiosulfate molecule the un-even distribution of radioactivity (reaction A)was probably due to the exchange occurringbetween the sulfonate sulfur atom and bisulfite.

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DISSIMILATORY REDUCTION OF BISULFITE 919

TABLE 1. Reduction ofpossible intermediates in dissimilatory bisulfite reduction by CE and USSaProduct.s (,punol) .utlieExtract assayed Substrate Prdt S)H utiilzed

S306 S203203amol)CE HS03- 0 0 2.74 7.51CE S2032- _b - 1.38 2.36CE S3062- 1.65 2.25 5.94CE HS03- + S3062- - 2.57 3.26 10.70USSC HS03- 0 1.48 0.46 3.78USS S2032- - - 2.30 2.55USS S3062- 2.73 0.68 3.68USS HSO3- + S3062- - 2.73 0 4.22USS + membranesd HS03- 0 0.13 2.60 6.10

a Standard assay conditions; CE concentration, 17.8 mg/ml. All substrate concentrations were 5.0 ymol each.Incubation time, 20 min.

b Not done.c USS concentration, 15 mg/ml.d Membrane concentration, 1.5 mg/ml. No dissimilatory reductase(s) activities were associated with the

purified membranes (methyl viologen as carrier). When the membrane preparation was heated in a boiling-water bath for 10 min and then added to USS plus bisulfite, the reaction took on the characteristics of the USSsystem without membranes; i.e., sulfide was not formed immediately as in the USS-plus-membranes system.

TIME (minutes)

FIG. 2. Effect oftime on bisulfite reduction by USS.Standard assay conditions. USS concentration, 17.6mg; NaHSO3, 5.0 pmol. Symbols: trithionate, A; thi-osulfate, 0; sulfide, O; H2 consumption, broken lines.

However, even with the exchange, it is seen thata substantial incorporation of 3 S occurred intothe sulfane atom.

Effect of membranes on the recycling ofthe sulfonate group of thiosulfate. It hasbeen proposed that thiosulfate is reduced bythiosulfate reductase according to the followingequation (10, 12, 13): S-S*032-+S2 + S*0Q2-3The sulfonate group, released as sulfite,

should subsequently be reduced (recycled) toform doubly labeled thiosulfate (10). When CEwas incubated with [35S]sulfonate-labeled thio-sulfate for varying time intervals and the resid-

ual thiosulfate was isolated and degraded, thesulfane atom remained unlabeled (Table 4).When the same experiment was performed withUSS, the sulfane atom became increasingly ra-dioactive with time. When [35S]thiosulfate wasreduced by USS, sulfide was preferentially de-rived from the sulfane atom (Table 5). As thereaction time was increased, the rates for thereduction of the sulfane and sulfonate sulfuratoms approached unity. With CE, both thesulfane and sulfonate sulfur atoms were reducedto sulfide at equal rates. This phenomenon wasreproducible in the USS system by the additionof membranes.Effect of a-BR on bisulfite reduction.

When CE was treated with a-BR, its ability toreduce bisulfite was inhibited; trithionate andthiosulfate reductions were unaffected. Thesame pattern was observed when USS wastreated with a-BR. Normal serum controlsshowed no activity against bisulfite reduction byeither USS or CE. The requirement for bisulfitereductase activity in these extracts for sulfideformation from bisulfite was demonstrated bythe addition of purified bisulfite reductase to a-BR-treated USS and CE (Table 6).

Effect of a-BR on "S-labeled thiosulfate.It was previously noted (Table 5) that CE re-duced both sulfur atoms of thiosulfate at equalrates. If the [t3S]sulfonate groups are released as"free" [3S]sulfite, as predicted for thiosulfatereductase activity, and subsequently recycled toform doubly labeled thiosulfate, the presence ofan unlabeled bisulfite pool should result in adilution of the [36S]sulfonate group. Table 7shows that this was not the case. The reduction

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TABLE 2. Effect of dissimilatory intermediates on the specific activity of[6SJsulfide formed by CE"

Reaction Substrate (pmol) H2 utilized Products (MOI)b [S36S]sulfide ap actno. W"S03 S2Oa- S062- (JAmoI) 5232- S2- Sulfide (cpm) (cpm/,ol)

1 5 0 0 3.19 0.09 1.22 188,252 154,302 (1.00)2 5 5 0 3.73 1.3 157,500 120,229 (0.78)3 5 5 0 3.41 1.08 80,611 74,639 (0.48)4 5 0 1 3.93 0.91 0.86 110,521 128,512 (0.83)5 5 0 5 4.05 1.88 0.34 20,346 59,841 (0.39)

aStandard assay conditions. Each reaction contained 18.9 mg of CE. NaH3SO3 specific activity was 1.6 x 105cpm/,umol. Reaction time was 20 mi.

b No appreciable trithionate formation was detected.c Numbers in parentheses represent the specific activities of each reaction in reference to the specific activity

of reaction no. 1.

TABLE 3. Incorporation of3S into thiosulfate andtrithionate by USS'Unla- % Distribution of

Incuba- beled 358Reac- tion com- Total radio-tion time pound activity(lntmin added (cpm) S S03

and iso-lated

A 25 S2032- 768,435 31 69B 10 S3062- 120,750 38 31, 31

aStandard assay conditions: USS concentration, 20mg/ml. Reaction A contained 5.0 ,umol of H3SO3- (2.2x 10' cpm/umol) plus 5.0 ,umol of unlabeled thiosul-fate. Reaction B contained 5.0 Umol of H'SO3 (1.6x 10' cpm/,mol) plus 5.0,mol of unlabeled trithion-ate.

TABLE 4. Recycling of the sulfonate sulfur atom ofthiosulfate by CE and USSa

Thiosulfate cpm (% distribu-Incubation tion)bExtract time (min)

S S03

CE 15 5.3 94.7CE 30 3.5 96.5CE 45 3.5 96.5USS 7.5 5.8 94.2USS 15 8.3 91.7USS 30 14.3 85.7USS 50 24.4 75.6

a Standard assay conditions. Protein concentrationswere: CE, 20.0 mg; USS, 19.0 mg. Each flask contained5.0 pmol of [t3S]-sulfonate-labeled sodium thiosulfate,1.46 x 106 cpm/umol in CE experiment and 1.30 x 105cpm/,unol in USS experiments.

b Each experiment was performed in duplicate; thepercent distribution represents the average of bothexperiments. The percent recovery varied from 92.0 to105.5.

of both [3S]sulfur atoms of thiosulfate to[3S]sulfide occurred at equal rates even in thepresence of exogenous unlabeled bisulfite. WhenCE was treated with a-BR, only the sulfane

TABLE 5. Effect ofmembranes on the reduction of[3S]thiosulfatea

Mem- Sulfide formed (cpm)from thiosulfate tiContents banded _________RtoAadded [36S] sulfo- ["S]sWul B

(mg) nate (A) fane (B)

CE 48,976 47,424 1.03USS 0 14,219 37,590 0.38USS 0.23 21,068 41,976 0.50USS 0.46 27,885 42,511 0.66USS 0.92 38,767 47,933 0.81USS 1.91 47,792 47,858 1.00USSb 0 64,587 85,936 0.75

a Standard assay conditions. CE concentration, 18.0mg. USS concentration, 12.0 mg. Each reaction con-tained 3.0 pmol of Na2S203, 3.34 x 104 cpm/pmol,sulfonate or sulfane label. Incubation time, 20 min.

b Incubation time, 80 min.

TABLE 6. Reconstitution of bisulfite reduction inextracts treated with bisulfite reductase antiserum"

Products (umol)Contents

S3062- S203 S2-

CE 0.05 0.14 1.00CE + antiserumb 0 0 0.09CE + antiserum + BRC 0.40 0.10 0.97USS 0.10 1.15 0.29USS + antiserumb 0 0 0.03USS + antiserum + 0.92 0.50 0.41BRC

a Standard assay conditions. CE concentrations,17.6 mg. USS concentration, 19.6 mg. All reactionscontained 5.0 umol of NaHSO3. Incubation time, 30mm.

bAntiserum concentrations were 33.0 mg in CEexperiment and 40.0 mg in USS experiment.

c Bisulfite reductase (BR) concentration, 3.2 mg.

atom of thiosulfate was reduced to sulfide (Table7); i.e., the reduction (recycling) of the sulfonategroup was significantly decreased.

Effect of Triton X-100 on the USS-mem-branes system. The requirement for mem-

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DISSIMILATORY REDUCTION OF BISULFITE

TABLE 7. Formation of[3S]sulfide from [3S]-thiosulfate by CE treated with bisulfite reductase

antiserumaSubstrate Sulfide (cpm)

Contents [3 S]thio- Unlabeledsulfate bisulfite Expt 1 Expt 2

(innol)CE + normal Sulfane 0 23,932 25,527

serumnbCE + normal Sulfonate 0 23,254 24,222serum

CE + normal Sulfane 5.0 16,525 18,197serum

CE + normal Sulfonate 5.0 16,057 16,442serum

CE + antise- Sulfane 0 26,523 32,633rum

CE + antise- Sulfonate 0 2,354 6,210rum

CE + antise- Sulfane 5.0 19,246 27,002rum

CE + antise- Sulfonate 5.0 2,024 3,665rum

'Standard assay conditions. Both experiments contained20.0 mg of CE. Serum concentrations were 40.0 mg each.Thiosulfate concentration was 5.0 p,mol (2.0 x 104 cpm/Amol,experiment 1; 2.2 x 10' cpm/pmol, experiment 2. Both sulfaneand sulfonate were equivalently labeled in each experiment.)Incubation times for both experiments were 20 min.

bSimilar results were obtained with untreated CE; normalserum had no affect on diwimilatory activities in D. vulgarisextracts.

branes by USS in reducing bisulfite to sulfidewas shown earlier. A 3.6% concentration of Tri-ton X-100 in reaction mixtures caused a decreasein the amount of sulfide formed from bisulfiteby the USS-membranes system. These resultsprovided additional evidence that membranesare somehow involved in the dissimilatory re-duction of bisulfite to sulfide.

Specificity of the membrane effect. Theparticulate fractions of several other microor-ganisms were tested for their ability to associatewith D. vulgaris USS to reduce bisulfite tosulfide. These were from Desulfotomaculum ni-grificans, Desulfotomaculum ruminis, Clostrid-ium pasteurianum, and Bacillus coagulans. Allof the membrane fractions were capable of func-tioning with D. vulgaris USS to form sulfidefrom bisulfite. The best activity was noted withD. vulgaris membranes, and the least effectivewere membranes from B. coagulans.

DISCUSSIONIshimoto and Yagi (16) first postulated that

several reductases may be involved in the dissi-milatory reduction of sulfite by sulfate-reducingbacteria. They proposed that a sequence of threetwo-electron reductions may result in the for-mation of sulfide. Subsequent work by Kobay-ashi et al. (21), Suh and Akagi (38), and Findley

and Akagi (10) suggested that trithionate andthiosulfate were intermediates in the dissimila-tory reduction of bisulfite to sulfide. The reduc-tases currently believed to be involved in thisprocess are bisulfite reductase (7, 9, 17, 20, 22,23), the thiosulfate-forming enzyme TF (8), andthiosulfate reductase (12, 13, 27). Work in thislaboratory suggests that a trithionate reductasemay also be involved (unpublished data). It hasalso been suggested (5) that trithionate and thi-osulfate are not intermediates in bisulfite reduc-tion.The data reported in this study present evi-

dence that trithionate and thiosulfate are inter-mediates during bisulfite reduction to sulfide byextracts of D. vulgaris. Membranes were shownto play a fundamental role in the dissimnilatoryprocess. The CE reduced bisulfite to sulfidewithout the formation of any detectable inter-mediate compounds. When CE was subjected tohigh-speed centrifugation, the resulting super-natant fraction (USS) was observed to reducebisulfite sequentially to trithionate, thiosulfate,and sulfide. When the particulate fraction, con-taining membranes, was added back to USS, theCE type of bisulfite reduction was restored; i.e.,trithionate and thiosulfate were not detected asintermediate.With partially purified membranes, more evi-

dence for their participation in the dissimilatoryprocess was obtained. When [3S]sulfonate-la-beled thiosulfate was incubated with crude ex-tracts of D. vulgaris, the residual thiosulfategradually became enriched with 36S in the sul-fane atom (10). This introduced the recyclinghypothesis for the sulfonate group of thiosulfateduring the dissimilatory reduction of bisulfite.The present study showed that a recycling proc-ess was apparent only in the absence of mem-branes. USS reduced the sulfane atom of thio-sulfate to sulfide and recycled the sulfonategroups, as free bisulfite, to thiosulfate. In thepresence of membranes, extracts reduced boththe sulfane and sulfonate sulfur atoms to sulfideat equal rates. This occurred even in the pres-ence of an exogenous pool of unlabeled bisulfite(Table 7). When membranes are present, thesulfonate group of thiosulfate is not released asfree (bi)sulfite. In the previous study (10), mem-branes were apparently removed by centrifuga-tion during the preparation of crude extracts.By using antiserum directed against bisulfite

reductase, the requirement for bisulfite reduc-tion through the dissimilatory pathway wasdemonstrated. Bisulfite reduction (Table 6) andthe recycling of the sulfonate group of thiosul-fate (Table 7) were found to require bisulfitereductase activity. Since bisulfite reductase re-

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922 DRAKE AND AKAGI

duces bisulfite to trithionate (7, 23), the first stepin dissimilatory bisulfite reduction must be theformation of trithionate.A general model involving a sequential ar-

rangement of bisulfite reductase (Aase), tri-thionate reductase or TF (Base), and thiosulfatereductase (Case) on a membrane surface is pro-posed (Fig. 3). Having the enzymes arrangedsequentially (linear or otherwise) could result inthe reduction of bisulfite to sulfide without the"release" of the intermediates trithionate or thi-osulfate. The model also explains how both thesulfane and sulfonate sulfur atoms of thiosulfateare apparently reduced to sulfide at equal rates.The sulfonate group is not released as free sulfitebut is immediately reduced by a neighboringbisulfite reductase (Aase). The addition of un-labeled intermediates to the "dissimilatory com-plex" lowered the specific activity of the sulfideformed from [3S]bisulfite (Table 2). This sug-gested that although the intermediates are notreleased, they can enter the complex ("respira-tory tunnel") and retard (or interfere with) thecatalysis of the preceding step. Lynen (26) andKempner (19) postulated that, in a tightly cou-pled system, intermediates can neither enter norleave the multienzyme complex. We concludethat our in vitro dissimilatory complex is notanalogous to a tightly coupled system, i.e., closedtunnel, although it is possible that within thecell a closed sstem is operating.Although S-labeling studies demonstrated

that bisulfite was reduced successively to tri-thionate, thiosulfate, and sulfide by the solublesystem (USS), we have consistently observedthat the formation of thiosulfate from bisulfiteoccurred without appreciable formation of tri-thionate. Furthermnore, purified bisulfite reduc-tase (Aase) plus TF (Base) catalyzed the reduc-tion of bisulfite primarily to thiosulfate withoutforming significant quantities of trithionate (8).These observations could be interpreted to meanthat Aase and Base are closely associated in theform of a complex. Further association of theAase-Base complex with the terminal catalyst,Case, may require the presence of a membrane.Whatever the case may be, the interaction be-

AASE BASE

*HS03 LS3.6J2-SS2J32-TC.

//// 4MEMBRANES/ / / /

FIG. 3. Proposed model for membrane-associateddissimilatory pathway.

J. BACTERIOL.

tween the enzymes and membrane must be rel-atively weak since they are dissociated in thecentrifugal field.The cell would obviously benefit by having a

dissimilatory pathway structurally organized. Asdiscussed by Lynen (26), Srere and Mosback(37), and Racker (35), the efficiency of a meta-bolic pathway depends on the distance betweenthe individual enzymes comprising the pathway.Another advantage for having the pathwaymembrane associated would be to provide anefficient mechanism for eliminating toxic prod-ucts from the cell. It is not likely that the cellcould maintain itself if large amounts of sulfideaccumulated in the cytoplasm. The fonnation ofsulfide at the membrane site would allow sulfate-reducing bacteria to excrete sulfide rapidly intothe surrounding environment.Peck and co-workers (4, 28, 31) demonstrated

that sulfate reduction by Desulfovibrio was cou-pled to anaerobic oxidative phosphorylation.Since electron transport phosphorylation is amembrane-associated phenomenon, couplingwith the dissimilatory pathway would mostlikely occur at the site of phosphorylation, i.e.,the membrane.

ACKNOWLEDGMENTSThis study was supported in part by a National Science

Foundation grant, PCM 76-80496, by a University of KansasGeneral Research Fund grant, and by a Biomedical SciencesSupport grant. H. L. D. is a recipient ofa Wakaman Fellowshipfrom the American Society for Microbiology.We thank J. C. Brown for help during the preparation of

bisulfite reductase antiserum.

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