compounds' - mmbr.asm.org · symposium on metabolism of inorganic compounds' v....

SYMPOSIUM ON METABOLISM OF INORGANIC COMPOUNDS'

V. COMPARATIVE METABOLISM OF INORGANIC SULFUR COMPOUNDS IN MICROORGANISMS

H. D. PECK, JR.Biology Division, Oak Ridge National Laboratory,2 Oak Ridge, Tennessee

I. Introduction .............................................................................A. General Aspects of Inorganic Sulfur Metabolism.........................................B. The Activation of Sulfate...............................................................

II. Assimilatory Sulfate Reduction...........................................................A. The Reduction of Sulfate...............................................................B. The Reduction of Sulfite................................................................C. The Reduction of Thiosulfate.........................................................D. The Metabolism of Elemental Sulfur and Sulfide........................................

III. Dissimilatory Sulfate Reduction..........................................................A. General Aspects.......................................................................B. The Reduction of Sulfate...............................................................C. The Reduction of Other Sulfur Compounds ...........................................D. The Energy Metabolism of Desulfovibrio desulfuricans..................................

IV. The Distribution of APS-reductase and PAPS-reductase in Microorganisms.................V. The Oxidation of Reduced Sulfur Compounds ............................................

A. General Aspects.......................................................................B. The Oxidation of Reduced Sulfur Compounds by Heterotrophic Microorganisms.........C. The Oxidation of Reduced Sulfur Compounds by Photosynthetic Microorganisms.D. The Oxidation of Reduced Sulfur Compounds by the Thiobacilli........................E. The Energy Metabolism of the Thiobacilli.............................................

VI. Literature Cited...........................................................................

676869707072737373737477788081818283838889

I. INTRODUCTIONAlthough a voluminous literature exists con-

cerning the biology of microorganisms that oxi-dize and reduce inorganic sulfur compounds,until recently little was known about the enzy-matic mechanisms involved in the metabolismof these compounds. This is surprising when oneconsiders the role that these microorganisms haveplayed at one time or another in the develop-ment of our concepts concerning the coupling ofenergy-producing mechanisms with biosyntheticreactions, comparative biochemistry, and theevolution of life. In addition, these microorgan-isms and the transformations of sulfur com-pounds effected by them are important in certainbasic biological and geological phenomena that

1 This symposium was organized by Dr. HowardGest under the auspices of the Division of GeneralBacteriology and presented at the Annual Meetingof the American Society for Microbiology inChicago, Ill., on April 26, 1961. Dr. Jacques C.Senez served as honorary convener.

2 Operated by Union Carbide Corporation forthe U. S. Atomic Energy Commission.

are in some cases of great economic importance.The extent of the metabolism of sulfur com-pounds by microorganisms in nature is evidentto the most casual of observers by the wide-spread production of hydrogen sulfide and oc-currence of black iron sulfides. The biologicalaspect of inorganic sulfur metabolism has re-cently been the subject of an excellent review byPostgate (95) and is one of the most interestingand fascinating features of these organisms.The transformations of inorganic sulfur com-

pounds in nature have been formalized in the so-called sulfur cycle (see reference (132)) that iscomparable in many respects to the betterknown nitrogen cycle. The microorganisms thatparticipate in the sulfur cycle are physiologicallydiverse and comprise both heterotrophic andautotrophic organisms, the latter exhibitingunique modes of existence. The metabolism ofinorganic sulfur compounds has been the sub-ject of reviews by Butlin and Postgate (13) andFromageot and Senez (30). In this discussion,the enzymes involved in the metabolism of in-organic sulfur compounds will be emphasized

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So4 ORGANIC SULFATES

Th/bbocidllll Ds\ vi

C\Custrk/d n ripffonsd9oe S03

C/rom/ium S20i' (INDIRECTLY MANY

HETEROGRAPHIGa1Wown S40 / ORGANISMS)

HETEROTROPHICORGANISMS so S-- CELLULAR MATERIAL

FIG. 1. Simplified diagram of the sulfur cycle.The major microorganisms that participate in thereduction of sulfur are listed on the right hand side;those that take part in the oxidation of elemental sul-fur and sulfide to sulfate are listed at left.

and allusion made to the more biological aspectsof these reactions only where their interpretationis influenced by a knowledge of the enzymaticmechanisms.

A. General Aspects of Inorganic Sulfur MetabolismAlthough mammals can oxidize reduced sulfur

compounds, i.e., sulfide (7), thiosulfate (114),and sulfite (38), and incorporate sulfate intovarious organic molecules (67), they are unableto reduce sulfate to the level of sulfide (23) andmust depend upon plants and bacteria to providethem with their reduced sulfur compounds. InFig. 1 the basic essentials of the sulfur cycle are

shown with most of the ancillary reactionsomitted. The sulfur compounds included in thecycle are those that accumulate or are readilydetectable in nature. Elemental sulfur has notbeen included as an intermediate in the sulfurcycle since it does not seem to be an intermediatein either the reduction of sulfate (93) or oxida-tion of sulfide (9).Although many heterotrophic microorganisms

reduce sulfate, the sulfur does not usually appear

as sulfide but rather is incorporated into cellularmaterial and returned to the sulfur cycle byother reactions. The main group of organismsproducing large amounts of sulfide directly fromsulfate are the so-called "sulfate-reducing bac-teria" (see Fig. 1, right).

Thiobacillus species are chemoautotrophic or-

ganisms that derive the energy required forgrowth from the oxidation of reduced sulfur com-pounds (Fig. 1, left) and are the most well knownof the nonphotosynthetic sulfur bacteria (130).Beggiatoa species and allied organisms also carry

out the oxidation of reduced sulfur compounds;

however, these organisms will not be discussedsince almost nothing is known about their nutri-tion or metabolism (24).The second large physiological group of micro-

organisms that oxidize reduced compounds ofsulfur are photosynthetic organisms belongingto the families Thiorhodaceae and Chlorobac-teriaceae and typified by the genera Chromatiumand Chlorobium. The enzymatic reactions con-cerned with the oxidation of reduced sulfur com-pounds are essentially unknown in these organ-isms; however, recent observations suggest thatthe enzymes of sulfur oxidation are intimatelyrelated to the photosynthetic apparatus (44).Some heterotrophs are also capable of oxidizing

reduced inorganic sulfur compounds and it hasbeen suggested that these organisms are themajor group of organisms responsible for theoxidation of sulfur compounds in soils. Theseheterotrophic organisms are incompletely de-scribed; however, the isolation of a facultativeautotroph, Thiobacillus novellus, may indicatethat there is a gradation in the ability of theseorganisms to oxidize reduced sulfur compoundsand to derive energy from these oxidations, acapability rdaching its greatest development inthe autotrophic thiobacilli (130).

Until recently the intermediates in the oxida-tion and reduction of inorganic sulfur compoundshave been unknown. In the center of the sulfurcycle shown in Fig. 1 are listed some of the morestable sulfur compounds that have at one timeor another been implicated or suggested as inter-mediates in inorganic sulfur metabolism. A verysimplified scheme for the oxidation of sulfide orreduction of sulfate (59) is shown in Fig. 2 withthe valence states of the sulfur atoms indicated.This scheme, which is an initial formulation ofthe pathway of sulfate reduction, indicates thatthere may be four steps in the oxidation or reduc-tion of these sulfur compounds. However, simpleinorganic sulfur compounds with an oxidationlevel of +2 and 0 (with the exception of ele-mental sulfur) are extremely unstable and prob-ably could not exist under physiological condi-tions. Alternatively, it might be postulated that

(+6)m (_+4) S -(42L-l)S_(0)

S(4g" --SO3-- --SOj -*---SO--O-- S-(

FIG. 2. Postulated inorganic intermediates in thereduction of sulfate. Valence states of the sulfuratoms are shown in parentheses.

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at some oxidation level, the inorganic sulfur isincorporated into an organic molecule and thenoxidized or reduced. A third group of compoundsthat might function as intermediates in the oxi-dation and reduction of sulfur compounds arethe complex inorganic compounds of sulfur, suchas the polysulfides or polythionates (S306`, S406,S506-). The polythionates, in particular, areformed by many microorganisms, and schemesfor the oxidation of reduced sulfur compoundshave been proposed that utilize known chemicalreactions of the polythionates (65, 130).

Another question of interest is whether thepathway of sulfate reduction is enzymologicallythe same as the pathway of sulfide oxidation,and even more specifically whether the pathwaysof sulfate reduction or sulfide oxidation are iden-tical in the various physiological types of micro-organisms that carry out these reactions. On aphysiological basis, organisms that are capableof reducing sulfate can be classified into twodistinct types (93). Most organisms can reducesulfate, as evidenced by their ability to grow onsulfate as a sole source of sulfur; however,they do not form detectable amounts of sul-fide directly from sulfate. This small-scale re-duction of sulfate has been termed "assimila-tory sulfate reduction" by analogy to Kluyver'sclassification of the various types of nitrate re-duction (58). Another, and much smaller, groupof microorganisms utilizes sulfate as the terminalelectron acceptor in anaerobic respiration andproduces massive amounts of hydrogen sulfide.This large-scale reduction of sulfate has beentermed "dissimilatory sulfate reduction," againby analogy to Kluyver's classification. Organ-isms that might be classified as "incidental dis-similatory sulfate reducers" have not been de-scribed; however, many organisms that are not"dissimilatory sulfate reducers" can reduce in-organic sulfur compounds other than sulfate (31,80). Organisms that oxidize reduced sulfur com-pounds can be categorized into three groups onthe basis of the role that sulfur plays in theirphysiology; "heterotrophic sulfur oxidizers,""autotrophic sulfur oxidizers" that include thethiobacilli, and the "photosynthetic sulfur oxi-dizers."The transformation of inorganic sulfur com-

pounds is intimately connected with the energymetabolism of the dissimilatory sulfate reducersand the autotrophic and photosynthetic sulfur

oxidizers. This aspect has been the subject ofconsiderable speculation and interest in thepast, and recent observations on the metabolismof sulfate in higher animals suggest the possibil-ity that there is a closer relationship betweenenergy and sulfur metabolism than has beenpreviously suspected.

B. The Activation of Sulfate

Although mammals are unable to reduce sul-fate to the level of sulfide, they are capable ofutilizing sulfate for the synthesis of various sul-fated carbohydrates, lipids, and phenols. Thereactions and enzymes involved in the esterifica-tion of sulfate have been recently reviewed byLipmann (67) and Gregory and Robbins (33).The reaction requires adenosine triphosphate(ATP) for the initial activation of the sulfate,and two sulfur-containing nucleotides have beenshown to be intermediates. The structures ofthese nucleotides, adenosine 5'-phosphosulfate(APS) and 3'-phosphoadenosine-5'-phosphosul-fate (PAPS) are shown in Fig. 3. The first stepin the metabolism of sulfate is the formation ofAPS and pyrophosphate (PPi) by the reactionof ATP and sulfate in the presence of the en-zyme, ATP-sulfurylase (equation 1). The en-

ATP + S04- = APS + PPi (1)

zyme requires Mg++ for activity and has beencharacterized and purified from baker's yeastby Robbins and Lipmann (98). The equilibriumof this reaction lies far toward ATP and sulfate(K = 10-8, pH 8.0, 37 C) and to observe a netformation of APS it is necessary to "pull" the

-O-;t-O-IP-0-CH2 EADENINE

OH OH

ADENOSINE-5-PHOSPHOSULFATE (APS)

0 0-O-S-°-P-°- (>12 ADENINE

0 -0

?OH0S P-O-

0~9-PH0SPHOADENOSINE-5!-PHOSPHOSULFATE (PAPS)

FIG. 3. Structures of APS and PAPS

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reaction by removing the other product, PP ,

with inorganic pyrophosphatase. It is interestingthat the group potential of the sulfate (18 to 19kcal) is much higher than that of phosphate.Under certain conditions, ATP-sulfurvlase cata-lyzes another reaction, in which orthophosphate(Pi) is produced from ATP in the presence ofpyrophosphatase and group VI anions (M1oO4\,SeO4=, W024, CrO4=, and S03=. The mechanismof these reactions seems to be similar to the wellknown arsenolysis reactions (135).The next step in the metabolism of sulfate is

the phosphorylation of APS in the 3'-positionby ATP to yield PAPS and adenosine diphos-Ihate (ADP) (99), as shown by equation 2. The

APS + ATP = PAPS + ADP (2)

enzyme responsible for the phosphorylation hasbeen termed APS-kinase. PAPS is the so-called"active sulfate" of mammalian tissue and, inthe presence of the appropriate enzymes (termedsulfokinases), can transfer sulfate to phenols,lipids, and carbohydrates. The other product ofthis transfer is 3',5'-diphosphoadenosine (PAP).The formation of PAPS has been observed in

extracts of Escherichia coli (42) and many fungi(22, 41, 117). The formation of a sulfur-contain-ing nucleotide having the same electrophoreticmobility as PAPS has been observed in extractsof many bacteria (Peek, unpublished data). Re-cently, it has been suggested that specializedbacteria are the agents responsible for the reduc-tion of sulfate in many insects (34). There is apaucity of data concerning the occurrence ofsulfated compounds in bacteria, and the transferof sulfate from PAPS (or possibly APS) to forma sulfated compound has not been described inbacteria. In addition, the transfer of sulfatefrom PAPS to phenols has not been observed,and the extracts examined seem to be lackingthe specific sulfokinase (117). Recently a sulfate-containing polysaccharide has been isolated andcharacterized from bacteria and this compoundmay be useful in studying the mechanism ofsulfate transfer in bacteria (125).The occurrence of the choline ester of sulfuric

acid in fungi has been known for a number ofyears (138). Kaji and Gregory (56) have shownthat the sulfate donor for the synthesis of sulfo-choline is PAPS and it appears that the transferis catalyzed by a specific sulfokinase (117). Al-though some fungi accumulate fairly large

amounts of sulfocholine, the exact role of thiscompound in the metabolism of fungi is un-known. It is of interest that the occurrence ofsulfocholine in certain groups of fungi does seemto have some taxonoimic significance (5, 35, 52).One other enzyme that utilizes APS as sub-

strate has been described from yeast. This en-zyme, ADP-sulfurylase, catalyzes the phospho-rylation of APS to form ADP and sulfate (99), asshown in equation 3.

APS + Pi - ADP + SO4= (3)

This enzymatic activity has been separatedfrom ATP-sulfurylase and therefore appears tobe catalyzed by a specific enzyme. The formationof APS by this enzyme has not been observedand, consequently, an equilibrium constant hasnot been calculated. However, the equilibrium ofthis reaction is probably quite similar to theequilibrium of the reaction catalyzed by ATP-sulfurylase, since both reactions involve the hy-drolysis of a single phosphate bond. In contrastto ATP-sulfurylase, a requirement for MIg+ hasnot been reported. The enzyme catalyzes anarsenolysis of APS (99) but does not catalyzethe liberation of Pi from ATP in the presence ofgroup VI anions (Peck, unpublished data). Froma consideration of these observations, it does notseem that ADP-sulfurylase is involved in theformation of APS, and its role in the metabolismof APS in yeast is at present unknown.

II. ASSIMILATORY SULFATE REDUCTION

A. The Reduction of SulfateAlthough the production of hydrogen sulfide

from some inorganic and organic compounds isfrequently reported (2, 12, 17, 31, 80, 97, 137),the production of sulfide directly from sulfate isnot commonly observed in microorganisms evenwhen they are apparently reducing and incorpo-rating sulfur into cellular materials. In the ab-sence of sulfide production, other criteria can beemployed to demonstrate that a given microor-ganism can reduce sulfate to the level of sulfideand is an assimilatory sulfate reducer.The incorporation of S504= into the reduced

sulfur compounds of cellular material constitutesa direct demonstration of the ability of a givenorganism to reduce sulfate to the level of sulfide.When observations of this type are made withorganisms grown in complex media, the enzymesresponsible for the reduction of sulfate may be

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repressed (10), and negative results obtained;however, if these organisms are grown underother cultural conditions, it might be possible todemonstrate the presence of sulfate-reducing en-zymes.Many yeasts grow well on a defined medium in

which the main source of sulfur is sulfate (106)and also incorporate sulfur from S3504= into cys-teine and methionine (136). These organismstherefore seem to be in all respects assimilatorysulfate reducers. A cell-free system from yeastthat reduces sulfate to sulfite and sulfide in thepresence of Mg++, ATP, and reduced triphos-phopyridine nucleotide (TPNH) has recentlybeen described independently by Wilson andBandurski (134) and by Hilz, Kittler, andKnape (42). These investigators have furthershown that the actual intermediate for the reduc-tion of sulfate to sulfite is PAPS, formed as indi-cated previously (equations 1 and 2). Recentevidence obtained with partially purified yeastsulfate reductase indicates that this enzyme maybe capable of reducing APS; however, the ac-tivity observed with APS is much less than thatobserved with PAPS (133). Hilz et al. (42) haveshown that MoO4J, which interferes with ATP-sulfurylase, the first enzyme involved in theactivation of sulfate, inhibits the formation ofsulfite from ATP and sulfate. To demonstratethat the effect of MoO4 was on the formationand not on the reduction of PAPS, this substratewas formed from labeled p-nitrophenylsulfateand PAP by a purified sulfokinase, as shown inequation 4. The fact that MoO4= did not inhibit

of S3047. Sulfite itself may inhibit the formationof PAPS by its effect on ATP-sulfurylase (135).The PAPS-reducing enzyme (hereafter called

PAPS-reductase) is inhibited completely byarsenite (3 X 10-4 M) and seems therefore torequire an intact vicinal dithiol for activity (42).The enzyme has been purified by both Hilz et al.(42) and Bandurski, Wilson, and Asahi (6) fromyeast; however, there is some disagreement asto the nature of the sulfhydryl involvement.Hilz et al. (42) reported that lipoic acid will re-store activity of PAPS-reductase when added toinactive preparations and recently showed thatreduced lipoic acid or its amide will replaceTPNH as electron donor (40).On the other hand, Bandurski et al. (6) sepa-

rated PAPS-reductase from yeast into two heat-labile components (fractions A and B) and aheat-stable component (PrSS) that seems to bea protein. All three components are required forthe reduction of PAPS with TPNH. However, apartial reaction, the oxidation of TPNH andappearance of sulfhydryl groups in PrSS, can beobserved in the presence of fraction A. SinceHilz et al. (42) postulated that lipoic acid is in-volved in this reductase, it is surprising thatPrSS contains almost no lipoic acid (6). FractionA will act as a diaphorase, coupling TPNH oxi-dation with ferricyanide reduction. From theseconsiderations, the enzyme described by Ban-durski et al. (6) resembles in many respects di-hydrothioctyldehydrogenase (70, 107). The reac-tion mechanism has been postulated essentiallyas follows by Hilz et al. (42):

NO2 OSO3- + PAP =

PAPS + NO2 OH

(4)

the reduction of PAPS under the latter conditionindicated the effect of MoO4t was solely on ATP-sulfurylase, and that PAPS was formed by thepreviously described reaction sequence for theactivation of sulfate. Employing the p-nitro-phenylsulfate system, it was also shown thatthiosulfate competitively inhibits the activationof sulfate but has no effect on the production ofsulfite from PAPS. This is of interest in connec-tion with the data of Cowie, Bolton, and Sands(21), who indicated that thiosulfate as well asother sulfur compounds inhibited the utilization

RSS + TPNH + H+ =

R(SH)2

PAP-OSO3- + R(SH)2 =

PAP-OH +

SS03-

R

SH

+ TPN (5)

SS03-R

SH

= RSS + HSO3-

where R may be either lipoic acid or the heat-stable protein of Bandurski et al. (6). Althoughthe reactions are indicated as being reversible,evidence pertaining to this aspect has not yetbeen reported. PAPS-reductase has been reported

(6)

(7)

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to be present in extracts of E. coli (42, 69); how-ever, the electron donor in this case for the re-duction of PAPS is claimed to be reduced diphos-phopyridine nucleotide (D1PNH) rather thanTPNH (69).

It has previously been postulated that sulfitewas an intermediate in the reduction of sulfatefrom results obtained with isotopes (21, 62) andbiochemical mutants (18, 43, 111). Recent enzy-matic studies completely support this view sincesulfite is one of the products of the reaction cata-lyzed by PAPS-reductase. These enzymatic ob-servations also confirm results, obtained with bio-chemical mutants, which indicate that reductionof sulfate to sulfite is a complex reaction requir-ing at least three factors (18).

B. The Reduction of Sulfite

Interpretation of results obtained with bio-chemical mutants leads one to believe that at theoxidation level of sulfite, further reduction ofsulfite may depend upon its incorporation intoan organic molecule (111). Enzymological evi-dence pertinent to this aspect is scanty; however,data have been presented that indicate sulfitecan be incorporated into cysteine to form cysteicacid and hydrogen sulfide (16). For this reactionto be of importance in the reduction, presumablythe cysteic acid would be reduced to cysteine byreversing the cysteinesulfinic acid pathway ofcysteine oxidation (112). However, this pathwaydoes not seem to be involved in the reduction ofsulfite since the over-all reaction is not reversi-ble (112). In addition, Cobey and Handler (19)have shown that extracts of E. coli do not in-corporate sulfite labeled with sulfur-35 intocysteinesulfinic acid. Although these particularcompounds of cysteine do not seem to be inter-mediates in the reduction of sulfite, similar typesof compounds may be involved. Regarding theinterpretation of nutritional studies, it should becautioned that, in addition to the fact that manyof these putative intermediates of sulfur metabo-lism are unstable, the organic sulfur may be con-verted to inorganic sulfur before oxidation or re-duction (29). On an enzymatic level there is littleevidence to suggest that sulfite is incorporatedinto a small organic molecule before reduction(69).An inorganic pathway of sulfite reduction has

been indicated in studies with sulfur-35 (21) andby some of the results obtained with biochemical

mutants (18). An inorganic pathway involvingthe formation of thiosulfate as an intermediatein the reduction of sulfite has been postulatedsince mutants can be obtained that utilize neithersulfate nor sulfite but can utilize thiosulfate astheir sole source of sulfur. Similar conclusionswere made from studies on the incorporation ofS350,= into cellular materials in the presence ofthiosulfate. Since recent studies (described inSection IIC) indicate that thiosulfate can bemetabolized by a reductive cleavage to sulfiteand sulfide (the presumptive product of sulfatereduction), it is not necessary to place thiosulfatedirectly on the pathway of sulfate reduction.A TPN-specific sulfite reductase, which reduces

sulfite to sulfide, was found in extracts of E. coli(69). It is described by the over-all reactionshown in equation 8. This enzymatic activity

S03 + 3 TPNH + 5 H+-8H2S + 3 TPN+ + 3 H20 ()

from E. coli has been enriched about 150-fold,requires flavin adenine dinucleotide for activity,and is inhibited by KCN. Several interestingaspects of this enzyme were reported by Mager(69). He noted that preparations of hydroxyla-mine reductase also exhibited sulfite-reductase ac-tivity and that the ratio of these two activitieswas constant throughout purification and undervarious adverse treatments. Since sulfite in lowconcentrations inhibits hydroxylamine reductionand the K. for sulfite is 100-fold lower than thatfor hydroxylamine, Mager postulated that sulfitereduction is the "true physiological function" ofthe enzyme and that the reduction of hydroxyl-amine is an "incidental" capacity of the sameenzyme. Furthermore, the formation of bothsulfite reductase and hydroxylamine reductaseby proliferating cells of E. coli is inhibited specifi-cally by the presence of cysteine or methioninein the growth medium. It is postulated that thisinhibition reflects a feedback repression by cyste-ine and methionine of sulfite reductase. Thisresult agrees with the data of Cowie et al. (21)that were obtained from studies on the biosyn-thesis of cysteine from sulfite with whole cellsof E. coli. The repression of sulfite reductase,rather than PAPS-reductase or the enzymes re-quired for the activation of sulfate, by cysteineand methionine (69), may indicate that sulfiteitself is required for the formation of some cellu-lar components.

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The fact that sulfite reductase has been puri-fied 150-fold, with apparently no evidence forth-coming to indicate that more than one enzymeis involved in this reduction, suggests that asingle enzyme catalyzes the reduction of sulfiteto sulfide. In addition, no factors except flavinadenine dinucleotide (FAD) are required for thereduction. The enzymological data indicate thatsulfide is an intermediate in the reduction ofsulfite and therefore sulfide is probably the in-organic form of sulfur that is incorporated intoorganic cellular materials.

C. The Reduction of ThiosulfateThiosulfate can serve as the sole source of

sulfur for the growth of many organisms, andthe production of hydrogen sulfide from thio-sulfate has been frequently observed (17). Al-though the production of sulfite and sulfide fromthiosulfate has been less frequently observed(78), the reductive cleavage of thiosulfate tosulfite and sulfide seems to be the first step inthe metabolism of thiosulfate. The reduction oftetrathionate, a proposed intermediate in theoxidation of inorganic sulfur compounds, in-volves its initial conversion to thiosulfate bytetrathionase (87).An enzyme, isolated from yeast and charac-

terized by Kaji and McElroy (55), catalyzes thereductive cleavage of thiosulfate to sulfite andsulfide. The electron donor for this reductionmay be either glutathione (GSH), cysteine, orhomocysteine. The reaction for the reductivecleavage of thiosulfate with glutathione is givenby equation 9. This enzyme is remarkably stable

2 GSH + S203-. GSSG + S03- + H2S (9)

to heat and is inhibited by sulfite. In view ofthis latter observation, a mechanism for thereaction has been proposed that is similar to onesuggested for the oxidation of sulfite (28). Thio-sulfate reacts with a disulfide group, presumablyon the enzyme, to yield

SSS03-1

Enzyme 0

S _

which is reductively cleaved to yield sulfite andsulfide.A second pathway of thiosulfate reduction

may be present in some organisms (18) since mu-

tants, unable to grow on low concentrations ofthiosulfate, will grow in the presence of high con-centrations of thiosulfate. This growth does notseem to result from the nonenzymatic cleavageof thiosulfate to sulfite and sulfide or elementalsulfur because these mutants cannot grow onany of these three compounds. Studies withmutants (77) suggest that thiosulfate and serinecan react to form cysteine-S-sulfonate, whichcan then be cleaved to yield cysteine as one ofthe products. However, there is no enzymologicalevidence to support the presence of this secondpathway of thiosulfate reduction.

D. The Metabolism of Elemental Sulfurand Sulfide

The evidence available, and particularly thatobtained with cell-free systems, indicates thatsulfur compounds are reduced to sulfide andthen incorporated into cellular materials. An en-zyme, which has been isolated from yeast, formscysteine from serine and hydrogen sulfide (105),as shown in equation 10.

H2COH H2CSH

HCNH2 + H2S = HCNH2 + H20 (10)

COOH COOH

This enzyme, serine-sulfhydrase, requires pyri-doxal phosphate for activity. Thus, enzymeshave been described for the reduction of sulfateto sulfide and the incorporation of the sulfurinto organic materials.

Elemental sulfur does not seem to be an inter-mediate in this reaction sequence but is probablynonenzymatically reduced to sulfide and thenmetabolized. Thus, GSH is capable of reducingelemental sulfur to sulfide in the absence of anenzyme (118). Nevertheless, in some instances(the thiobacilli and certain photosynthetic or-ganisms), this reduction may be catalyzed by aspecific enzyme.

III. DISSIMILATORY SULFATE REDUCTION

A. General Aspects

The general physiology and taxonomy of thedissimilatory sulfate-reducing bacteria have re-cently been reviewed in detail by several au-sors (93, 119, 139). Therefore, the more enzy-mological aspects of sulfate reduction are em-phasized in this section. Surprisingly few bonafide dissimilatory sulfate reducers have been de-

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scribed, and a large portion of the physiologicaland enzymological observations concerning thereduction of sulfate have been made with theclassic sulfate reducer, Desulfovtibrio desulfuricans.Thermophilic sporeforming sulfate-reducing bac-teria have been identified as Clostridium nigrifi-cans (15). Most of the other dissimilatory sulfatereducers seem to be species of Desulfovibrio.Many of the older species of Desulfovibrio arenow regarded as D. desulfuricans; however, ithas not been possible to reisolate all of theolder species for examination (108). The applica-tion of newer techniques in the cultivation ofthese organisms and the use of biochemical prop-erties in taxonomy, such as the presence or ab-sence of various pigments (94), should greatlyaid in classifying these organisms. Recently, em-ploying both taxonomic and biochemical criteriafor identification, two apparently new species ofsulfate-reducing bacteria were described (1, 20).The sulfate-reducing bacteria can no longer

be regarded as organisms that can grow hetero-trophically with sulfate and an organic electrondonor or autotrophically with sulfate, hydrogen,and carbon dioxide. Sulfate cannot be consideredan obligatory electron acceptor for the growthof D. desulfuricans under all conditions, sincePostgate has shown that it is possible to growthis organism on pyruvate in the absence of sul-fate (90). The presence of a clostridal "phos-phoroclastic" reaction has been demonstrated incell-free extracts. Thus, pyruvate is metabolizedto hydrogen, carbon dioxide, and acetyl phos-phate (74) and high-energy phosphate from thislatter compound can be utilized to produce ATP(84). Independently, both Postgate (96) andMechalas and Rittenberg (71) showed that incontrast to earlier views (14), D. desulfuricansis not a true autotrophic organism since the car-bon for the synthesis of cellular materials is notderived in large measure from carbon dioxide.They presented evidence that the carbon mole-cules of the cells are derived mainly from eitherimpurities in the media or added yeast extract.These organisms are also unique among hetero-trophic organisms since carbon from certain or-ganic substrates is not assimilated into cellularmaterial during growth, i.e., the organic electrondonor can function solely as an energy source. Asimilar situation has been described in the photo-synthetic bacteria (25). Nevertheless, D. desul-furicans can obtain the energy required for

growth from the reduction of sulfate with hy-drogen (109).

Although, from these considerations, one mightexpect to find eventually a graded series of sul-fate-reducing organisms that vary from "inci-dental sulfate reducers" to "autotrophic sulfatereducers," enzymological and physiological stud-ies suggest the possibility that growth with sul-fate as the sole electron acceptor may constitutea unique type of metabolism. Evidence support-ing this idea has been forthcoming from themechanism of sulfate reduction and from thestudies on the energy metabolism of these organ-isms.

B. The Reduction of SulfateWhole cells of D. desulfuricans rapidly reduce

sulfate, sulfite, and thiosulfate to sulfide in thepresence of molecular hydrogen (89). The over-all reaction for the reduction of sulfate is shown

4 H2+ S04 -- S + 4 H20 (11)

by equation 11. Most strains of this organismcontain a very active hydrogenase that is presenteven when an organic electron donor is utilizedfor growth. The hydrogenase has been purifiedand studied by Sadana and Jagannathan (100)and more recently by Krasna, Riklis, and Ritten-berg (61). The purified enzyme requires Fe++ formaximal activity (101), a fact suggested by thelow hydrogenase content of cells grown in iron-deficient media (91). The observation that sul-fate-reducing organisms that do not contain hy-drogenase grow satisfactorily in the presence oforganic electron donors and sulfate, demon-strates that the presence of hydrogenase is notessential for the reduction of sulfate.

D. desulfuricans is the first nonphotosyntheticanaerobic organism in which the presence ofcytochrome was demonstrated (47, 92). Thiscytochrome, identified as cytochrome c3, has alow potential (Eo' = -0.205) (92) and wasshown to function as an electron carrier for theproduction of hydrogen and carbon dioxide fromformate and for the reduction of sulfite and thio-sulfate with hydrogen in extracts (48). It has alsobeen implicated as an electron carrier for thereduction of sulfate with hydrogen, since reducedintracellular cytochrome cs is oxidized in the pres-ence of sulfate (92).

Although cell-free extracts of D. desulfuricanscontain enzymatic systems that reduce thiosul-

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1METABOLISM OF INORGANIC SULFUR COMPOUNDS

fate, sulfite, and tetrathionate to sulfide withmolecular hydrogen (48), initial attempts to ob-tain the reduction of sulfate in extracts were un-

successful. The reduction of sulfate with hydro-gen by whole cells is competitively inhibited byselenate; however, the reduction of sulfite or

thiosulfate is not affected by this anion (88).Moreover, molybdate ion similarly inhibits sul-fate reduction but not the reduction of sulfite(46). Since Wilson and Bandurski (135) observedthat the enzyme ATP-sulfurylase catalyzes a

rapid liberation of Pi from ATP in the presence

of inorganic pyrophosphatase and group VIanions, the inhibition of sulfate reduction, butnot sulfite reduction, by MoO47 and SeO4- sug-

gested that ATP-sulfurylase was involved inthe reduction of sulfate by D. desulfuricans. Cell-free extracts of this "sulfate reducer" containATP-sulfurylase, as indicated by the release ofPi from ATP in the presence of MoO47, WO4t,and CrO4= (82). When extracts were supple-mented with ATP and sulfate and placed underan atmosphere of hydrogen, hydrogen was uti-lized, as shown in Table 1, and, in the presenceof S3504, radioactive acid-volatile sulfur (sulfideor sulfite) was produced (50, 82). If either ATPor sulfate was omitted, there was no utilizationof hydrogen. ATP was not required by wholecells for the reduction of sulfate and actuallyseemed to inhibit the reduction. The fact thatMoO4-= inhibited sulfate reduction in whole cellsas well as in extracts indicates that the reductionof sulfate observed in extracts is identical withthat observed in whole cells.When cell-free extracts were passed over a

column of Amberlite resin to remove cytochromec3 (48), the ability to reduce sulfate with hydro-gen was lost as well as the abilities to reducethiosulfate and decompose formate to hydrogenand carbon dioxide. The addition of purifiedcytochrome c3 restored all three activities. Thisconstitutes evidence that cytochrome c3 functionseither directly or indirectly in the reduction ofsulfate. The activity of Amberlite-treated ex-

tracts could not be restored by the addition ofDPN, TPN, or flavin; however, the addition ofmethyl viologen stimulated hydrogen utiliza-tion with sulfate and ATP about 8-fold over thatobserved in crude extracts. Methyl viologen alsofacilitates the reduction of sulfite and thiosulfatewith molecular hydrogen, and this one-electrondye presumably functions as an efficient electron

TABLE 1. Effect of A TP and molybdate on sulfatereduction with hydrogen in whole cells

and cell-free extracts

Activity, jAl ofH2/15 min

Reaction mixture Cell-free Wholeex- cellst

tract*

Complete ........................ 86 175Minus Na2SO4................... 0 0Minus ATP...................... 0 314Plus 10 Mmoles of molybdate 0..... 0t

* Complete system: 125 jAmoles of phosphatebuffer (pH 7.75), 10 Mmoles of MgCl2, 10 jAmolesof ATP, 40 Moles of Na2SO4, 0.8 smole of methylviologen, and 15 mg of protein. Total volume,2.0 ml; 0.2 ml of 20% KOH in center well; tem-perature, 30 C; gas phase, hydrogen.

t Complete system: 125 jsmoles of phosphatebuffer (pH 7.15), 10 jsmoles of MgCl2, 40 jsmolesof Na2SO4, 10 Moles of ATP, 0.5 ml of a 20%suspension of cells. Total volume, 2.0 ml; 0.2 mlof 20% KOH in center well; temperature, 30 C;gas phase, hydrogen.

t ATP omitted.

carrier between hydrogenase and these reductases(48). These observations indicate that the reac-tion sequence for electron transport between hy-drogenase and each of these reductases is proba-bly identical.The stoichiometry for the reduction of sulfate

in the presence of ATP and hydrogen indicatedthat APS was the form in which sulfate was re-duced by D. desulfuricans (82), rather than PAPS(the form in which sulfate is reduced in assimila-tory sulfate reducers). The formation of a singlelabeled nucleotide when cell-free extracts of D.desulfuricans are incubated with ATP and S3504=(82) has been reported. This nucleotide has thesame electrophoretic mobility as APS, movingslightly faster than ADP, and is hydrolyzed by0.1 M HCO at 37 C for 30 min to AMP and sul-fate. In Fig. 4 the effects of various treatmentson the formation of this labeled nucleotide bythe extract are shown. Heating the extract for 3min in a boiling water bath completely destroysthe ability of extracts to form the nucleotide.ATP is required, and the presence of MoO4= in-hibits formation of the nucleotide. The observedinhibition by MoO4- also indicates that the la-

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PPAPS

PPAPS_t

ORGIN

FIG. 4. Formation of APS and stability ofPAPS in extracts of Desulfovibrio desulfuricans.Each reaction mixture contained: tris buffer (pH8.0Ou), 300 ,umoles; MgC12, 10 ,imoles; and, whereindicated, A TP, 20 ,imoles; Na2S3504, 2.5 ,umoles(2.5 X 104 counts per minute per ,mole);Na2.MoO4,2 ,umoles; PAPS35, 0.018 ,umole (1 .0 X 106 counts per

minute per ,umole); and 5.0 mg of crude extract in a

volume of 1 ml. After 15 min, the reaction was stoppedby placing in a boiling water bath for 3 min. Thenucleotides were adsorbed on charcoal, eluted, andseparated by electrophoresis. The outlined areas rep-

resent ultraviolet quenching, and the shaded areas

represent radioactivity as determined by radio-autography.

beled nucleotide, APS, is formed by the enzyme,

ATP-sulfurylase.The absence of a labeled nucleotide corre-

sponding to PAPS suggests that the extracts are

incapable of forming this compound. However,the failure to observe the formation of PAPScould be caused by the rapid hydrolysis or me-

tabolism of this nucleotide. PAPS, labeled withsulfur-35, was prepared and shown to be reducedto acid-volatile sulfur in extracts of yeast supple-mented with TPNH. As shown in Fig. 4, afterincubation of the labeled nucleotide with D. de-sulfuricans extract, it was still possible to demon-strate the presence of labeled PAPS. This resultindicated that the failure to detect PAPS was a

consequence of the absence of APS-kinase ratherthan the rapid destruction of the nucleotide. Theability of extracts to form acid-volatile sulfurfrom APS35, PAPS35, and ATP plus S504= was

examined and the results are shown in Table 2.The electron donor for these reductions was

methyl viologen reduced by the enzyme, hy-drogenase, that is present in crude extracts ofD. desulfuricans (100). No acid-volatile sulfur is

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produced from APS hydrolyzed in 0.1 M HCl for30 min at 37 C; however, APS itself does giverise to the production of acid-volatile sulfur. Theamount of acid-volatile sulfur produced repre-sents complete reduction of the added APS toacid-volatile sulfur. The low activity exhibitedby ATP and S3504" indicates that APS is neitherphosphorolyzed to ADP and S3504= nor pyro-phosphorolyzed to ATP and S35042 and theS504= then reduced by another pathway. Thisconclusion is also substantiated by the observation that the label of APS35 is not diluted duringreduction by a large pool of unlabeled S04-.These results conclusively demonstrate that APSrather than PAPS is the substrate for sulfate re-duction by D. desulfuricans. The enzyme cata-lyzing the reduction of APS to adenosine mono-phosphate (AMP) and sulfite has been termedAPS-reductase (equation 14).The reduction of sulfate in these extracts has

been shown to require at least two enzymaticcomponents (Peck, unpublished data). Whencrude extracts of D. desulfuricans are heated for2 min at 64 C, the abilities to reduce sulfate withhydrogen in the presence of ATP and to liberatePi from ATP in the presence of MOO4= are de-stroyed. Inorganic pyrophosphatase, ADP-sul-furylase, and APS-reductase were not apprecia-

TABLE 2. Activity of PAPS, APS,and sulfate in crude extracts

Additions Acid-volatile sulfurAdditions ~~~producedrn,unoles

Hydrolyzed APS15............... 2APS'5......................... 100S'504=+ ATP .................. 16PAPS35 .0

Methods: Each flask contained: tris(hydroxy-methyl)aminomethane buffer (tris), pH 8.0, 300Mgmoles; MgCl2, 10 pmoles; methyl viologen, 0.8Mumole; EDTA, 10 Mmoles; and, where indicated,APS35, 0.1 jmole (3,015 counts per min); hydro-lyzed APS35, 0.107 /Amole (3,230 counts per min);Na2S'504, 2.5 MAmoles (63,700 counts per min);ATP, 5 umoles; PAPS35, 0.018 Mmole (8,330 countsper min). Each reaction mixture contained 4.56mg of crude extract heated at 60 C for 3 min.After incubation of reaction mixtures at 30 C for20 min under H2, 0.3 ml of 12 N H2SO4 was tippedfrom a side arm and acid-volatile S35 absorbed in0.2 ml of N KOH.

0 00AMP ADP ATP

APS

76

STANDARDS

35o

S5O- WTH ATP

S3504 WITH ATPAND MoOr-

ATP WITH SO4--(BOILED)

PAPS35

PAPS35(BOILED)


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bly affected by this exposure to heat. WhenATP-sulfurylase, prepared from yeast (98), was

added to these heated extracts, the ability to re-

duce sulfate in the presence of ATP and hydro-gen was restored. These data therefore indicatethat ATP-sulfurylase is the enzyme responsiblefor the formation of APS in the reduction of sul-fate. These two activities, ATP-sulfurylase andAPS-reductase, also have been separated by elec-trophoresis with Geon (98). Since heated extractsdo not reduce sulfate but do have ADP-sulfuryl-ase, it appears that this enzyme cannot formAPS to any extent under the conditions of theexperiment. The fact that group VI anions in-hibit the reduction of sulfate in whole cells indi-cates that this conclusion is also valid for wholecells.

Although evidence has not been presented, a

third enzyme, inorganic pyrophosphatase, isprobably required for maximal activity since theequilibrium of the reaction catalyzed by ATP-sulfurylase lies far toward ATP and sulfate. Re-moval of PPi would serve to "pull" the reactiontoward APS formation. The pathway of sulfatereduction in D. desulfuricans can be summarizedby the following equations:

ATP + SO4-= APS + PPi (12)

PPi + H20-- 2 Pi (13)

APS + 2e = AMP + S03 (14)

Although the substrates for the reduction of sul-fate by yeast and D. desulfuricans are analogous,the enzymes catalyzing these reactions seem tobe entirely different. APS-reductase does notproduce acid-volatile sulfur from PAPS, althoughPAPS-reductase does show slight activity towardAPS (133). In addition, TPNH will not functionas electron donor for the reduction of APS;neither will arsenite inhibit nor lipoic acid stimu-late the reduction of APS in partially inactivatedpreparations. Another interesting property ofAPS-reductase is that the reaction is reversible(85). In the presence of extract, sulfite, AMP,and ferricyanide, ferricyanide is reduced andAPS formed. The stoichiometry of the reactionindicates that the over-all reaction can be repre-

sented by equation 15. The reaction is unique in

S03- + AMP + 2Fe(CN)6- (15)APS + 2Fe(CN)64

that the biologically utilizable energy is producedin the form of a high-energy sulfate rather than

TABLE 3. Nucleotide specificity of APS-reductase

Nucleotide Activity(per cent activity of AMP)

None 10AMP 100IMP 36GMP 28CMP 0UMP 6dAMP 102dGMP 9dCMP 10TMP 10PAP 9

Materials: The complete system contained:tris (pH 8.0), 300 jsmoles; nucleotide, 5 jsmoles;Fe(CN)6¶, 3 ;moles; Na2SO3, in 5 X 103 M EDTA,10 M&moles. A charcoal-treated extract of Desulfo-vibrio desulfuricans, 0.4 mg in a volume of 3.0 ml,was used.

Abbreviations: AMP, IMP, GMP, CMP. UMP,TMP, the 5'-phosphates of ribosyl adenine, hy-poxanthine, guanine, cytosine, and uridine, re-spectively; d, deoxy. PAP, 3', 5', diphosphoadeno-sine. TMP, thymidylic acid.

high-energy phosphate. The reaction is not spe-cific for AMP and, as shown in Table 3, othersulfate-containing nucleotides can be formed.The formation of the sulfate derivatives ofguanosine 5'-phosphate (GMP), inosine 5'-phos-phate (IMP), and 2'-deoxyribosyl adenine 5'-phosphate (dAMP) have also been demonstratedby the use of S35-labeled sulfite. The importanceof these sulfur-containing nucleotides in the re-duction of sulfate by D. desulfuricans is unknown.

It is of interest that neither TPN, DPN, flavin,or cytochrome c3 will function as electron ac-ceptor in this oxidation. Reduced cytochrome c3does not supply electrons for the reduction ofAPS by dilute or purified preparations of APS-reductase and therefore does not seem to be theimmediate electron donor for APS-reductase.

Thus, the pathway of sulfate reduction in D.desulfuricans is quite different from that de-scribed in yeast, and the data indicate that atleast two pathways of sulfate reduction exist inbacteria.

C. The Reduction of Other Sulfur Compounds

The role of sulfite as an intermediate in the dis-similatory reduction of sulfate has been well es-

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tablished by data obtained with both intact cellsand extracts of D. desulfuricans. The formation ofsulfite during the reduction of sulfate by wholecells was reported by Millet (75). In extracts, theformation of sulfite from ATP and sulfate hasbeen observed (82). Cell-free extracts (95) reducesulfite to sulfide with molecular hydrogen andthe reaction requires cytochrome C3 , hydrogenase,sulfite reductase and possibly other components(51, 76) for the over-all reduction shown byequation 16. Postgate (89) presented evidence

S03= + 3H2 >* S= + 3H20 (16)

which indicates that thiosulfate, althoughreduced to sulfide by intact cells, is not anintermediate in the reduction of sulfite. As in thecase of sulfate reduction, methyl viologen willcouple sulfite reductase with hydrogenase in theabsence of cytochrome C3 (48). Recently, an assaymethod, which employs the oxidation of reducedmethyl viologen as an index of activity, was de-vised for this sulfite reductase (51). This methodseems to be a direct assay of sulfite reductase andshould eliminate many of the difficulties en-countered when hydrogen utilization is used forassay. When assayed in partially purified prepa-rations, cytochrome C3 will not couple sulfite re-ductase with hydrogenase and, as in the case ofAPS-reductase, such inactivity indicates thatother components are required for the transfer ofelectrons from reduced cytochrome C3 to the sul-fite reductase. Similarly TPN, DPN, and flaxindo not seem to be active in this system.The sulfite reductase of D. desulfuricans has not

been characterized well enough to decide con-clusively whether it is similar to the sulfite re-ductase of E. coli, and the nature of the electrondonor should be investigated much more thor-oughly in this system. The one major differenceis the greater sensitivity of sulfite reductase of E.coli to KCN. The unique property of the sulfitereductase from E. coli is its ability to reduce hy-droxylamine as well as sulfite. Although cell-freeextracts of D. desulfuricans reduce hydroxylaminewith hydrogen and cytochrome C3 or methyl violo-gen as electron carrier (110), this reduction seemsto be a nonenzymatic process. The observations ofMager (69) do indicate that the question of thepresence of hydroxylamine-reducing activityshould be re-examined in extracts of D. desul-furicans. Whole cells and extracts reduce bothtetrathionate and thiosulfate to sulfide (95). The

SULFIE OXKATZN SULFATE REDUCTION

ADPPL4S.-ATP PAlPS-APP RAI

50jX-4 APS ,PAPS (@) SULFATES

AMP TP NH

02 02

GSH

ORGANIC SULFUR

(5) COMPOUNDS

FIG. 5. Intermediates in the interconversion ofsulfate and sulfide. (1) ATP-sulfurylase: (2) APS-kinase; (3) sulfokinases; (4) PAPS-reductase; (5)APS-reductase; (6) sulfite reductase; (7) serinesulfhydrase; (8) enzyme unknown, may be nonenzy-matic; (9) sulfide oxidase; (10) tetrathionase; (11)thoisulfate reductase; (12) sulfite oxidase; and (13)ADP-sulfurylase.

reduction of tetrathionate seems to involve pre-liminary cleavage to thiosulfate by the enzymetetrathionase, and then reduction of the thiosul-fate. Thiosulfate is reductively cleaved to sulfiteand sulfide, as in yeast, and its reduction withhydrogen requires cytochrome C3 The latter can-not be replaced by DPN, TPN, or flavin. Methylviologen will also function as electron carrier be-tween hydrogenase and thiosulfate in this reac-tion; however, the role of GSH and other sulfhy-dryl reagents in this system have not beeninvestigated. In contrast to sulfite reductase andAPS-reductase, cytochrome C3 seems to donate itselectrons directly to thiosulfate reductase (48).

In Fig. 5, the sulfur cycle is again shown, andthe intermediates that have been demonstratedto function in the reduction of sulfate to sulfiteare inserted. Although two pathways for the re-duction of sulfate to sulfite have been reported(the PAPS-pathway in yeast and the APS-path-way in D. desulfuricans) the reduction of sulfiteand other oxidized sulfur compounds to sulfideseems to be catalyzed by similar enzymes.

D. The Energy Metabolism ofDesulfovibrio desulfuricans

In spite of the fact that D. desulfuricans is notan autotrophic organism, it can obtain the energyrequired for growth from the oxidation of hydro-gen with sulfate (109). The increased incorpora-tion of P32-labeled Pi during this oxidation of hv-drogen with sulfate suggests that the organism

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can transform some of the chemical energy liber-ated into high-energy phosphate (49, 72, 116).These observations are particularly interestingsince the organism requires energy to utilizesulfate as terminal electron acceptor. The factthat high-energy phosphate is required for thereduction of sulfate but not the reduction of sul-fite or thiosulfate, makes this system (the reduc-tion of sulfate, thiosulfate, or sulfite with hydro-gen) ideally suited for inhibitor studies on themechanism of phosphorylation that has beenpostulated to occur concomitantly with the oxi-dation of hydrogen.

In all probability, the inhibition of sulfate re-duction in whole cells by group VI anions indi-cates that only the enzyme ATP-sulfurylase isinvolved in the formation of APS. This impliesthat two high-energy phosphates are required foreach sulfate reduced to sulfide, or each four hydro-gen molecules oxidized, if it is assumed that PPicannot give rise directly to high-energy phos-phate. This implication is probably correct, be-cause the formation of APS depends upon theremoval of PPi.

It has been shown that 2,4-dinitrophenol(DNP) has no effect on the reduction of thio-sulfate or sulfate with hydrogen in cell-free ex-tracts (84). Therefore, it can be concluded thatDNP does not have an inhibitory effect on theenzymes or electron transport sequence involvedin these reductions. Since in whole cells, DNPcompletely inhibits the reduction of sulfate butdoes not affect the reduction of thiosulfate, it wasapparent that DNP prevented the cells from pro-ducing the high-energy phosphate required forthe reduction of sulfate but not thiosulfate. Thus,it was postulated that an oxidative phosphoryla-tion was occurring during the oxidation of hydro-gen with sulfate (84), and uncoupling of theoxidative phosphorylation with DNP manifesteditself by failure to reduce sulfate but not thio-sulfate.Another interesting inhibitor of sulfate reduc-

tion in whole cells is methyl viologen. As notedpreviously, methyl viologen efficiently coupleshydrogenase with sulfite, thiosulfate, and APS-reductase in cell-free extracts. The fact that withwhole cells methyl viologen inhibits sulfate reduc-tion but stimulates thiosulfate reduction with hy-drogen is ascribed to the ability of methyl viologento shunt electrons around the natural electroncarriers participating in the oxidative phospho-

rylation. Thus the generation of high-energyphosphate, required for the activation of sulfatebefore its reduction, is prevented.Whole cells of D. desulfuricans can reduce

DNP to aminophenols that are much less inhibi-tory for sulfate reduction. When the DNP isreduced, the restoration of sulfate reduction doesnot occur. However, if, after the reduction ofDNP, a catalytic amount of pyruvate, thiosul-fate, or sulfite is added, sulfate reduction isalmost immediately restored to the rate observedin the controls. Since pyruvate can supply ATPby the formation of acetyl phosphate, and sincesulfite and thiosulfate, by virtue of their abilityto act as electron acceptors, participate in thepostulated oxidative phosphorylation, it seemslikely that these three compounds can supplyenough ATP to initiate sulfate reduction andthereby cause this "sparking" phenomenon (84).All these observations are consistent with theoccurrence of oxidative phosphorylation duringthe oxidation of hydrogen in the presence ofvarious sulfur compounds.

Nevertheless, it was a disturbing fact that cells,grown heterotrophically on lactate and sulfate,were capable of oxidative phosphorylation duringthe oxidation of hydrogen. Inspection of theequations of the reactions involved in the lactate-sulfate fermentation (equations 17 to 21) reveals

2CH3CHOHCOOH 172CH3COCOOH + 4H- (1)

2CH3COCOOH + 2HOP03-2CH3COOP035 + 2CO2 + 4H.

2CHCOOPP03 + AMP- + 2H+ -*2CH3COOH + ATP4-

S04- + ATP4- + 8H -

S= + 2H20 + AMP-+ 2HOP03= + 2H+

(18)

(19)

(20)

2CH3CHOHCOOH + 2041)2CH3COOH + 2CO2 + S= + 2H20 )

that there is no net production of high-energyphosphate from substrate-level phosphorylationduring this fermentation. Therefore, even whengrown on organic electron donors, oxidativephosphorylation is probably necessary to obtainthe energy required for growth (84).Not only is the pathway of sulfate reduction

unique in the sulfate-reducing bacteria, but theenergy requirement for sulfate reduction neces-sitates that these organisms obtain energy by

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some means other than substrate-level phos-phorylation during growth on many electrondonors and sulfate. Thus many electron donorscan probably be viewed as the equivalent ofmolecular hydrogen in the metabolism of theseorganisms, since their carbon is not incorporatedinto cellular material (6). Although cytochromeC3 presumably functions in some manner in theoxidative phosphorylation, other sulfate-reducingbacteria do not possess this cytochrome. It willbe of great interest both to investigate the elec-tron transport carriers and energy metabolism inthese organisms and to determine whether or notthere is an oxidative phosphorylation in each ofthe four reduction steps postulated in the reduc-tion of sulfate. Thus anaerobic growth withsulfate as sole electron acceptor is a unique me-

tabolism with respect to the role of organic elec-tron donors, energy metabolism, and the pathwayof sulfate reduction.

IV. THE DISTRIBUTION OF APS-REDUCTASE AND

PAPS-REDUCTASE IN MICROORGANISMS

Since yeasts, assimilatory sulfate reducers,reduce sulfate in the form of PAPS, and D.desulfuricans, a dissimilatory sulfate reducer,reduces sulfate in the form of APS, it seemed thatthe PAPS-pathway of sulfate reduction mightbe characteristic of assimilatory sulfate reductionand the APS-pathway of sulfate reduction charac-teristic of dissimilatory sulfate reduction. Table4 lists the results of a survey designed to ascertainwhether the pathway of sulfate reduction in a

given organism could be correlated with the roleof sulfate in the metabolism of that organism.The reduction of both APS and PAPS was ex-

amined in the presence of TPNH (generatedfrom glucose 6-phosphate by Zwischenferment)or reduced methyl viologen as electron donor.Activity was indicated by the formation of acid-volatile sulfur and varied in the various prepara-tions from 0.1 to 30 mjtmoles of acid-volatilesulfur per hr per mg of protein.The electron donor for the reduction of PAPS

varies considerably in different organisms. Yeastutilizes only TPNH as electron donor for thisreduction, whereas E. coli will utilize reducedmethyl viologen, TPNH, and DPNH (69) forthe reduction. In extracts of Clostridium pas-

teurianum and Rhodopseudomonas spheroides, onlymethyl viologen will function as electron donor.Thus, there seems to be a variability in the

TABLE 4. Pathway of sulfate reductionin assimilatory sulfate reducers

APS- PAPS- Assimila-Organism reduc- reduc tory sul-

tase tase- fate re-tase tase duction*

Escherichia coli ........-... + +Baker's yeast.............- _ - +Aerobacter aerogenes.......- + +Proteus mirabilis..........- + +Proteus vulgaris..........- + +Pseudomonas hydrophila -.- + +Aeromonas punctata.......- + +Clostridium kluyveri.......- + +Clostridium pasteurianum. - + +Rhodopseudomonas

spheroides .............-.. + +Rhodopseudomonas

palustris ........... ... - - +Bacillus subtilis........... - - +Bacillus polymyxa ........ - - +Bacillus megaterium ..... - - +Bacillus terminalis........- ± +Leuconostoc mesenteroides.. - _Staphylococcus aureus......Streptococcus faecalis ......

* Plus signs in this column indicate that anorganism can either incorporate S3 04= into cellu-lar material containing reduced sulfur compoundsor can grow on sulfate as the sole source of sulfur.

specificity of PAPS-reductase for the electrondonor from exclusively pyridine nucleotide(yeast) to exclusively some unknown electrondonor that is replaceable by methyl viologen (C.pasteurianum). R. spheroides reduced PAPS to alimited extent only with reduced methyl viologenas electrondonor, and Rhodopseudomonas palustrisshowed no reducing activity toward PAPS orAPS. Ibanez and Lindstrom (44) demonstratedthe photoreduction of sulfate by chromatophoresof Rhodospirillum rubrum. The failure to observegood reduction of these sulfur-containing nucleo-tides with photosynthetic organisms may be dueto the fact that no special precautions were takenwith respect to the competence of chromatophoresor to the presence of light during the incubationperiod. However, the only nucleotide that wasreduced was PAPS.

Although very slight PAPS-reductase activitywas found in extracts of Bacillus terminalis,extracts of Bacillus subtilis, Bacillus polymyxa,and Bacillus megaterium did not reduce eitherPAPS or APS when grown on a complex or

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minimal medium. These- organisms will grow ona defined medium with sulfate as the sole sourceof sulfur (60) and, in addition, the formation ofsmall amounts of radioactive sulfide from S3504-has been observed with whole cells of B. mega-terium (11). In these respects, the Bacillus speciesseem to be assimilatory sulfate reducers. Whetherthe failure to observe the reduction of PAPS orAPS is due to technical difficulties, such as rapidhydrolysis of added sulfur-containing nucleotides,or to differences in the sulfate metabolism of theseorganisms is at present unknown.From these limited observations it therefore

seems likely that the PAPS pathway of sulfatereduction is characteristic of microorganisms thatreduce sulfate solely for assimilatory purposes.However, it must be cautioned that exceptionsto this idea may be found in the future.

Organisms that reduce APS but not PAPS arelisted in Table 5. APS is reduced only with re-duced methyl viologen as electron donor and thespecific activity of APS-reductase is usually 20to 100 times that observed for PAPS-reductasein assimilatory sulfate reducers. Other strains ofD. desulfuricans seem to also utilize the APS-pathway of sulfate reduction (Peck, unpublisheddata).

C. nigrificans is a dissimilatory sulfate reducer(15) and extracts of this organism, when supple-mented with ATP and S3504- under an atmos-phere of molecular hydrogen, reduce sulfate toacid-volatile sulfur. Since this reduction was com-pletely inhibited by MoO47, the first step in thereduction of sulfate by this organism seemed tobe the formation of APS by the enzyme ATP-sulfurylase. Both ATP-sulfurylase and ADP-sulfurylase have been shown to be present inthese extracts (Peck, unpublished data). As shownin Table 5, these extracts formed acid-volatilesulfur from APS and the specific activity of APS-reductase is high compared to the specific activityof PAPS-reductase in assimilatory sulfate re-ducers. In all respects, the mechanism of sulfatereduction in C. nigrificans appears identical tothat in extracts of D. desulfuricans.Another organism, Vibrio cholinicus, isolated

by Hayward and Stadtman (36), seems to be adissimilatory sulfate reducer. Growth on cholineis stimulated when sulfate is added to the me-dium, and in addition radioactive sulfide isproduced from S35047 during the oxidation ofcholine by extracts of this organism (37). As

TABLE 5. Pathway of sulfate reduction in dissim-ilatory sulfate reducers and organisms that oxi-dize reduced sulfur compounds

Organism APS- PAPS-reductase reductase

Desulfovibrio desulfuricans +Clostridium nigrificans ......... +Vibrio cholinicus............... +Thiobacillus thioparus. ....... +Thiobacillus thiooxidans........ +Thiobacillus denitrificans . +

shown in Table 5, extracts of V. cholinicus ex-hibit only APS-reductase with reduced methylviologen in high specific activity and show noPAPS-reductase activity. These results indicatethat V. cholinicus is a dissimilatory sulfate re-ducer and recently it has been claimed that V.cholinicus was incorrectly named and the organ-ism is actually D. desulfuricans (4). Thus, alldissimilatory sulfate reducers that were examinedreduced sulfate in the form of APS and exhibitedno activity for PAPS.

In addition to microorganisms that reducesulfur compounds, extracts of organisms thatoxidize compounds of sulfur more reduced thansulfate were examined for the presence of APS-reductase and PAPS-reductase. Three species ofThiobacillus grown on thiosulfate as energy sourceshowed APS-reductase, in some instances equal inspecific activity to that observed in the dis-similatory sulfate-reducing bacteria, and noPAPS-reductase. Since cell-free extracts of E.coli and Proteus vulgaris did not contain APS-reductase when grown heterotrophically withadded thiosulfate (Peck, unpublished data), it didnot seem that the presence of APS-reductase is ageneral response of microorganisms to growth inthe presence of thiosulfate. These are extremelyinteresting observations in view of the fact thatthiobacilli obtain the energy required for growthby the oxidation of reduced sulfur compounds.The role of APS-reductase in the sulfur metab-olism of these organisms is discussed subse-quently.

V. THE OXIDATION OF REDUCEDSULFUR COMPOUNDSA. General Aspects

The formation of sulfate and other sulfur com-pounds from more reduced sulfur compounds,

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both organic and inorganic, is commonly ob-served in cultures of single microorganisms (30)and in mixed cultures (32). In addition, sulfatemay be formed from the hydrolysis of organicsulfates by enzymes termed sulfatases (33). Thepathway for the oxidation of cysteine to sulfatehas been described by Singer and Kearney (112)in extracts of P. vulgaris. The reaction sequenceinvolves the formation of a series of oxidizedderivatives of cysteine that terminate in theproduction of sulfite; the sulfite thus formed isoxidized to sulfate. Many organisms can appar-ently oxidize cysteine to sulfate; however, theremay be some differences in the intermediatesinvolved (27, 79). Generally speaking, it is quitepossible that inorganic sulfur compounds, suchas sulfide, can react with the proper organicacceptor to form an organic intermediate andbe oxidized to sulfate. The pathway, described bySinger and Kearney (112) for the oxidation ofcysteine, does not seem to be of major importancefor the oxidation of inorganic sulfur compoundssince some organisms that have been shown tooxidize cysteine to sulfate do not oxidize reducedinorganic sulfur compounds to sulfate. Therefore,the enzymatic reactions discussed in this sectionwill mainly concern the formation of sulfate andother inorganic sulfur compounds from sulfideby an inorganic pathway.The stable sulfur compounds reportedly formed

during the oxidation of reduced sulfur compoundsare sulfite, thiosulfate, and elemental sulfur. Inaddition, various polythionates, in particulartetrathionate, are commonly formed from theoxidation of reduced sulfur compounds (130). Theformation of polythionates has not been observedduring the reduction of sulfate although someorganisms may reduce polythionates to sulfide.Many heterotrophic organisms are capable of

oxidizing reduced sulfur compounds, and theproducts are usually sulfate or polythionates.However, neither the organisms nor the en-zymatic reactions involved have been charac-terized to any extent. The thiobacilli and thephotosynthetic bacteria are the best studied withregard to the over-all oxidation of reduced sulfurcompounds. Until recently, little informationhas been available concerning the enzymaticmechanisms involved in these oxidations.

B. The Oxidation of Reduced Sulfur Compoundsby Heterotrophic Microorganims

It has been known for many years that certainheterotrophic organisms can oxidize reducedsulfur compounds (30, 130). These organisms canbe separated into two general physiologicalgroups. The first group can oxidize elementalsulfur and thiosulfate to polythionates, and thesecond group can convert the polythionates tosulfate. In mixed cultures these organisms canconvert reduced sulfur compounds to sulfate.The only enzyme studied that is possibly in-volved in this transformation is tetrathionase,which forms tetrathionate from thiosulfate (87,129). Evidence has been presented that indicatesthe oxidation of thiosulfate to tetrathionateprobably does not supply energy for the growthof cells capable of catalyzing this oxidation (7,129). The mechanism for the formation of otherpolythionates is at present unknown; however, itmay be possible that tetrathionase can also formother polythionates by a mechanism similar tothat suggested by Lees (65) for the oxidation ofthiosulfate.Very little is known concerning the oxidation

of elemental sulfur, sulfide, or sulfite in thesemicrobial systems. Elemental sulfur is probablynonenzymatically reduced to sulfide (118), andthe sulfide is either reoxidized to elemental sulfuror oxidized to thiosulfate. The formation ofthiosulfate from sulfide has been observed inextracts of various mammalian tissues (7, 45, 115)and Thiobacillus thiooxidans (121) and seems tobe a unique reaction. Protein fractions, ferritin,and various metalloprotein complexes (88, 115)can catalyze this oxidation of sulfide and amechanism for this oxidation has been pro-posed. Although intermediates, if any, havenot been isolated, it seems that free polythionatesare not involved (9, 121). It is interesting thatin mammals, thiosulfate is either excreted assuch or metabolized in such a manner that thetwo sulfur atoms are not equivalent (114).The oxidation of sulfite to sulfate is nonen-

zymatically catalyzed by various metal ions (38).In the presence of various chelating agents, it ispossible to observe the enzymatic formation ofsulfate from sulfite and the oxidation has beenmost extensively studied in mammalian tissue(28). This enzyme, sulfite oxidase, requireshypoxanthine as a cofactor and probably ilt-

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volves a disulfide group, possibly lipoic acid,since the reaction is inhibited by arsenite. Atpresent, the reactions involved in the oxidationof these inorganic compounds by heterotrophicmicroorganisms can only be inferred from thenature of these reactions in other organisms.

C. The Oxidation of Reduced Sulfur Compoundsby Photosynthetic Microorganisms

The chlorobacteriaceae and the thiorhodaceaegrow photosynthetically under anaerobic condi-tions and require reduced sulfur compounds (orcertain other compounds) that are oxidized tosulfur or sulfate. Although little is known aboutthe actual pathway of the metabolism of in-organic sulfur compounds, these organismsappear not to derive energy from the oxidationof such reduced sulfur compounds (63). Grossly,the pathway of oxidation of reduced sulfur com-

pounds seems to be similar to that in other micro-organisms. Elemental sulfur is formed as a

transitory intermediate in the oxidation of thethiosulfate (68), and polythionates are utilized(63) and may be formed during the oxidation ofsulfide (57). Hendley (39) has observed some

reduction of S35-labeled sulfate to sulfide in wholecells of Chromatium sp. and APS-reductase hasbeen found in extracts of this organism; however,the activity was low compared to that observedin the thiobacilli and the dissimilatory sulfatereducers (Peck, unpublished data). These ob-servations suggest that the oxidation of reducedsulfur compounds proceeds by way of a non-

phosphorylative pathway in these organisms.However, the oxidation of reduced sulfur com-

pounds is closely coupled to photosynthesis (68)and maximal activity may depend upon thepresence of light and an intact photosyntheticapparatus (49).

D. The Oxidation of Reduced Sulfur Compoundsby the Thiobacilli

The thiobacilli are a small group of micro-organisms whose energy metabolism is uniquelyadapted to obtain all the energy required forgrowth from oxidation of inorganic sulfur com-

pounds to sulfate, and which utilize carbondioxide as the source of carbon for the synthesisof cellular materials. In addition to being auto-trophic, the thiobacilli also exhibit other inter-esting biological phenomena. These organisms are

obligately autotrophic with the exception of T.novellus, which will grow on organic substratesand is therefore a facultative autotrophic organ-ism (130). One species, T. thiooxidans, is tolerantof extremely acid environments, and is capableof growing at pH values less than 1; however,other strains vary in their tolerance toward acid.T. denitrificans can utilize nitrate anaerobicallyin place of oxygen as electron acceptor and pro-duces molecular nitrogen (3). Some of the thio-bacilli can oxidize thiocyanate or ferrous ion andthus obtain the energy required for growth (130).In view of these differences, it is interesting thatrecently Johnstone, Townshend, and White (54)claimed that various strains of thiobacilli canbe derived from a single species of these organ-isms. The general physiology of the thiobacillihas been reviewed by Vishniac and Santer (130),Larsen (64), and Umbreit (128). Lees (65) hasrecently reviewed the energy metabolism of theseorganisms.Most of the thiobacilli are capable of oxidizing

sulfide, elemental sulfur, thiosulfate, tetra-thionate, and, in some cases, sulfite to sulfate(81). The usual substrates for growth, however,are either elemental sulfur or thiosulfate, thio-sulfate being the preferred substrate for technicalreasons. When grown on thiosulfate, thiobacilliform elemental sulfur either as a transitory inter-mediate or in quantities approaching the amountof sulfate produced (T. thioparus); however, theamount of sulfur produced seems to depend notonly upon the species but also upon the condi-tions employed to culture the organism (130).During the oxidation of thiosulfate by wholecells of T. thioparus, polythionates consistingmainly of tetrathionate are rapidly formed (130),and schemes for the oxidation of reduced sulfurcompounds involving polythionates as inter-mediates have been proposed by Tamiya, Haga,and Huzisige (124), Vishniac and Santer (130),and more recently by Lees (65). Lees proposedthat the polythionates are enzyme-bound inter-mediates and that the reactions involving poly-thionates take place at the surface of the cell.Only at the level of sulfide does the sulfur atomenter the cell, at which time it is oxidized tosulfate. Thus the problem of the oxidation ofdifferent reduced sulfur compounds can be viewedas the problem of the oxidation of sulfide. En-zymological evidence to support this scheme is

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scanty, consisting mainly of the observation thatcell-free extracts will oxidize thiosulfate to tetra-thionate (126). This reaction seems similar tothe reaction catalyzed by the enzyme tetra-thionase (87). 'Iost of the evidence supportingthe polythionate pathway is derived from obser-vations made with whole cells. Although not a

universal observation (81), many strains of thio-bacilli form and oxidize tetrathionate and otherpolythionates. In addition, the rapid formationof polythionate from radioactive thiosulfate hasbeen observed (127) and Vishniac and Santer(130) have reported the formation of thiosulfateand polythionate during the oxidation of S35_labeled sulfide. Other observations have sug-

gested a role of phosphate in the oxidation ofthiosulfate (104).The presence of Pi is required for the complete

oxidation of thiosulfate and tetrathionate tosulfate, and the phosphate can be replaced byarsenate. This result indicates that phosphatemay be involved in the oxidation of reduced sulfurcompounds at the substrate level. In addition,the phosphate requirement for both tetrathionateand thiosulfate oxidation indicates that at some

point these compounds are oxidized by the same

pathway. Santer (103) recently presented evi-dence that further substantiates the direct role ofPi in the oxidation of thiosulfate. When wholecells oxidized thiosulfate in the presence of 018-labeled P1, 018 was transferred from the Pi to thesulfate formed during the oxidation. The transferis of sufficient magnitude to indicate that one ofthe oxygen atoms of each of the sulfate moleculesformed is derived from the phosphate. Theseresults suggested the possibility that APS or

PAPS might be intermediates in the oxidation ofreduced sulfur compounds by these organisms.

Since extracts of the thiobacilli had only beenobserved to oxidize thiosulfate to tetrathionate,it was possible that the failure to observe thecomplete oxidation of thiosulfate to sulfate was

due to the fact that the initial step in the oxida-tion of thiosulfate was a reduction, i.e., the reduc-tive cleavage of thiosulfate to sulfide and sulfite,as previously described in other organisms.Several observations indicated that the reductivecleavage of thiosulfate was involved in thio-sulfate metabolism of these organisms. Skar-zynski and Ostrowski (113) have reported thatintact cells of T. thioparus incorporate the ligandor outer sulfur atom of thiosulfate into cellular

materials and elemental sulfur. However, Santeret al. (104) were unable to confirm this observa-tion and indicated that both sulfur atoms ofthiosulfate are metabolized in the same manner.

Other evidence indicates that, in the presence ofan unlabeled pool of elemental sulfur, the prefer-ential incorporation of the outer sulfur atom ofthiosulfate into elemental sulfur can be observed(Peck and E. Fisher, unpublished data). The pres-

ence of thiosulfate-reductase in extracts of T. thio-oxidans is suggested by the observation thatsulfide is produced by reaction mixtures contain-ing GSH and thiosulfate (121) and the presence

of thiosulfate reductase, as indicated by theformation of both sulfide and sulfite from GSHand thiosulfate, has been demonstrated in ex-

tracts of T. thioparus. The products of this reduc-tion could be the precursors of the sulfate andelemental sulfur which are formed during thegrowth of this organism. Thus sulfide is oxidizedto elemental sulfur by extracts of T. thiooxidans(121) and T. thioparus (82), and sulfate couldbe produced by the oxidation of sulfite, possiblyby a sulfite oxidase similar to that described inmammalian tissues (28) and T. denitrificans(73). However, such a reaction sequence does notoffer an explanation for the role of Pi in theoxidation of thiosulfate.

Extracts of T. thioparus have been shown tocontain other enzymatic activities that may beconcerned in the oxidation of reduced sulfur com-

pounds (83). Extracts of this organism reduceAPS to sulfite and AMIP in the presence of re-

duced methyl viologen, and the specific activityof APS-reductase in these extracts is comparableto that observed in the dissimilatory sulfate-reducing bacteria. In contrast, PAPS is notreduced with either reduced methyl viologen or

TPNH as electron donor. ATP-sulfurylase,ADP-sulfurylase, and adenylic kinase are alsopresent in high specific activity in these extracts.From a consideration of results obtained withboth whole cells and extracts, the reaction se-

quence (equations 22 to 27) for the oxidation ofthiosulfate to sulfate has been proposed (83).

4H+ + 4e + 2S203 thiosulfate reductase

2SO3= + 2H2S(22)

2H2S + 02 sulfide oxidase 2S° + 2H20 (23)

2SO3 + 2AMP PS-reductase (24)

2APS + 4e

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2APS + 2P; ADP-sulfxurylase (25)

2ADP + 2SO4-

2ADP adenvlic kinase I AMP + ATP (26)

2S203- + 02 + AMP + 2Pi + 4H+

2SO + 2SO4- + ATP + 2H20

Thiosulfate is postulated as being reductivelycleaved to sulfide and sulfite by thiosulfatereductase and the sulfide oxidized to elementalsulfur by sulfide oxidase. Sulfite is oxidized in thepresence of AMP to yield APS and the high-energy sulfate exchanged for phosphate to yieldADP by the action of ADP-sulfurylase. ATP can

then be formed from ADP by adenylic kinase.The sum of equations 22 to 26 (equation 27)indicates that a net production of high-energyphosphate should occur during the oxidation ofthiosulfate. ADP- and ATP-sulfurylase can ex-

change the high-energy sulfate for phosphate or

pyrophosphate, respectively; however, the extentof participation of each enzyme in this exchangeprobably depends upon the availability of Piand PPi. Since Pi is required for the completeoxidation of thiosulfate, ADP-sulfurylase hasbeen proposed as the enzyme most likely involvedin the formation of high-energy phosphate fromAPS.The reaction sequence explains the effects of

Pi and arsenate observed with whole cells. Adeficiency of Pi will result in an accumulation ofAPS and consequent depletion of the AMP pres-ent in the cells and, thus, inhibition of the oxida-tion of sulfite or thiosulfate. Arsenate may func-tion to replace Pi in two ways. First, arsenatewill replace Pi by catalyzing the arsenolysis ofAPS (99), thereby regenerating AMP for theoxidation of sulfite. A second possible action ofarsenate may be to liberate phosphate from phos-phate-containing organic compounds in the cells,thereby replenishing the Pi pool in these cells.A cell-free system that oxidizes thiosulfate in

the presence of GSH has been described (83).Further observations with this system (86)demonstrate that in the presence of GSH, thio-sulfate is reductively cleaved to sulfite and sulfide,and the sulfite oxidized to sulfate with the produc-tion of one high-energy phosphate per sulfateformed. To observe phosphorylation, it is neces-

sary to supplement reaction mixtures with NaFand ethylenediaminetetraacetate (EDTA) to

inhibit hydrolytic enzymes and to add AMP andPi as substrates for the phosphorylation. As.shown in Table 6, the phosphorylation was notinhibited by DNP and therefore is probably notthe result of oxidative phosphorylation. This isfurther supported by the observation that, in thepresence of P32-labeled Pi, almost all of theesterified phosphate was found to be present inADP. This result is to be expected from a con-sideration of the postulated reaction sequence(equations 22 to 26) since NaF is known to inhibitadenylic kinase. Furthermore, it was observedthat even in the presence of a pool of unlabeledATP, almost all of the esterified phosphate waslocated in the ADP. From these results, it wasconcluded that ADP was the initial product ofthis phosphorylation and could not have beenformed in some manner from ATP. It was alsopossible to demonstrate a dependency on AMPfor phosphorylation and oxygen utilization, andAMP is the only nucleotide with which significantphosphorylation has been observed. Arsenatewill stimulate the formation of sulfate whenconditions are such that there is an absence of Piand only a catalytic amount of AMP. Thus itseems that arsenate can stimulate the oxidationof thiosulfate by the arsenolysis of APS. Thenature of the phosphorylation and the productsof the oxidation demonstrated that sulfate isproduced and ADP formed in a 1:1 ratio inaccord with the postulated reaction sequence.Oxygen utilization and the effect of arsenate

TABLE 6. Esterification of orthophosphate (Pi) duringthe oxidation of thiosulfate by cell-free

extracts of Thiobacillus thioparus

Esteri- Esteri-Additions Pi 02 fled -fiedAdditions utilized utilized phos- phos

phate

jimoles Mmoles Mmoles jsmoles

Complete ............. 2.53 3.47 2.69 2.26Complete plus 2,4-di-

nitrophenol (2 X104M).3.06 4.66 3.72 3.22

Materials: The complete system contained:tris (pH 8.0), 300 jzmoles; EDTA, 10.umoles; NaF,100,umoles; AMP, 10 smoles; Na2S203, 40 ,umoles;GSH, 40 izmoles; TPN, 0.67 jmole; p,32, 5 Mmoles(containing 2.28 X 106 counts:min:jsmole). Thio-bacillus thioparus extract, 26 mg in a total volumeof 2.0 ml. Time, 30 min.

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can also be explained by this scheme. Similarresults were obtained using sulfite rather thanthiosulfate and GSH as substrate for this oxida-tion (Table 7).

Extracts of T. thioparus will form APS in thepresence of enzyme, AMP, SOt=, and Fe(ClN)t7,as was observed with extracts of D. desulfuricans(85). IMP, GMP, and dAMP were the onlynucleotides tested that substitute for AMP in

this reaction and form the corresponding sulfur-containing nucleotides. The facts that PAP willnot function in this system, that arsenite doesnot inhibit the production of APS, and that ex-

tracts do not reduce PAPS, indicate that APS-reductase is quite different from PAPS-reductase(85). Studies on the stability, inactivation, andfractionation of APS-reductase activity, whichwas measured with reduced methyl viologen as

electron donor and ferricyanide as electron ac-

ceptor, indicate that these activities are catalyzedby a single enzyme. However, some differenceshave been observed between the APS-reductaseof D. desulfuricane and that of T. thioparus. Theenzyme is much more labile in extracts of T.thioparus than in extracts of D. desulfuricans. Inaddition, the APS-reductase of T. thioparus, butnot that of D. desulfuricans, catalyzes a rapidexchange between sulfite and the sulfate of APS(Peck and W. J. Payne, unpublished data). Thisdifference may be ascribed to the unknown elec-tron carriers, which may differ with the physio-logical function of the enzyme, in the oxidation or

reduction of APS. The demonstration of the re-

versibility of APS-reductase, the critical and

TABLE 7. Oxidation of S203, SOr, and S

System 0, Pi S04cSystem utilized esterified produced

pmoles umoles pmoles

Extract ....... ..... 0 0 0+ S203 ............. O 0 0

+ GSH............ 0 0 0

+ S203-+ GSH ....... 2.7 2.0 4.2SO03................2.5 4.5 7.-8

+ S...................3.3 1.8 4.0

Materials: Each reaction mixture contained:tris (pH 8.0), 300 pmoles; AMP 10 pmoles; EDTA,10 pmoles; NaF, 100 pmoles; Pi, 5 pmoles; and,where indicated, Na2S2,O, 10 jumoles; GSH, 40pmoles; Na2SO3, 10 pmoles; Na2S, 10 pmoles; andThiobacillus thioparus extract, 29.5 mg, in a totalvolume of 2.0 ml. Time, 30 min.

TABLE 8. Oxidation of thiosulfate andtetrathionate by extracts

Addiion.Pi S04Additions esterified produced

pnmwtlets pn

S2003 0GSH.0 0S203- + GSH.1.7 2.0S40r........................ 0 0S406 + GSH.1.9 2.1

Materials: Each reaction mixture contained:tris (pH 8.0), 300jumoles; AMP, 10 ptmoles; EDTA,10 jsmoles; NaF, 100 umoles; Pi, 5 pmoles; and,where indicated, Na2S203, 10lsmoles; K2S406, 10usmoles; GSH, 40 pmoles. Thiobacillus thioparusextract, 40 mg in a total volume of 2.0 ml. Time,40 min.

novel reaction in the oxidation of thiosulfate, in-dicates that the thiobacilli can produce a high-energy sulfate at the substrate level from theoxidation of reduced sulfur compounds. All theevidence available indicates that sulfite is an in-termediate in the oxidation of thiosulfate (andother reduced sulfur compounds) and is oxidizedto sulfate by a phosphorylative pathway ratherthan by the sulfite oxidase described in mamma-lian tissue. An effect of AMP on the oxidation ofsulfite was observed previously in plants, butneither phosphorylation or APS formation was re-ported (123).

Although sulfite can be produced from thereductive cleavage of thiosulfate, it is possiblethat sulfite can be formed by other mechanisms,namely, the postulated polythionate cycle (65).The main evidence for the involvement of thio-sulfate reductase in the oxidation of thiosulfate,aside from the presence of the enzyme in extracts,is the absolute requirement for substrate amountsof GSH for oxygen utilization, sulfate production,and ADP formation. Extracts are capable ofoxidizing thiosulfate to tetrathionate; however,added tetrathionate will not function as substratefor sulfate production or ADP formation unlessGSH is added (Table 8). GSH rapidly and non-enzymatically reduces tetrathionate to thio-sulfate, which is then oxidized as previouslydescribed.. Therefore, it appears that, in theseextracts, sulfite is prbduced solely from thiosulfateby thiosulfate reductase.The possible involvement of sulfide in the

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by Starkey (120), who observed the formation ofsmall amounts of hydrogen sulfide from elementalsulfur. When sulfide was added to reaction mix-tures designed to permit phosphorylation andsulfate formation with an extract of T. thioparus,the formation of sulfate and ADP was observed.In addition, it was also possible to detect theformation of thiosulfate but not polythionate.Since the oxidation of sulfide to thiosulfate inmammalian tissues and T. thiooxidans does notseem to involve the formation of polythionates, areaction sequence for the oxidation of elementalsulfur and sulfide to sulfate can be proposed. Thescheme utilizes known reactions and does notinvolve the polythionates as intermediates, asindicated by equations 28 and 29. The thiosulfate

S0+ 2GSH -* H2S + GSSG

2SH- + 202 -f S203 + H20

(28)

(29)

is then oxidized to sulfate, as described in equa-tions 22, 24, and 25. The second reaction has beendiscussed previously. Tetrathionate and otherpolythionates do not function as intermediates inthis scheme, and their occurrence during theoxidation of reduced sulfur compounds by thethiobacilli as well as by heterotrophic organismsis regarded as incidental. With regard to theelemental sulfur formed by many of these organ-isms during the oxidation of thiosulfate, sulfideoriginating from the reductive cleavage of thio-sulfate may be oxidized to elemental sulfur tosupply electrons for the regeneration of GSHthat was oxidized during the reductive cleavageof thiosulfate.The reactions postulated for the oxidation of

thiosulfate (equations 22 to 26) also offer anexplanation for the 018 data obtained by Santer(103), if the transfer of 018 is assumed to occurby established mechanisms. Since the pool ofintracellular AMP is small compared to theamount of substrate oxidized, AMP would rapidlybecome labeled with 018 from Pi as shown inequations 30 and 31.

O 0

2Ado-P-O-S-0- + 2PO418=-=

0-(30)

2Ado-P-08-PO318- + 2S04-

011II2Ado-P-O'8-P0318-0-

0

Ado-P-018- + ATP

0-

(31)

During subsequent cycles of oxidation, 018 willbe transferred from the AMP to sulfate, as shownby equations 32 and 33.

011

S03- + Ado-P-018- =

0-

011

Ado-P-08-SO- + 2e

0-0

Ado-P-0'8-S030-

(32)

+ P04

018

ADP + 0--S-O

0-

(33)

Santer (103) observed that 22 or 23% of the018 present in the Pi was transferred to sulfateduring the oxidation of thiosulfate. Consideringthat these results were obtained with whole cells,his data are in excellent agreement with the 25%value expected if sulfite were oxidized by thereaction sequence just described. Furthermore,the results of Santer can now be interpreted toindicate that the APS pathway or phosphoryla-tive pathway is the major, if not sole, pathwayfor the oxidation of sulfite to sulfate in theseorganisms, since each sulfate formed must havebeen at some time covalently bonded through anoxygen atom to phosphate.The mechanism of the transfer of 018 has been

studied in cell-free extracts of T. thioparus (Peckand M. P. Stulberg, unpublished data). In thepresence of 0'8-labeled P,, 018 is transferred tosulfate during the oxidation of thiosulfate (Table9). However, owing to a dilution of the labeled Piduring the course of the reaction, it was notpossible to calculate the amount of 018 thatshould have been observed in the sulfate pro-duced. Consequently, each of the reaction cycles

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TABLE 9. 018 transfer during the oxidation of thiosulfate in extracts of Thiobacillus thioparuts

Atoms % excess 018Substrates AMP 018 compound added Product isolated

Calculated Observed

eMmolesS203= + GSH................. 4 P04O S04 0.03S03- + Fe(CN)6= ............ 40 P04O ADP 1.66 1.45S203 + GSH ................. 20 AMP S04= 0.11 0.08

required for the transfer of 0'1 from Pi to sulfatewas studied separately. For 018 to be transferredto sulfate, 08 must first be incorporated intoAMP, as shown in equations 30 and 31. Sulfite,AMP, ferricyanide, and 0'8-labeled Pi were incu-bated with a high concentration of sodiumfluoride and the ADP formed was isolated anddegraded to AMP by myokinase. As shown inTable 9, the observed and calculated values forthe 018 content of the AMP are in good agree-ment. The transfer of 018 from AMP to sulfate(indicated by equations 32 and 33) was demon-strated by preparing AMP labeled with 018 andobserving the transfer of the 018 to the sulfatethat was produced during the oxidation of thio-sulfate in the presence of GSH. Again, goodagreement was obtained between the calculatedand observed value for the 018 content of thesulfate produced during the oxidation (Table 9).These results are in agreement with those ob-tained by Santer with whole cells of T. thioparus.They demonstrate conclusively that sulfite isoxidized to sulfate by the APS pathway in ex-tracts as well as in whole cells. Further work isnecessary to define more clearly the pathway(s)and intermediates involved in the oxidation ofsulfide to sulfite.

Figure 5 indicates the most probable inter-mediates in the oxidation of sulfide and otherreduced sulfur compounds to sulfate. A numberof features of this scheme should be emphasized.First, elemental sulfur and polythionates (indi-cated as tetrathionate) are not indicated as beingintermediates in this oxidation. Secondly, thio-sulfate is an intermediate in the oxidation ofsulfide, and the reaction is catalyzed by a sulfideoxidase. Thirdly, in the oxidation of thiosulfateto sulfate, the initial attack on the thiosulfate isthe reductive cleavage of the thiosulfate to sulfideand sulfite. Lastly, sulfite can be oxidized tosulfate by a phosphorylative pathway, involving

APS, and by a nonphosphorylative p)athway,involving sulfite oxidase.

E. The Energy Metabolism of the Thiobacilli

The uniqueness of chemoautotrophism as amode of existence has prompted a search for thebiochemical phenomena uniquely characteristicof chemoautotrophic organisms. However, asdata became available concerning the biochemis-try of these organisms, particularly of the thio-bacilli, the similarity of these forms of life to otherforms, rather than their uniqueness, has beenemphasized. Thus, the thiobacilli contain theusual amino acids (26) and phosphorylated inter-mediates (66) found in most tissues, and they fixcarbon dioxide by a reaction sequence apparentlycommon to all autotrophic organisms (65). Theproduction of a high-energy sulfate at the sub-strate level from the oxidation of sulfite is a reac-tion that at present is physiologically unique tothe thiobacilli. Although it is doubtful that thisreaction plays an important role in the patternof chemoautotrophism, as exhibited by the thio-bacilli, its role in the energy economy of theseorganisms should be investigated in detail. Theinhibition of oxidation by carbon monoxide,cyanide, and azide (53), the stimulation of oxida-tion by DNP (131), and the presence of cyto-chromes (126) suggest that these organisms cangenerate biological energy by means of oxidativephosphorylation. On the other hand, the observa-tion that T. thiooxidans does not contain cyto-chromes (122), the failure of Santer to observe aneffect of DNP on the transfer of 018 from Pi tosulfate (103), and the mere presence of this sub-strate sulfurylation suggest that oxidative phos-phorylation may not be as important in theenergy metabolism of these organisms as pre-viously suspected. Considering the apparent com-plexity of the oxidation of sulfide to thiosulfate,it may be possible that the electrons produced

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during this oxidation are at too high a potentialto participate in oxidative phosphorylation. Thisproblem might be approached by studying themolecular growth yields of these organisms whengrown on different substrates and determining ifenough energy could be produced from this sub-strate sulfurylation to account for the observedgrowth of these cells. It would also be of interestto observe the changes in the enzymes concernedwith the metabolism of thiosulfate when hetero-trophically grown cells of T. novellus (102) areadapted to the chemoautotrophic mode of life.

It is possible that the thiobacilli may be foundto have other unique reactions involving sulfur-containing nucleotides. Sulfur-containing nucleo-tides other than APS can be formed during theoxidation of sulfite to sulfate and these nucleo-tides apparently cannot be phosphorylated byADP-sulfurylase. It is tempting to speculatethat such sulfur-containing nucleotides may bedirectly utilized by these cells in reactions involv-ing nucleotide activation (e.g., amino acid activa-tion). In this respect it is interesting that high-energy sulfate has about twice the energy ofhigh-energy phosphate, and utilization of a high-energy sulfate rather than PP1 for biosyntheticreactions could result in the conservation of mostof the energy in the sulfate bond.The postulations of Umbreit (128) concerning

the separation of the generation of biologicalenergy in the form of high-energy phosphate andits utilization for the fixation of carbon dioxideseem to be essentially correct, although perhapsnot warranted by the data existing at the time(64). The stimulation of carbon dioxide fixationby the addition of ATP to cell-free extracts andthe independent production of high-energy phos-phate during the oxidation of thiosulfate has beendemonstrated (65). Thus, it should now be possi-ble to reproduce these experiments in a cell-freesystem. Reducing power, if needed, can probablybe obtained from the oxidation of reduced sulfurcompounds.

Although the thiobacilli may be considered asunique with regard to the specific mechanisminvolved in the production of biological energy,and possibly in other respects, they conform tothe general pattern of autotrophic metabolism inthat they have independent reaction sequences forproducing high-energy phosphate and reducing

power, and for utilizing these products for thefixation of carbon dioxide.

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