Abstract Sulfite-oxidizing enzyme activities were ana-lyzed in cell-free extracts of aerobically grown cells ofAcidianus ambivalens, an extremely thermophilic andchemolithoautotrophic archaeon. In the membrane andcytoplasmic fractions, two distinct enzyme activities werefound. In the membrane fraction, a sulfite:acceptor oxi-doreductase activity was found [530 mU (mg protein)–1;apparent Km for sulfite, 3.6 mM]. In the cytoplasmic frac-tion the following enzyme activities were found and areindicative of an oxidative adenylylsulfate pathway: adeny-lylsulfate reductase [138 mU (mg protein)–1], adenylylsul-fate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)–1], adenylate kinase [650 mU (mgprotein)–1], and rhodanese [thiosulfate sulfur transferase,9.2 mU (mg protein)–1]. In addition, 5′,5′′′ -P1,P4-di(aden-osine-5′) tetraphosphate (Ap4A) synthase and Ap4A py-rophosphohydrolase activities were detected.

Key words Archaea · Sulfite: acceptor oxidoreductase · Adenylylsulfate: phosphate adenyltransferase · ADP sulfurylase · Adenylylsulfate reductase · 5′,51,P4-di(adenosine-5′) tetraphosphate synthase · 5′,5-P1,P4-di(adenosine-5′) tetraphosphate pyrophosphohydrolase · Adenylate kinase

Abbreviations APS Adenylylsulfate (= adenosine-5′-phosphosulfate) · APAT Adenylylsulfate:phosphate adenyltransferase ·Ap4A 5′,5′′′ -P1,P4-di(adenosine-5′) tetraphosphate · SAOR Sulfite:acceptor oxidoreductase · PAPS 3′-Phosphoadenosine-5′-phosphosulfate

Introduction

The extremely thermophilic and acidophilic archaeonAcidianus ambivalens grows optimally at 80°C and pH2.5 in a mineral medium supplemented with elementalsulfur under a gas phase of either CO2-enriched air orCO2/H2 (Zillig et al. 1985, 1986). The organism is obligatelychemolithoautotrophic; the only energy source for growthand CO2 fixation is the oxidation or reduction of sulfur. Alarge amount of sulfuric acid is produced during aerobicgrowth, causing the pH of the medium to drop below pH1 during the stationary phase. A. ambivalens is phyloge-netically related to other extremely thermophilic aci-dophiles such as Sulfolobus, Stygiolobus, and Metal-losphaera (Fuchs et al. 1996).

The oxidation of elemental sulfur in A. ambivalensproceeds in (at least) two steps: sulfur is first oxidized tosulfite and then to sulfate [for reviews on sulfur oxidationsee Takakuwa (1992), Kelly and Wood (1994), Friedrich(1998), and Kelly (1999)]. The initial S0 oxidation is me-diated by a soluble, cytoplasmic sulfur oxygenase reduc-tase that catalyzes an oxygen-dependent disproportiona-tion reaction yielding sulfite and hydrogen sulfide in a 1:1stoichiometry (Kletzin 1989, 1994). Neither substrate-level phosphorylation nor the generation of an electro-chemical gradient is supported by this reaction. Conse-quently, energy conservation is restricted to the oxidationof sulfite and hydrogen sulfide.

In various thiobacilli and other bacteria, sulfite oxida-tion is catalyzed by a molybdenum-containing membrane-bound or a periplasmic sulfite: cytochrome c oxidoreduc-tase [summarized by Takakuwa (1992), Kelly and Wood(1994), Friedrich (1998), and Kelly (1999)]. In mammals,another type of sulfite oxidase is used for sulfite detoxifi-cation (Kisker et al. 1997). Alternatively, many thiobacilliand phototrophic sulfur bacteria can oxidize sulfite via anoxidative adenylylsulfate (= adenosine-5′-phosphosulfate;APS) pathway in which the substrate is oxidized by anAPS reductase with AMP and an electron acceptor. Theproduct APS is used for substrate-level phosphorylation

Peter Zimmermann · Simone Laska · Arnulf Kletzin

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Arch Microbiol (1999) 172 :76–82 © Springer-Verlag 1999

Received: 17 August 1998 / Accepted: 29 April 1999

ORIGINAL PAPER

P. Zimmermann · S. Laska · A. Kletzin (Y)Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstraße 10, D-64287 Darmstadt, Germanye-mail: kletzin@bio.tu-darmstadt.deTel.: +49-6151-16-3555, Fax: +49-6151-16-2956

by an ATP sulfurylase or by an adenylylsulfate:phosphateadenyltransferase (APAT). APAT has been termed “ADPsulfurylase” in the literature; however, it has been recentlyrenamed to acknowledge the fact that the reaction be-tween APS and phosphate to ADP and sulfate is irre-versible [for equations, see Table 1; C. Dahl and T. Brüser,Institute of Microbiology, University of Bonn, Bonn, Ger-many, personal communication; Brüser et al. (1999)].

Sulfite oxidation or reduction has not yet been studiedin Acidianus or Sulfolobus. Since no c-type cytochromeshave been detected in any of the Sulfolobales, a putativesulfite oxidase must use electron acceptors other than cy-tochrome c. For this study, we analyzed various enzy-matic activities that could be involved in sulfite oxidationin aerobically grown cells of A. ambivalens. We presentevidence for the existence of at least two different sulfiteoxidation pathways.

Materials and methods

Organism, growth conditions, and preparation of cell-free extracts

A. ambivalens (DSM 3772) was grown aerobically at 80°C as de-scribed (Zillig et al. 1986). For lysis, the cell pellets were sus-pended in double-distilled water (5 ml per gram of cells, wet mass)and the pH was adjusted to 7.5 with 1 M potassium phosphatebuffer (Kpi buffer; pH 8.0). The cells were disrupted by sonicationfor 20 min on ice with a sonifier equipped with a microtip (BransonUltrasonic, Danbury, Conn., USA). Sulfur particles and intact cellswere pelleted by low-speed centrifugation (20 min at 2,000 × g).Membrane and cytoplasmic fractions were separated from the re-sulting supernatant by centrifugation for 60 min at 120,000 × g(SW41 rotor; Beckmann, Palo Alto, Calif., USA). The supernatant(cytoplasmic fraction) was dialyzed against 0.1 M Kpi buffer. Thesediment containing the membrane fraction was resuspended afterone washing step in 1% (w/v) sodium deoxycholate dissolved inKpi buffer. The membrane proteins were solubilized by sonication(20 min) and clarified by centrifugation for 60 min at 120,000 × g.The pellet was discarded. The supernatant was dialyzed against de-oxycholate-containing Kpi buffer.

Enzyme assays

The sulfite:acceptor oxidoreductase activity (SAOR) was mea-sured by following the sulfite-dependent reduction of ferricyanidephotometrically at 420 nm and 80°C in a temperature-controlledcuvette holder. The reaction mixture (500 µl) contained 50 mM Bis-Tris [Bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane;Roth, Karlsruhe, Germany], 2.5 mM K3Fe(CN)6, and 2.5 mM cit-ric acid. The pH was adjusted to pH 6 with HCl. Various amountsof cell-free extracts were added, and the absorption was followedfor 2 min. Then the reaction was started by adding 2.5 mMNa2SO3. The changes in absorbance were corrected for the non-en-zymatic background reaction. One unit of activity was defined asthe oxidation of 1 µmol of sulfite per minute. Similarly, the APSreductase activity was followed as the sulfite-dependent ferri-cyanide reduction in the presence of 1 mM AMP [modified in ac-cordance with Dahl and Trüper (1994)] in the same buffer. Thechanges in absorbance were corrected for the non-enzymatic back-ground reaction and for SAOR activity. The adenylate kinase ac-tivity was determined in a coupled enzymatic assay as describedwith minor modifications (Lacher and Schäfer 1993). The reactionmixture contained 50 mM triethanolamine/HCl (pH 6), 2.5 mMMgCl2, 75 mM KCl, 0.15 mM phosphoenolpyruvate, 0.5 mMNADH, 1 mM AMP, 0.8 mM ATP, and 5 U of pyruvate kinaseand lactate dehydrogenase (Roche Diagnostics, Mannheim, Ger-

many). The reaction mixture was equilibrated in a temperature-controlled cuvette holder at 44°C. The reaction was started by theaddition of cell-free extract from A. ambivalens, dialyzed against100 mM Bis-Tris-HCl buffer (pH 6). The changes in absorbancewere followed at 340 nm.

For APAT, an enzyme assay based on the APS-dependentphosphate consumption was developed. The mixtures (1 ml) con-tained 0.1 M Tris-HCl (pH 8.0), 5 mM EDTA, and cell-free extract(50 µl) dialyzed against 100 mM Tris/HCl buffer (pH 8), 50 µMNa2HPO4, and 0.1 mM APS or no APS. After incubation in a cu-vette at 80°C for 5 min, the phosphate was complexed with 200 µlammonium molybdate solution (25 mg/ml) in 15% H2SO4 (byvol.). The resulting complex was subsequently reduced with 100 µlof an SnCl2 solution in 0.1 M HCl (2.5 mg/ml), and the absorbancewas read immediately at 700 nm [modified in accordance withDane and Wille (1985)]. The absorption coefficient ε determinedwith various phosphate concentrations under these conditions was1.01 · 104 M–1 cm–1. One unit of activity was defined as the con-sumption of 1 µmol phosphate per minute. In addition, the APATactivity was measured in an assay similar to the one describedabove for adenylate kinase except that 1 mM APS and 1 mMK2HPO4 were added instead of AMP and ATP. Rhodanese activitywas measured either by the thiosulfate- and cyanide-dependent re-duction of dichloroindophenol at 75°C (Kelly and Wood 1994) orby colorimetric determination of the product SCN– (Wood andKelly 1981).

APS reductase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS)reductase, 5′,5′′′ -P1,P4-di(adenosine-5′1) tetraphosphate (Ap4A) syn-thase, Ap4A pyrophosphohydrolase, APS phosphokinase, APAT,ADP sulfurylase, ATP sulfurylase, and adenylate kinase activitieswere assayed by incubation of the reaction mixture for 10–15 minat 75–80°C and subsequent analysis of the products at varioustime points by high-performance thin-layer chromatography(HPTLC). The reaction mixtures (100 µl) contained 50 mM Tris-HCl (pH 8.0; 100 mM for the APAT, ADP sulfurylase, ATP sul-furylase, and adenylate kinase assays), 30 µl cell-free extract dia-lyzed against 20 mM Tris-HCl buffer (pH 8), 1 mM of the appro-priate nucleotide substrate, and 10 mM of the inorganic cosub-strates (sulfite, thiosulfate, or sulfate; see Table 1). For the Ap4Apyrophosphohydrolase assay, 1 mM CoCl2 or MgCl2 was added.Aliquots of 5 × 1 µl of each reaction mixture were pipetted suc-cessively onto an HPTLC plate containing a fluorescence marker(Silica gel 60 F254; Merck, Darmstadt, Germany) and were imme-diately dried with a fan. The plates were developed with iso-propanol, ammonia, and water (6:3:1 by vol.; Kelly and Wood1994) and photographed under UV illumination. The productswere identified by comparison of their Rf values with those of thepure substances incubated without cell extracts under otherwiseidentical conditions. For control of non-enzymatic background re-actions, assays were also performed with proteinase-K-digestedcell extracts. All nucleotides were obtained from Sigma (Munich,Germany). All chemicals were from Merck (Darmstadt, Germany)unless stated otherwise.

Results

Sulfite:acceptor oxidoreductase

Cytoplasmic and membrane fractions of aerobicallygrown A. ambivalens cells were assayed for the presenceof sulfite-oxidizing enzyme activities. In the membranefraction, an SAOR activity was found in an assay thatcouples the oxidation of sulfite to the reduction of ferri-cyanide, which can be followed photometrically at 420nm. The specific activity of this SAOR determined at pH6 and 80°C was 530 mU (mg protein)–1 in the solubilizedmembrane fraction and 62 mU (mg protein)–1 in the cyto-plasmic fraction (Table 1). The activity increased with

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temperature. The optimum temperature was above 90°Cand no activity was observed below 55°C. Both the en-zyme activity and the non-enzymatic sulfite oxidationwere pH- and temperature-dependent (Fig. 1). At 80°Cand pH 6, the sulfite oxidation rate was 220 nmol ml–1

min–1 with 400 µg of membrane solubilisate per milliliter.The background reaction rate was 50 nmol ml–1 min–1,

which equals 22% of the total sulfite oxidation rate. At pH7, the background rate was 39% of the total sulfite oxida-tion rate. At pH 8.3, the enzymatic and nonenzymaticrates were indistinguishable.

No activity was observed when the solubilized mem-brane fraction was digested with proteinase K prior to theassay. This result confirmed that the observed activity wasprotein-dependent. The optimal sulfite concentration was5 mM. The apparent Km value for sulfite, determined at85°C, was 3.6 mM. The calculated theoretical maximumvelocity of sulfite oxidation, Vmax, was 0.89 µmol min–1

(mg protein)–1. No enzyme activity was found in cyto-plasmic and in membrane fractions with thiosulfate assubstrate or with methylene blue, decyl ubiquinone, orcaldariella quinone as electron acceptors instead of ferri-cyanide (Cohen and Fridovich 1971).

Adenylylsulfate reductase

An APS reductase activity was found in cytoplasmic frac-tion, but not in the membrane fraction. In sulfate-reducingbacteria and archaea, the enzyme catalyzes the reductivecleavage of APS to AMP and sulfite (Lampreia et al.1994; Speich et al. 1994). In thiobacilli and sulfur-oxizid-ing phototrophic bacteria, the reverse reaction (the forma-tion of APS from AMP and sulfite) is catalyzed duringsulfite oxidation (Dahl and Trüper 1989, 1994; Taylor1994). APS reductase activity was measured photometri-cally as a sulfite- and AMP-dependent ferricyanide reduc-tion in a way similar to that of measurements of AMP-in-

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Table 1 Results of the measurement of the enzyme activities ofcell-free extracts of Acidianus ambivalens. + Indicates that the cor-responding activity was found in the HPTLC analysis; – indicatesthat it was not found. Specific activities in the extracts are given

when known [mU (mg protein)–1] [nd not determined, APS adeny-lylsulfate, PAPS 3-phosphoadenosine-5-phosphosulfate, and Ap4A5′,5′′′ P1,P4-di(adenosine-5′) tetraphosphate]

Enzyme activity Reaction catalyzed Activity in

cyto- mem-plasmic branefraction fraction

Sulfite:acceptor oxidoreductasea SO3– + 2 Fe(CN)6

3– → SO42– + 2 Fe(CN) 6

4– 62 530Thiosulfate:acceptor SSO3

2– + 2 Fe(CN)63– → O3S–S–S–SO3

4– + 2 Fe(CN) 64– < 1 < 1

oxidoreductasea

Rhodanese SSO32– + CN– + H+ → SCN– + HSO3

– 9.2 < 1APS reductasea AMP + HSO3

– + 2 Fe(CN)63– APS + 2 Fe(CN)6

4–/a + H+ 138 < 1Reverse reaction APS + 2 Fe(CN)6

4–/a + H+ AMP + HSO32– + 2 Fe(CN)6

3– + –APAT APS + HPO4

2– → ADP + SO42– + H+ 43/86b < 1

Reverse reaction ADP + SO42– + H+ → APS + HPO4

2– –c ndATP sulfurylase APS + HP2O7

3– → ATP + SO42– + H+ –c/d nd

Reverse reactione ATP + SO42– + H+ → APS + HP2O7

3– –c ndAdenylate kinase AMP + ATP 2 ADP 650 < 1APS phosphokinase APS + ATP → PAPS + ADP – ndPAPS reductasea PAPS + 2 Fe(CN)6

4-/a + H+ HSO3– + 3′-Phospho–AMP + 2 Fe(CN)6

3– – ndAp4A synthase APS + ATP → Ap4A + SO4

2– + ndAp4A pyrophosphohydrolase Ap4A + H2O → 2 ADP + nd

a The natural electron carrier is not knownb Results of the measurement based on phosphate consumption andon the coupled enzymatic assayc With and without Mg2+

d Same result as with APS and inorganic phosphatee The reaction is driven by pyrophosphatase-catalyzed pyrophos-phate hydrolysis

Fig. 1 pH-Dependence of the sulfite:acceptor oxidoreductase(SAOR) activity. All reactions were done at 80°C in a volume of500 µl. P Specific SAOR activity (left axis); p sulfite oxidationrate with enzyme added (right axis); m non-enzymatic sulfite oxi-dation rate in the reaction mixture (right axis). The specific activi-ties were calculated from the difference of the two other curves.Buffers used: pH 3–6, 50 mM Bis-Tris/citrate adjusted with HCl;pH 7 and 8.3, 50 mM Tris/citrate adjusted with HCl; pH 6.5, 25mM Bis-Tris (pH 6) in a 1:1 mixture with 25 mM Tris-HCl (pH 7)

←→←→

←→

←→

dependent SAOR activity. The enzymatic oxidation rateof sulfite was corrected against the nonenzymatic reactionrate and the residual SAOR activity present in the cyto-plasm. The specific activity in the cytoplasmic fraction at80°C was 138 mU (mg protein)–1 (Table 1).

Adenylylsulfate:phosphate adenyltransferase

Sulfate is released from APS in a nucleophilic substitutionreaction either with phosphate catalyzed by APAT or withpyrophosphate catalyzed by a reverse ATP sulfurylase(Dahl and Trüper 1989, 1994). The production of AMPand ADP was observed after the incubation of the cyto-plasmic fraction with APS and phosphate at 75°C andsubsequent analysis of the products with HPTLC. The re-sults indicated the presence of an APAT (Table 1; Fig. 2).No activity was found in the membrane fraction. The re-sults were independent of the addition of Mg2+ ions (2.5mM) to the reaction mixture. No APS production wasseen in the HPTLC analysis after incubating ADP or ATPwith sulfate and cell-free extracts, indicating that the re-verse reactions were not catalyzed (ADP or ATP sulfury-lase reaction; not shown). In addition to ADP, ATP andAMP were also present in significant amouts on theHPTLC plates as secondary products due to an adenylate

kinase activity in the cell-free extract (see below). Whenthe cytoplasmic fraction was incubated with APS and py-rophosphate, the product patterns on the HPTLC plateswere identical to those after incubation with APS andphosphate (not shown).

Two different photometric assays were used to quan-tify the APAT activity. A novel method based on the APS-dependent phosphate consumption was developed. Thespecific APAT activity in the cytoplasmic extract was cal-culated to be 43 mU (mg protein)–1. The APAT activitywas independent of divalent cations; it was neither en-hanced by the addition of 1 mM MgCl2 to the enzyme as-say nor inhibited by the addition of 5 mM EDTA. How-ever, the activities varied up to 90% with each determina-tion. This was probably due to fact that the assay wasbased on an end-point measurement of one of the reactionpartners, which led to considerable inaccuracy.

In a different approach, the adenylate kinase assay (seeMaterials and methods and below) was modified to followAPAT activity. The ADP formation by APAT from APSand phosphate was coupled to the oxidation of NADH byadding phosphoenolpyruvate, pyruvate kinase, and lactatedehydrogenase to the reaction mixture. Any ADP forma-tion led to a release of pyruvate, which was then reducedwith NADH to give lactate (Lacher and Schäfer 1993).When using APS and phosphate as the substrates for theassay, the specific activity of the APAT was 86 mU (mg pro-tein)–1 at 44°C. When using APS and pyrophosphate, thespecific activity was 90 mU (mg protein)–1. In the absenceof magnesium, the specific activities were 80 and 85 mU(mg protein)–1, respectively.

Adenylate kinase

When performing the HPTLC analysis for APAT, addi-tional bands corresponding to ATP and AMP were ob-served (Fig. 2). The occurrence of AMP and ATP can beexplained by a weak nonenzymatic hydrolysis of ADP toAMP and by the action of an adenylate kinase, respec-tively. Adenylate kinase is a ubiquitous enzyme that con-verts 2 mol of ADP into 1 mol each of AMP and ATP thatalso performs the reverse reaction. An increase of the in-

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Fig.2 Separation of nucleotides, derivatives, and enzymatic reac-tion mixtures by HPTLC. All reactions were incubated at 75°C. A Adenylylsulfate:phosphate adenyltransferase assay. Cytoplas-mic extract of Acidianus ambivalens was incubated with adenylyl-sulfate (APS) and phosphate; samples were removed from the re-action mixture at various time points and separated. The decreasein the relative intensity of the APS band and the increase of ADPand AMP bands was followed. In the proteinase-K-digested con-trol, the intensity of the APS band was not different from that ofthe APS reference. Left lanes reference substances, E extract with-out APS, E* cytoplasmic extract incubated with AMP and phos-phate for 10 min, and PK proteinase-K-digested cytoplasmic ex-tract incubated for 10 min with APS and phosphate. B Adenylatekinase assay. Cytoplasmic extract was incubated with ADP andanalyzed as described above; the decrease in the relative intensityof the ADP band and the increase in the intensity of the AMP andATP bands was followed. E Cytoplasmic extract incubated for 10min without ADP and PK, proteinase-K-digested cytoplasmic ex-tract incubated for 10 min with ADP

tensity of the AMP and ATP spots was observed withtime, when cytoplasmic A. ambivalens extracts were incu-bated with ADP and the products were subsequently sep-arated by HPTLC (Fig. 2).

The activity of the reverse reaction of the adenylate ki-nase was followed in a coupled enzymatic assay at 44°Cusing phosphoenolpyruvate, pyruvate kinase, and lactatedehydrogenase as described above for APAT. The specificactivity was 650 mU (mg protein)–1 in the cytoplasmicfraction. No activity was found in the membrane fraction.The activity was strictly magnesium-dependent; no activ-ity was observed without the addition of Mg2+ ions. Theoxidation rate of NADH under the assay conditions withcytoplasmic extract of A. ambivalens (300 µg protein perml) was 218 nmol ml–1 min–1. The background oxidationrate of NADH under the assay conditions without A. am-bivalens extract was 38 nmol ml–1 min–1.

Other enzyme activities

The rhodanese activity was assayed either by the thiosul-fate- and cyanide-dependent reduction of the artificial dye2,6-dichloroindophenol, or by incubation of extract withthiosulfate and subsequent colorimetric determination ofthe product thiocyanate. It was 0.8 mU (mg protein)–1 incytoplasmic extracts when assayed with 2,6-dichloroin-dophenol, and 9.2 mU (mg protein)–1 when thiocyanateproduction was followed colorimetrically. However, the2,6-dichloroindophenol-based assay had a high nonenzy-matic background reaction. The oxidation rate of sulfiteunder the assay conditions was 6.8 nmol ml–1 min–1 withcell extracts and 5.8 nmol ml–1 min–1 without. Therefore,the determination of the reaction rate was probably not veryaccurate. No activity was found in the membrane fraction.

From assimilatory sulfate reduction via the PAPS path-way, two other enzymes (APS phosphokinase and PAPSreductase) are known. APS phosphokinase phosphoryl-izes APS at the 3′-position with ATP to PAPS. After theincubation of APS and ATP with cytoplasmic extracts, anadditional band that did not migrate as far as the PAPSstandard (Fig. 3) appeared on HPTLC plates. The sub-stance had an Rf value identical to that of Ap4A, pointingto an Ap4A synthase activity. Since the band was oftenweak, we screened for the presence of an Ap4A-degradingenzyme activity. When Ap4A was incubated with cyto-plasmic cell extracts, a Co2+-dependent production ofAMP, ADP, and ATP by Ap4A pyrophosphohydrolase wasobserved after HPTLC separation of the products (Fig. 3).

Discussion

In our studies with cell-free extracts of A. ambivalens wefound enzyme activities from two independent pathwaysof sulfite oxidation, an SAOR activity in the membranefraction and an APS reductase and APAT activity in thecytoplasmic fraction (Fig. 4). The ferricyanide-basedSAOR and APS reductase assays had a high nonenzy-

matic background reaction rate depending on the pH andthe buffer used (Fig. 1). The effect was enhanced whenphosphate buffers were used (data not shown). The strongreducing potential of sulfite is generally known in thechemical literature (SO3

2–/SO42–: E′0 = –550 mV; Taka-

kuwa 1992; Wiberg 1995). The nonenzymatic sulfite oxi-dation rate decreased with the pH; it was neglible at pH 4and below. The natural electron acceptor of the SAOR isnot known. From the location of the enzyme in the mem-brane, we would assume that it reduces caldariellaquinone. To date we have been unable to demonstrate adirect reduction of quinone compounds with sulfite. Atpresent, we do not know whether this is due to the exper-imental conditions, e.g., the lack of an additional electrontransfer factor in the assay. Sulfite is produced in the cyto-plasm by a soluble sulfur oxygenase/reductase, whereas theend product of the sulfur oxidation, sulfate, is accumulated

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Fig.3 A Synthesis of 5′,5′′′ P1,P4-di(adenosine-5′) tetraphosphate(Ap4A) from ATP and adenylylsulfate (APS) by cytoplasmic ex-tract of Acidianus ambivalens. Left lanes Separation of the refer-ence substances by HPTLC as in Fig. 2, E separation of productsafter incubation of cytoplasmic extract with APS and ATP for 10 min, and M the same with the membrane fraction. After incu-bation, no 3′-phosphoadenosine-5′-phosphosulfate (PAPS) wasformed, as expected, but an additional band appeared with an Rfvalue identical to that of Ap4A (arrow). B Hydrolysis of Ap4A byAp4A pyrophosphohydrolase. Left lanes Reference substances;lanes 1 Ap4A incubated with buffer and 1 mM Co2+, 2 proteinase-K-digested cytoplasmic extract incubated with Ap4A and Co2+, 3cytoplasmic extract incubated with Ap4A and Co2+, 4 proteinase-K-digested cytoplasmic extract incubated with Ap4A and 1 mMMg2+, and 5 cytoplasmic extract incubated with Ap4A and Mg2+.The Ap4A pyrophosphohydrolase formed AMP, ADP, and ATPfrom Ap4A in the presence of Co2+ (lane 3) and to a lesser degreewith Mg2+

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outside of the cell (Fig. 4, Kletzin 1989, 1992, 1994). Wedo not know yet whether the SAOR also transports sulfateacross the membrane during the oxidation or whether anadditional sulfate-transporting protein is present.

In the cytoplasm, we found an APS reductase and anAPAT activity that would allow the sulfite-dependentADP formation in an oxidative APS pathway. The substi-tution of sulfate by phosphate from APS by the enzymaticactivity of the APAT is an exergonic process [∆G°′: –55.9kJ mol–1, calculated in accordance with values given byThauer et al. (1977)]. The reverse reaction, the activationof sulfate with ADP to APS and phosphate, is thereforethermodynamically unfavorable (“ADP sulfurylase”). Theexistence of an APAT or an “ADP sulfurylase” has beenquestioned for a long time (Renosto et al. 1991) and it hasbeen purified to homogeneity only recently (Schwarz etal. 1998; Brüser et al. 1999).

With a combination of different methods we couldshow that there is no ATP sulfurylase but an APAT activ-ity present in A. ambivalens extracts. The addition ofphosphate and APS to the assay mixture was always suf-ficient for APAT activity. We found no change in the ac-tivity with or without addition of Mg2+. Thus, it was ex-cluded that a reverse ATP sulfurylase was responsible forthe observed activity since these enzymes are strictlymagnesium-dependent (Dahl and Trüper 1994). The ADPproduction with pyrophosphate can be explained by thepresence of minor amounts of phosphate in the commer-cially available pyrophosphate (0.1%; value given by themanufacturer; Merck, Darmstadt, Germany) and/or by py-rophosphate conversion to phosphate by a pyrophos-phatase activity. A pyrophosphatase has been purifiedfrom the phylogenetically related and extremely ther-mophilic archaeon Sulfolobus acidocaldarius (Meyer etal. 1995; Schäfer et al. 1996). An alternative route forADP formation from APS and Pi via the combined action

of a reverse pyrophosphatase, an ATP sulfurylase, and aadenylate kinase has been suggested by Renosto et al.(1991). However, from the absence of an ATP sulfurylaseactivity and the thermodynamical consideration men-tioned above, this route can be excluded. The use of anAPAT instead of reverse ATP sulfurylase has the advan-tage that it is independent of the energy-rich pyrophos-phate molecule. This is the first time that APAT activityhas been found in an archaeon.

The ADP formed by the APAT is converted to ATP andAMP by adenylate kinase (Figs. 2, 4; Kath et al. 1993;Lacher and Schäfer 1993; Bonisch et al. 1996). The cou-pled enzymatic assay used for the measurement of theadenylate kinase and the APAT activities was developedfor the adenylate kinase from S. acidocaldarius (Kath etal. 1993; Lacher and Schäfer 1993; Bonisch et al. 1996;Schäfer et al. 1996). The upper temperature limit of theassays (44°C) was defined by the thermal stability of theauxiliary enzymes from mesophilic organisms. However,this temperature was approximately 36 °C below the op-timal growth temperature of A. ambivalens. Under theseconditions, the APAT activity was low but still measur-able. In contrast, the adenylate kinase activity was muchhigher [the method has been discussed in greater detail byLacher and Schäfer (1993)].

When assaying for additional enzymes that metabolizesulfated nucleotides, an Ap4A synthase and an Ap4A py-rophosphohydrolase activity but no PAPS metabolizingenzyme activities were found. Ap4A is formed from APSand ATP. It cannot be decided at present whether theAp4A pyrophosphohydrolase cleaves the substrate Ap4Asymmetrically into two molecules of ADP, or asymmetri-cally into AMP and ATP (Fig. 3). The enzyme assaysbased on the incubation of extracts with their substratesand subsequent analysis of the mixture by HPTLC areend-point measurements, which do not provide accuratekinetic data. We could show that the enzymes exist, buttheir physiological role in A. ambivalens cells is unre-solved. They do not play a role in the sulfur metabolism[for reviews, see Rauhut et al. (1985 and Plateau andBlanquet (1994)]. Significant levels of Ap4A and Ap4Asynthase activities had been found in virtually all bacteriainvestigated to date and in the archaeon Methanosarcinabarkeri. Only in one study has an archaeon (Pyrodictiumoccultum) been investigated for an Ap4A hydrolase (Gu-ranowski et al. 1983). As with the Ap4A hydrolase fromA. ambivalens, the enzyme from P. occultum is stronglyactivated by Co2+ and to a lesser degree by Mg2+.

A redundancy of sulfite oxidation pathways inolvingboth an SAOR and an APS reductase has been reportedpreviously for thiobacilli and for some phototrophic sulfurbacteria (Brune 1989; Dahl and Trüper 1989; Takakuwa1992; Taylor 1994). With A. ambivalens cells, weachieved results that point to a similar situation: one sul-fite oxidation pathway via APS would allow substrate-level phosphorylation. In contrast, the SAOR found in themembrane fraction would allow an electron transportfrom sulfite to cytochrome aa3 provided that sulfite oxi-dation can be coupled directly or indirectly to the reduc-

Fig.4 Tentative scheme of sulfur oxidation pathways in Acidi-anus ambivalens as suggested by the enzyme activities found. Theinitial sulfur oxidation is mediated by a soluble sulfur oxygenasereductase (Kletzin 1989, 1992, 1994). Sulfite can be oxidized viathe membrane-bound sulfite:acceptor oxidoreductase (SAOR) andvia the adenylylsulfate (APS) pathway. It is not known whether theSAOR can reduce caldariella quinone (CQ) directly or whether ad-ditional redox factors are necessary. It is also not known whethersulfate is transported across the membrane by the SAOR orwhether an additional sulfate-transporting protein is present

tion of caldariella quinone, the reduced form of which isthe natural electron donor of the terminal oxidase(Anemüller et al. 1994).

Acknowledgements We wish to thank F. Pfeifer for her generos-ity and encouragement, and also J. Gmeiner for help with the bu-reaucracy. This work was supported by grant no. Kl-885/3 fromthe Deutsche Forschungsgemeinschaft to A. Kletzin.

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