Vol. 172, No. 11JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6435 64410021-9193/90/116435-07$02.00/0Copyright X 1990, American Society for Microbiology

Purification and Characterization of the Reduced-Nicotinamide-Dependent 2,2'-Dithiodiethanesulfonate Reductase from

Methanobacterium thermoautotrophicum AHSCOTT G. SMITH AND PIERRE E. ROUVIEREt*

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 23 April 1990/Accepted 3 September 1990

A novel reduced nicotinamide-dependent disulfide reductase, the 2,2'-dithiodiethanesulfonate [(S-CoM)2]reductase (CoMDSR) of Methanobacterium thermoautotrophwcum was purified 405-fold to electrophoretichomogeneity. Both NADPH and NADH functioned as electron donors, although rates with NADPH were threetimes higher. Reduced factor F420, the deazaflavin electron carrier characteristic of methanogenic bacteria, wasnot a substrate for the enzyme. The enzyme was most active with (S-CoM)2 but could also reduce L-cystine at23% the (S-CoM)2 rate. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that theenzyme was monomeric with an Mr of about 64,000; spectral analysis showed that it was a flavoprotein withan estimated composition of one molecule of flavin per polypeptide. Maximal activity occurred at 64°C, and thepH optimum was 8.5. The apparent Km for both NADPH and (S-CoM)2 was 80 R,M. The enzyme wascompletely inactivated by oxygen in crude cell extracts but was oxygen stable in the homogeneous state. The lowactivity of the CoMDSR in cell extracts as well as its relatively low rate of reducing CoM-S-S-HTP (theheterodisulfide of the two thiol cofactors involved in the last step of methanogenesis) make it unlikely that itplays a role in the methylreductase system. It may be involved in the redox balance of the cell, such as theNADPH-dependent bis-,y-glutamylcystine reductase with which it shows physical similarity in anotherarchaebacterium, Halobacterium halobium (A. R. Sundquist and R. C. Fahey, J. Bacteriol. 170:3459-3467,1988). The CoMDSR might also be involved in regenerating the coenzyme M trapped as its homodisulfide, anonutilizable form of the cofactor.

Coenzyme M (2-mercaptoethanesulfonate, HS-CoM) is acofactor common to all methanogenic bacteria and carries aC1 unit as a methyl group [2-(methylthio)ethanesulfonate]prior to the formation of methane (19). When originallyisolated aerobically from cell extracts, HS-CoM was foundin the oxidized disulfide form, 2,2'-dithiodiethanesulfonate[(S-CoM)2], which was not the active form of the coenzyme(22). To form 2-(methylthio)ethanesulfonate, two proteincomponents were required for transfer of a methyl groupfrom methyl-cobalamin to (S-CoM)2 when NADPH wasused as the electron source (23). One component was shownto be a methyltransferase per se. NADPH and the secondcomponent (an acidic protein) could be replaced by sodiumborohydride, which reduced (S-CoM)2 to two HS-CoM. Thisevidence suggested that the second component was anNADPH-dependent (S-CoM)2 reductase (CoMDSR) andthat the transmethylation depended on the reduction of(S-CoM)2 to HS-CoM. This NADPH-dependent (S-CoM)2-reducing activity was first investigated in Methanospirillumhungatei but was not purified to homogeneity (J. G. Ferry,Ph.D. thesis, University of Illinois, Urbana, 1974).

In Methanosarcina barkeri, partially purified fractionscontaining hydrogenase and ferredoxin activities wereshown to reduce (S-CoM)2 in the presence of hydroxyco-balamin. The methanol:5-hydroxybenzimidazolylcobamidemethyltransferase enzyme from Methanosarcina barkeriwas also active in this hydrogenase- and ferredoxin-depen-dent reduction of (S-CoM)2. This reducing system func-

* Corresponding author.t Present address: Laboratoire de Biochimie Microbienne, Centre

D'Etudes Nucleaires-85X, 38041 Grenoble Cedex, France.

tioned chemically, however, and was not specific for (S-CoM)2 (24).The discovery of 7-mercaptoheptanoylthreonine phos-

phate (HS-HTP) (17), a second key sulfhydryl coenzyme inmethanogenic bacteria, has increased our interest in the rolethese sulfhydryl compounds play in anaerobic metabolism.Recently it was shown that the actual product of the methylreductase reaction is the heterodisulfide of HS-CoM andHS-HTP (6M-S-S-HTP) (2, 9). Earlier models for the func-tioning of the methyl reductase system had involved theformation of the disulfide of coenzyme M as a product of thedemethylation of 2-(methylthio)ethanesulfonate and sug-gested that CoMDSR could be the physiological disulfidereductase for this system. We report here the purificationand characterization of CoMDSR from Methanobacteriumthermoautotrophicum AH and suggest a role for the enzyme.(A preliminary report of this work has appeared [S. G.

Smith and P. E. Rouviere, Abstr. Annu. Meet. Am. Soc.Microbiol. 1987, 132, p. 177].)

MATERIALS AND METHODSMaterials. Phenyl-Sepharose CL-4B, fast protein liquid

chromatography Mono Q anion-exchange resin, agarose-hexane-NADP (linked through the C8 of the adenine ring viaa six-carbon spacer, AGNADP type 3), and Sephacryl S-200gel filtration resin were purchased from Pharmacia LKBBiotechnology Inc., Piscataway, N.J.; DE52 DEAE-cellu-lose was purchased from Whatman Ltd., Hillsboro, Oreg.;and Coomassie blue G-250 protein assay reagent was pur-chased from Pierce Chemical Co., Rockford, Ill. All otherchemicals were of reagent grade and purchased from SigmaChemical Co., St. Louis, Mo. (N-L-Lactyl-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-

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5-deazariboflavin-5'-phosphate (F420) was purified from cellsofM. thermoautotrophicum AH as previously described (6).

Preparation of (S-CoW2. Two grams of HS-CoM wasdissolved in 100 ml of aqueous 30%o NH40H. Oxygen wasbubbled through this solution at room temperature for 50 h.(S-CoM)2 powder was recovered by flash evaporation. Thi-ols were assayed by the procedure of Ellman (10) andshowed 99.96% conversion of HS-CoM to (S-CoM)2. Thepowder was lyophilized and stored dessicated at roomtemperature.Growth of cells and preparation of celi extracts (CFE).

M. thermoautotrophicum AH (ATCC 29096; DSM 1053) wasgrown in a 200-liter fermentor as previously described (27).Cells were suspended in 20 mM potassium phosphate (pH7.0) that contained 10 mM 2-mercaptoethanol and storedfrozen at -20°C. CFE were prepared by passing a thawedcell slurry through a French pressure cell at 69 MPa. Theextract was then centrifuged anaerobically at 15,000 x g for20 min to remove particulate debris. Cells and CFE werekept under an N2 atmosphere at all times.

Protein determination. Protein concentrations were mea-sured by the method of Bradford (4) with the Pierce ProteinAssay reagent (Pierce Chemical Co.). Bovine serum albuminwas used as a standard. When higher accuracy was requiredto estimate the amount of flavin bound to the enzyme,protein concentrations were also determined by measuringturbidity after trichloroacetic acid precipitation (15) and bythe Lowry method modified by Peterson (18). The proteincontent of a sample was analyzed by each of the threemethods, and the values were averaged. The assays werecalibrated by using the averages of three different standardproteins: bovine serum albumin, egg white lysozyme, andbovine pancreas RNase A. The standard deviation for pro-tein determination between between the three methods was0.03 mg/ml.Enzyme assays. In crude fractions, the CoMDSR activity

was measured spectrophotometrically by monitoring theproduction of HS-CoM with 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB) (10). Each reaction mixture (0.8 ml) containing60 mM Tris buffer (pH 8.5), 3.2 ,Lmol of (S-CoM)2, 1.6 p.molof NADPH, and crude enzyme as desired was placed in a1.5-ml vial; the vial was sealed, and its atmosphere wasexchanged for N2. The reaction was initiated by incubationof the vials at 64°C or by addition of enzyme with a syringeby anaerobic techniques. At intervals, 100-,ul samples werewithdrawn with a syringe and kept in a tube in air on ice priorto assay. Sulfbydryl content was assayed by adding 900 pl of20mM potassium-20 mM phosphate buffer (pH 7.6) contain-ing 2.5 mM EDTA, 1.3 mM'DTNB, and 1 mM NaHCO3 5 to10 min before reading the A412. All assays with CFE wereblanked against a reaction mixture lacking (S-CoM)2 tocorrect for thiols and disulfides contained in the extracts.This background rate of thiol production in crude extractsamounted to 33% of the activity found in assays containing(S-CoM)2, and it disappeared after chromatography onDEAE-cellulose. One unit of activity was defined as 1 ,umolof (S-CoM)2 reduced per min.

In homogeneous enzyme fractions, activity was measuredspectrophotometrically by monitoring the disappearance ofNADPH at 339 nm. These assays were performed by adding0.32 pumol ofNADPH and 4 ,umol of (S-CoM)2 to each oftwoplastic cuvettes (kept anaerobic by storage in an anaerobicchamber) containing 60 mM Tris buffer at pH 8.5 in a totalvolume of 1 ml. Each cuvette was then sealed with a rubberstopper, taken out of the anaerobic chamber, and placed intoa spectrophotometer (Lambda 3B; The Perkin-Elmer Corp.,

Norwalk, Conn.) in which the thermostated cell holder wasmaintained at 60°C (the maximum for the instrument). Thereaction was initiated by injecting enzyme into one cuvetteand an equal volume of anaerobic buffer into the second. Theenzyme sample was injected into the reference positioncuvette, and as the reaction proceeded, a net increase in theA339 was recorded. The assay performed in this mannerallowed for continuous correction for nonenzymatic degra-dation of NADPH. Although homogeneous enzyme frac-tions were air stable, all fractions were obtained and storedanaerobically.Enzyme purification. All steps of enzyme purification were

performed inside an anaerobic chamber at room temperatureas described previously (12). Fractions were assayed forCoMDSR activity by the DTNB assay scaled down to a finalvolume of 0.4 ml.CFE (1.023 g of protein) was loaded onto an 80-ml DE52

DEAE-cellulose column (5 by 4 cm) equilibrated with anaer-obic 20 mM potassium phosphate buffer (pH 7) in 1 mM2-mercaptoethanol (buffer I). Proteins were eluted with a570-ml increasing potassium acetate gradient (0.0 to 0.8 M)which was monitored by conductivity. The first nine frac-tions each contained 40 ml. The last seven contained 30 mleach. Activity was eluted with 0.75 M potassium acetate.Fractions 10 to 15 were combined and concentrated anaer-obically by ultrafiltration on a PM-30 membrane (AmiconDivision, W. R. Grace and Co., Danvers, Mass.) to a 20-mlfinal volume.

Eighty-two milligrams of active pooled protein from theDEAE-cellulose column was loaded onto a 25-ml phenyl-Sepharose CL-4B column (2.5 by 5 cm) equilibrated withanaerobic 1 M potassium acetate in buffer I. Proteins wereeluted with a 200-ml decreasing potassium acetate gradient(1.0 to 0.0 M) in buffer I, and fractions (4 ml each) werecollected. Activity was eluted with 0.15 M potassium ace-tate. Fractions 26 to 39 were combined as the active pool.

This pool from the phenyl-Sepharose column (16.8 mg ofprotein, 56 ml) was mixed batchwise with 10 ml of NADP-C8-agarose resin in 130 ml of buffer I and 0.5 M potassiumacetate. The enzyme was allowed to bind the resin for 2 hwith occasional swirling. The slurry was then poured into a20-ml column and washed with 0.5 M potassium acetate inbuffer I until all unbound proteins were eluted. Boundproteins were eluted with 10 ml of 1 M' potassium acetatefollowed by 20 ml of 2 M potassium acetate, 15 ml of 2 mM(S-CoM)2, and 15 ml of 1 mM NADP, all in buffer I. Thecolumn was washed before and after each elution step with10 ml of buffer I. A total of 100 ml was collected in 25 4-mlfractions. The fraction not retained (190 ml) contained 90%of the protein loaded and was discarded. Fractions 21through 24, corresponding to the NADP eluate, were com-bined as the final active pool. This pool was concentrated,mixed with an equal volume of 40% glycerol, and stored at-20°C to preserve activity.

In some cases, chromatography on a Mono Q anion-exchange column was necessary to achieve homogeneity orto remove NADP from the enzyme pool when the NADPHoxidation assay was used. In a typical case, 50 ,ug from theNADP-agarose active pool was loaded onto a 1-ml Mono Qanion-exchange column. Chromatography was performed byfast protein liquid chromatography (Pharmacia). The columnwas equilibrated with 20% glycerol in buffer I. The enzymewas eluted with a potassium acetate gradient from 0 to 1 Min 20% glycerol and buffer I, and 0.5-ml fractions werecollected. Active fractions were pooled and stored anaero-bically. This step resolved the CoMDSR from protein and

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nonprotein contaminants, leaving it in 20% glycerol andbuffer I, ready for use or storage.

Analytical gel filtration. Homogeneous enzyme (1 ml) froman NADP-agarose pool was diluted to 10% glycerol with 1 mlof 20 mM potassium phosphate buffer (pH 7) (buffer II) andloaded onto a Sephacryl S-200 column (1.4 by 90 cm)equilibrated with anoxic 0.3 M potassium acetate-10% glyc-erol in buffer II. The column was calibrated with bovine livercatalase (Mr, 245,000), yeast alcohol dehydrogenase (Mr,150,000), bovine serum albumin (Mr, 66,000), ovalbumin(Mr, 45,000), bovine pancreas chymotrypsinogen A (Mr,24,000), and horse heart cytochrome c (Mr, 13,500). Bluedextran (Mr, 2,000,000) and flavin adenine dinucleotide(FAD) were used to measure the excluded and includedvolumes, respectively.PAGE. Discontinuous native polyacrylamide gel electro-

phoresis (PAGE) was performed with 5.3% acrylamide at pH6.8 in the stacking gel and 10% acrylamide at pH 8.8 in theresolving gel (16). The gels were run at a constant current of10 mA for stacking of proteins and 20 mA for resolution.Sodium dodecyl sulfate (SDS)-PAGE was performed as

described above with 0.2% SDS in the acrylamide solutionsand buffer system. Molecular weight standards were fruc-tose-6-phosphate kinase (Mr, 84,000), pyruvate kinase (Mr,58,000), fumarase (Mr, 48,500), lactate dehydrogenase (Mr,36,500), and triose phosphate isomerase (Mr, 26,000). Thegels were stained with Coomassie brilliant blue R250.Pharmacia PhastGel medium (gradient, 8 to 25% acryl-

amide) was employed as a second method to determinenative molecular weights. The gels were run at 400 V and 10mA for 40 min. The molecular weight standards used were

bovine liver catalase, yeast alcohol dehydrogenase, bakers'yeast hexokinase (Mr, 102,000), bovine serum albumin, andovalbumin.

Flavin determination. Homogeneous CoMDSR from MonoQ chromatography was concentrated aerobically to 0.36 +

0.03 mg/ml in a Centricon ultrafiltration vessel equipped witha PM-30 membrane. Absorption spectra of this sample(oxidized by aerobic centrifugation) were taken with a

Perkin-Elmer Lambda 3B scanning spectrophotometer. Fla-vin concentration was measured by monitoring the decreasein A450 upon reduction of the enzyme with sodium dithionite.

Preparation of reduced F420. To reduce F420 and avoid thepresence of borohydride or hydrogenase, a crude immobi-lized F420-reducing hydrogenase fraction was prepared. Inan anaerobic chamber, CFE (192 mg) was added to 6 ml ofdecyl-agarose equilibrated in buffer I containing 0.1 Mpotassium acetate. After 1 h, the resin was washed exten-sively with buffer I. The resin was then equilibrated with0.75 M potassium acetate in buffer I (to maintain the boundhydrogenase in a high-salt environment, in which it is morestable). Treated resin (1 ml) was added to a solution of F420(5 mM) in a 10-ml serum vial kept under a stream of H2 atroom temperature. Flushing was continued until most of theyellow color of oxidized F420 disappeared (about 1 h). Thereduced F420 slurry was then taken back into the anaerobicchamber and passed through a small tube with filter paper atthe bottom to retain the agarose. The reduced F420 solution,separated from the resin and free of hydrogenase, was

collected in a 10-ml serum vial and stored under an N2atmosphere at 4°C. In the absence of added reducing agents,this solution remained reduced for several days. Each assay(1 ml) received 10 ,ul of this preparation of F420, andoxidation was monitored by measuring an increase in A401,401 nm being the isosbestic point of F420.

TABLE 1. Purification of CoMDSR fromM. thermoautotrophicum

Purification Protein Activity Sp act Purifi- %step (mg) (U)a.b (10-3 U/mg) cation Recov

(fold) ery

CFE 1,023 1.718 1.68 1.0 100DEAE-cellulose 82 0.697 8.5 5.1 41Phenyl-Sepharose 16.8 0.655 39.0 23.2 38NADP-agarose 0.4 0.272 680.0 405 16

a One unit of activity is 1 pLmol of (S-CoM)2 reduced per min.b Activities for this purification were measured at 55°C and at pH 7.0.

Under optimal conditions (64°C and pH 8.5), these activities should befourfold higher. The average specific activity of homogeneous enzyme as-sayed under optimal conditions was 2 to 3 p.mol of (S-CoM)2 reduced min-mg1 of enzyme protein.

RESULTSPurification of CoMDSR. Cell extracts of M. thermoau-

totrophicum AH contained CoMDSR activity (5 x 1i-' to 10X 10-3 U/mg of protein). Purification of the enzyme from 30g of cell paste proceeded in three steps (Table 1). The firststep was chromatography on DEAE-cellulose (Fig. 1A). Theenzyme was eluted at the end of the salt gradient with 0.75 Mpotassium acetate, which indicated a strongly anionic pro-tein. Nonspecific elution of the enzyme (fractions 1 to 9)accounted for 34% of the activity, and the peak (fractions 10to 15) accounted for 41%, leaving 25% of the original activityunaccounted for. Addition of boiled CFE or FAD back to theenzyme pool from DEAE-cellulose did not restore the miss-ing activity. This was most likely due to an overestimation ofthe activity in crude extracts that was due to the presence ofother disulfide reductase activities.The enzyme was eluted from phenyl-Sepharose with 0.15

M potassium acetate (Fig. 1B) and yielded a 4.6-fold purifi-cation with 93% recovery. Affinity chromatography (Fig. 1C)was the most effective step, resulting in an 18-fold purifica-tion which yielded a homogeneous enzyme, as judged by oneband on 10% native PAGE (Fig. 2). In some cases, anadditional round of fast protein liquid chromatography withMono Q was necessary to effect complete purification. Theenzyme was eluted in one peak at 0.8 M potassium acetateand was well separated from any remaining protein contam-inants (data not shown). More than 90% of the activity wasroutinely recovered at this step. This step was also used toseparate the enzyme from the NADP used in the elution ofNADP-C8-agarose affinity chromatography.

Activity of CoMDSR. The stoichiometry of the reactionwas determined by monitoring, in the same reaction, theoxidation of NADPH and the production of HS-CoM. In atypical experiment, 16 mol of NADPH yielded 36 nmol ofHS-CoM, indicating a 1:1 stoichiometry between NADPHand (S-CoM)2. The reverse reaction (NADP reduction byHS-CoM) was not detected. The DTNB assay measuring theproduction of thiol groups was linear for 40 min, whereas theNADPH assay was linear for only 20 min because of a lowerconcentration ofNADPH used in the latter reaction vials. Inboth cases, rates were proportional to the amount of enzymeadded.With homogeneous enzyme, the reaction did not proceed

in the absence of NADPH, (S-CoM)2, or enzyme. In crudeCFE but not with the pure enzyme, H2 could replaceNADPH, indicating that an H2-linked NADP-reducing sys-tem was present in the extracts. The CoMDSR activity inCFE was completely inactivated upon exposure to air for 60min; however, the homogeneous enzyme was air stable.

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12 3 4

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FIG. 1. Chromatographic purification ofCoMDSR (performed asdescribed in Materials and Methods). (A) DEAE-cellulose chroma-tography of CoMDSR. Only the high-salt CoMDSR active peak(fractions 10 to 15) was kept for further purification. (B) Phenyl-Sepharose chromatography of CoMDSR. The enzyme was elutedwith low-salt buffer, indicating a fairly high hydrophobicity. (C)NADP-C8-agarose chromatography of CoMDSR. The enzyme waseluted specifically with NADP. KAC, Potassium acetate.

Sodium chloride inhibited the activity by 50% at a concen-tration of 1.5 M. Potassium acetate, in contrast, did notinhibit the enzyme at concentrations as high as 1.5 M andwas therefore used in the purification procedure. Addition ofEDTA or Mg2' (10 mM each) did not affect the activity,suggesting that divalent cations were not involved in thereaction. Maximal activity occurred at 64°C, and the pHoptimum of 8.5 was independent of the buffer used (phos-

FIG. 2. Native PAGE of CoMDSR at successive steps in purifi-cation. Lanes: 1, CFE (50 p,g); 2, DEAE-cellulose active fraction (50p,g); 3, phenyl-Sepharose active fraction (30 ,ug); 4, NADP-C8-agarose active fraction (4 jLg).

phate or Tris); at pH 7, the enzyme was only 70% as active,and at pH 6, it was only 9% as active.When assayed under optimal conditions, homogeneous

enzyme preparations with specific activities of 2 to 3,umol * min-' - mg-' of protein were routinely obtained (Ta-ble 1). The homogeneous enzyme was unstable in buffer I at4°C, having a half-life of about 3 days. Storage in buffer Iwith 20o glycerol at -20°C preserved full activity forseveral months.

Kinetic studies. Kinetic properties of CoMDSR were stud-ied with a 319-fold-purified enzyme preparation (afterDEAE-cellulose, phenyl-Sepharose, and fast protein liquidchromatography with Mono Q chromatographic steps) bymeasuring the effect of increasing substrate concentrationson enzyme activity. A plot of the Hanes-Woolf equation([SI/VO = [S]/Vmax + Km/Vmax, where [S] is substrateconcentration) gave a Km for each substrate of 80 'PM.Under the DTNB assay conditions, 2 mM NADPH and 4mM (S-CoM)2 gave velocities of 96% and 98% of Vmn,respectively. Concentrations of NADPH above 2 mM werefound to be inhibitory. This had been previously observedfor the CoMDSR in Methanospirillum hungatei extracts(Ferry, Ph.D. thesis).

Molecular size. Analysis by 10% SDS-PAGE of theCoMDSR showed only one band with a relative mobility(Rm) of 0.41, corresponding to an approximate molecularweight of 51,000 (Fig. 3). Estimations of the Mr by tech-niques using the native protein yielded significantly highervalues. The enzyme eluted from Sephacryl S-200 gel filtra-tion with a partition coefficient (Kav) of 0.3, indicating anapproximate Mr of 64,000 (Fig. 4A). An identical value wasobtained with PhastGel native polyacrylamide gradient gels(Fig. 4B). Finally, when the SDS-PAGE was performed with8 M urea added to the gel, an estimated molecular weight of46,000 was obtained (data not shown).

Flavin content. The spectrum of CoMDSR was typical offlavoproteins (26), showing peaks at 268, 359, and 441 nm,minima at 309 and 394 nm, and a shoulder at 475 nm (Fig. 5).The ratios A26,/A,,1, A280A"1, and A359/A,l were, respec-tively, 7.96, 6.22, and 0.84. Upon reduction, the peak at 359nm decreased significantly and the peak at 441 nm almostdisappeared (Fig. 5, inset). Because of the small amount ofthe CoMDSR obtained, the nature of the flavin was notidentified. Assuming the prosthetic group to be FAD (as isthe case with all other characterized flavin-containing disul-

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FIG. 3. SDS-PAGE of homogeneous CoMDSR. Lanes: 1 and 3,molecular weight standards (5 and 10 ,ug per band, respectively); 2,NADP-C8-agarose active fraction (4 ,ug). For standards, see Mate-rials and Methods.

fide oxidoreductases), an extinction coefficient at 450 nm of11,300 was used (in other disulfide reductases, the extinctioncoefficient of bound FAD is identical to that of free FAD[26]); with a protein concentration of 0.36 mg/ml (see Mate-rials and Methods), an estimated molecular weight of 64,000,and a decrease of 0.074 U of A450 upon reduction (Fig. SB),a value of 1.16 mol of FAD per mol of polypeptide wasdetermined. Free FAD had peaks at 263, 375, and 450 nm inthe buffer solution described above (data not shown).

Substrate specificity. Various disulfides were tested assubstrates for the CoMDSR, including the heterodisulfide ofHS-CoM and HS-HTP. NADH and the reduced methano-genic cofactor F420 were also tested as electron donors. Thereductase had the highest specific activity with (S-CoM)2,although L-cystine was also significantly reduced (Table 2).NADH could be used as an electron donor, though muchless efficiently. Reduced F420 was not used as an electronsource.

DISCUSSIONA soluble CoMDSR from M. thermoautotrophicum was

purified 405-fold by three chromatographic steps, including

c;- 1 50

.- 80-60

X% 40

20

r

3oo r A S-200 Gel Filtration 300 B Native Gradient PAGE

150

80

40

201-

0.2 0.4 0.6 0.2 0.4 0.6 0.8 1.0

Kav Rm

FIG. 4. Molecular weight determination of native CoMDSR. (A)Sephacryl S-200 gel filtration chromatography. (B) Native gradientPAGE. The proteins used as standards are described in Materialsand Methods. CoMDSR is represented by open circles.

FIG. 5. Absorption spectrum of CoMDSR. The enzyme wasdissolved in a solution containing 0.8 M potassium acetate, 20%glycerol, and 20mM potassium phosphate buffer (pH 7.3). The samesolution without enzyme served as a blank. Inset: , oxidizedCoMDSR; - - -, CoMDSR reduced with a molar excess of 30% ofsodium dithionite. The spectrum was corrected for excess dithion-ite.

affinity chromatography on NADP-agarose. Under the con-ditions of purification employed, CoMDSR was a mono-meric enzyme. The apparent molecular weight of the nativeprotein by S-200 gel filtration chromatography as well as byelectrophoresis on nondenaturing polyacrylamide gradientgels was about 64,000. However, its molecular weight wasabout 51,000, as indicated by SDS-PAGE. This discrepancymight be due to the strong acidity and strong hydrophobicityof the enzyme.The monomeric CoMDSR contrasts with other disulfide

reductase enzymes, such as glutathione reductase, thiore-doxin reductase, and lipoamide dehydrogenase. These threeenzymes have been characterized from many sources, in-cluding yeast cells, Escherichia coli, mammalian cells, andplants. They are all dimeric, with subunit Mrs ranging from36,000 for E. coli thioredoxin reductase to 63,000 for thePseudomonas mercuric reductase (26). A11 of these enzymescontain one FAD as a prosthetic group per polypeptide. TheCoMDSR is also a flavoprotein, binding most likely onemolecule ofFAD per 64,000 Da. In archaebacteria, a dimericflavoprotein with an Mr of 122,000 which reduces bis-y-glutamylcystine via NADPH oxidation has been purifiedfrom Halobacterium halobium (21). The nature of its flavinwas not determined.The temperature and pH optima of CoMDSR are in good

TABLE 2. Enzymatic activity of CoMDSR fromM. thermoautotrophicum

Substrates% Activity

Electron donor Disulfide

NADPH (S-CoM)2 100.0NADPH Oxidized glutathione 0.6NADPH Oxidized lipoic acid 0.0NADPH L-Cystine 23.0NADPH Cys-S-S-CH3 7.0NADPH 7,7'-Dithiodiheptanoate 1.0NADPH CoM-S-S-HTP 35.7F42OH2a CoM-S-S-HTP 0.0F420H2 (S-CoM)2 0.0NADH (S-CoM)2 27.0a Reduced form of coenzyme F420.

350 400 450Wavelength (nm)

550

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agreement with the thermophilic nature of the organism(optimum growth at 65°C) and the pKa of cysteine residues.The catalytic mechanism most likely involves one or morecysteine residues, as is the case with all of the previouslymentioned disulfide oxidoreductases, and is optimized whenthe protonated and deprotonated forms of cysteine are inequilibrium. The enzyme from Methanospirillum hungateialso had a pH optimum of 8.5 (Ferry, Ph.D. thesis).The CoMDSR showed equal affinity for both (S-CoM)2

and NADPH, with a Km of 80 p,M for each substrate. TheseKm values are similar to those of other disulfide reductases(26). Flavin-containing disulfide oxidoreductases are usuallyvery specific, and CoMDSR also showed a high degree ofspecificity for (S-CoM)2. The significant reduction of L-Cys-tine reduction is most likely due to the similarity in structurewith (S-CoM)2. It should be noted that the value for CoM-S-S-HTP activity is probably misleading because of a veryrapid disulfide interchange at 60°C leading to the constantformation of (S-CoM)2 from CoM-S-S-HTP and HS-CoM.The enzyme constitutes about 0.25% of the soluble protein

of the cell. Its approximate specific activity in CFE is 2 nmolof (S-CoM)2 reduced min-' mg-1 of total protein at neutralpH, which is seven times greater than the activity reported inextracts of Methanospirillum hungatei (Ferry, Ph.D. thesis).Nevertheless, this value is still much lower than the 20 to 30nmol of CH4 produced min-1 mg-1 of CFE protein (8),suggesting that the enzyme is not involved in providingelectrons to the methyl reductase system.When the structure and the role of HS-HTP were found, it

was thought that the CoMDSR might be the physiologicaldisulfide reductase for the regeneration of HS-CoM andHS-HTP from CoM-S-S-HTP in the methyl-coenzyme Mreduction reaction, since in crude extracts, NADPH hadbeen shown to drive methanogenesis at 86% of the rate of theH2-driven reaction (7). We expected that the reductaseactivity with CoM-S-S-HTP, the proposed physiologicalsubstrate, would be greater than that with (S-CoM)2. Thiswas not the case, the enzyme being less active with CoM-S-S-HTP than with (S-CoM)2, its likely physiological sub-strate. This too rules out a direct role of the CoMDSR inmethanogenesis. The partial purification of the H2-drivenheterodisulfide reductase activity in M. thermoautotrophi-cum Marburg has been reported previously (14). This activ-ity probably corresponds to two enzymes, and the interme-diate electron carrier between the hydrogenase and thedehydrogenase is not known. A report of the further purifi-cation and partial characterization of the reductase per sewith reduced viologen dyes as electron donors has appeared(13), but the physiological electron donor in Methanobacte-rium spp. of the reaction is not known yet. The CoMDSRcould be linked indirectly to the methyl reductase by regen-erating the HS-CoM which might be trapped in the cell as itsdisulfide, a nonutilizable form of the cofactor.Another possible function could be linked to sulfur reduc-

tion. Cells of Methanobacterium spp. and other methano-gens were shown to actively reduce elemental sulfur (20).This process could be mediated by HS-CoM, which readilyreduces elemental sulfur, yielding (S-CoM)2 (W. Sheridanand R. S. Wolfe, unpublished data). In Methanococcusvoltae, an active transport system for HS-CoM is present(5). In Methanobrevibacter ruminantium, an auxotrophicstrain for HS-CoM,, (S-CoM)2 is taken up (1). HS-CoMcould leave the cell according to its concentration gradient,chemically reduce S', and be taken back into the form of(S-CoM)2 in an energy-dependent process. The CoMDSRwould therefore be involved in maintaining a low redox

potential crucial for methanogenesis (25). This could also ofcourse contribute to sulfur assimilation.The enzyme may play a regulatory role by maintaining the

redox balance inside the cell, as does glutathione in eubac-teria. It seems that methanogens lack glutathione and coulduse HS-CoM or (S-CoM)2 as a major thiol-disulfide redoxregulator (11). In this respect, the CoMDSR from M. ther-moautotrophicum would be analogous to the bis--y-glutamyl-cystine reductase from H. halobium, an archaebacteriumwhich lacks glutathione and contains -y-glutamylcysteine asthe major low-molecular-weight thiol (21). Recently, Bobikand Wolfe showed that HS-CoM and HS-HTP are thesubstrates for fumarate reductase in M. thermoautotrophi-cum (3); HS-CoM may be involved in other redox reactionsin the cell.The role of the CoMDSR is linked to the role of HS-CoM,

and evidence in this report and elsewhere suggests that theenzyme does not play a central role in methanogenesis butmost likely plays a secondary role by maintaining reducedpools of sulfur, regulating redox reactions, or simply main-taining HS-CoM in its active form.

ACKNOWLEDGMENTS

This work was supported by U.S. Department of Energy grantDE-FG02-87ER 13651 to R. S. Wolfe.We thank Victor Gabriel for helpful technical assistance.

LITERATURE CITED1. Balch, W. E., and R. S. Wolfe. 1979. Transport of coenzyme M

(2-mercaptoethanesulfonic acid) in Methanobacterium rumi-nantium. J. Bacteriol. 137:264-273.

2. Bobik, T. A., K. D. Olson, K. M. Noll, and R. S. Wolfe. 1987.Evidence that the heterodisulfide of coenzyme M and 7-mercap-toheptanoylthreonine phosphate is a product of the methylre-ductase reaction in Methanobacterium. Biochem. Biophys.Res. Commun. 149:455-460.

3. Bobik, T. A., and R. S. Wolfe. 1989. An unusual thiol-drivenfumarate reductase in Methanobacterium with the production ofthe heterodisulfide of coenzyme M and N-(7-mercaptohep-tanoyl)threonine-03-phosphate. J. Biol. Chem. 264:18714-18718.

4. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Dybas, M., and J. Konisky. 1989. Transport of coenzyme M(2-mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic acid] in Methanococcus voltae: iden-tification of specific and general uptake systems. J. Bacteriol.171:5866-5871.

6. Eirich, L. D., G. D. Vogels, and R. S. Wolfe. 1978. Proposedstructure for coenzyme F420 from Methanobacterium. Biochem-istry 17:4583-4593.

7. Ellefson, W. L., and R. S. Wolfe. 1980. Role of component C inthe methylreductase system of Methanobacterium. J. Biol.Chem. 255:8388-8389.

8. Ellefson, W. L., and R. S. Wolfe. 1981. Component C of themethylreductase system of Methanobacterium. J. Biol. Chem.256:4259-4262.

9. Ellermann, J., R. Hedderich, R. Bochner, and R. K. Thauer.1988. The final step in methane formation: investigations withhighly purified methyl-CoM reductase (component C) fromMethanobacterium thermoautotrophicum (strain Marburg).Eur. J. Biochem. 172:669-677.

10. Eliman, G. L. 1958. A colorimetric method for determining lowconcentrations of mercaptans. Arch. Biochem. Biophys. 75:443-450.

11. Fahey, R. C., and G. L. Newton. 1983. Occurrence of lowmolecular weight thiols in biological systems, p. 251-260. In A.Larsson, S. Orrenius, A. Holgren, and B. Mannervick (ed.),Functions of glutathione: biochemical, physiological, toxicolog-

J. BACTEPIOL.

on February 11, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

M. THERMOAUTOTROPHICUM COENZYME M DISULFIDE REDUCTASE 6441

ical, and clinical aspects: proceedings of the Fifth KarolinskaInstitute Nobel Conference. Raven Press, New York.

12. Gunsalus, R. P., S. M. Tandon, and R. S. Wolfe. 1980. Aprocedure for anaerobic column chromatography employing ananaerobic Freter-type chamber. Anal. Chem. 101:327-331.

13. Hedderich, R., A. Berkessel, and R. K. Thauer. 1989. Catalyticproperties of the heterodisulfide reductase involved in the finalstep of methanogenesis. FEBS Lett. 255:67-71.

14. Hedderich, R., and R. K. Thauer. 1988. Methanobacteriumthermoautotrophicum contains a soluble enzyme system thatspecifically catalyzes the reduction of the heterodisulfide ofcoenzyme M and 7-mercaptoheptanoylthreonine phosphatewith H2. FEBS Lett. 234:223-227.

15. Kunitze, M. J. 1952. Crystalline inorganic pyrophosphataseisolated from baker's yeast. J. Gen. Physiol. 35:423-450.

16. Laemmli, U. K. 1970. Cleavage of structural protein during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

17. Noli, K. M., K. L. Rinehart, Jr., R. S. Tanner, and R. S. Wolfe.1986. Structure of component B (7-mercaptoheptanoylthreoninephosphate) of the methylreductase system of Methanobacte-rium thermoautotrophicum. Proc. Natl. Acad. Sci. USA 83:4238-4242.

18. Peterson, G. L. 1983. Determination of total protein. MethodsEnzymol. 91:96-105.

19. Rouviere, P. E., and R. S. Wolfe. 1988. Novel biochemistry ofmethanogenesis. J. Biol. Chem. 263:7913-7916.

20. Stetter, K. O., and G. Gaag. 1983. Reduction of molecularsulphur by methanogenic bacteria. Nature (London) 305:309-311.

21. Sundquist, A. R., and R. C. Fahey. 1988. The novel disulfidereductase bis-y-glutamylcystine reductase and dihydrolipoam-ide dehydrogenase from Halobacterium halobium: purificationby immobilized-metal-ion affinity chromatography and proper-ties of the enzymes. J. Bacteriol. 170:3459-3467.

22. Taylor, C. D., and R. S. Wolfe. 1974. Structure and methylationof coenzyme M. J. Biol. Chem. 249:4879-4885.

23. Taylor, C. D., and R. S. Wolfe. 1974. A simplified assay forcoenzyme M. J. Biol. Chem. 249:4886-4890.

24. Van der Meijden, P., C. van der Lest, C. van der Drift, and G. D.Vogels. 1984. Reductive activation of methanol: 5-hydroxybenz-imidazolyl-cobamide methyltransferase of Methanosarcinabarkeri. Biochem. Biophys. Res. Commun. 118:760-766.

25. Widdel, F. 1988. Microbiology and ecology of sulfate- andsulfur-reducing bacteria, p. 544. In J. B. Zehnder (ed.), Biologyof anaerobic microorganisms. John Wiley & Sons, Inc., NewYork.

26. Williams, C. H., Jr. 1976. Flavin-containing dehydrogenases, p.89-173. In P. D. Boyer (ed.), The enzymes, 3rd ed., vol. 13.Academic Press, Inc., New York.

27. Zeikus, J. G., and R. S. Wolfe. 1972. Methanobacterium ther-moautotrophicum sp. n., an anaerobic, autotrophic, extremethermophile. J. Bacteriol. 109:707-713.

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