Vol. 175, No. 18JOURNAL OF BACrERIOLOGY, Sept. 1993, p. 5970-59770021-9193/93/185970-08$02.00/0Copyright © 1993, American Society for Microbiology

Methyl Viologen Hydrogenase II, a New Member of theHydrogenase Family from Methanobacterium

thermoautotrophicum AHGUN-JO WOO, ALAIN WASSERFALLEN,t AND RALPH S. WOLFE*Department ofMicrobiology, University of Illinois, Urbana, Illinois 61801

Received 29 March 1993/Accepted 7 July 1993

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum AH havebeen separated (resolution, R. at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel andSuperose 6 Prep Grade. The newly discovered MVH (MVHII ) was eluted at 0.5 M NaCl with a linear gradientof 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56M. The specific activities of MVI I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, whenenzyme activity was compared at pH 7.5, the optimal pH for MVHII . Gel electrophoresis in nondenaturingsystems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH IIshowed four protein bands (cm, 50 kDa; 13, 44 kDa; fy, 36 kDa; 8, 15 kDa), similar to MVH I. The N-terminalamino acid sequences of the a, -y, and 8 subunits of MVHIIH were identical with the sequences of the equivalentsubunits of MVH I. However, the N-terminal amino acid sequence of the 13 subunit of MVH II was totallydifferent from the sequence of the 13 subunit of MVH I. Both MVH I and MVHII had the same optimaltemperature of 60°C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5,respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activitywas only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8days at -20°C under anaerobic conditions. When exposed to air, 90% ofMVH I activity was lost within 2 min;however, MVH II lost only 50% of its activity in 3 h.

The archaeon Methanobacterium thermoautotrophicumAH (35) is a thermophilic strict anaerobe that uses H2 as thesole source of energy and reducing equivalents for thereduction of CO2 to methane. The pathway of methanogen-esis, involving six unusual coenzymes and several enzymes,has been thoroughly reviewed (6, 18). Hydrogenase plays akey role in the biogenesis of methane. Many bacterialhydrogenases are known to contain Fe-S clusters (1). Nickelis also required for the biosynthesis of certain hydrogenasesor for the growth of some species of bacteria (2, 8, 10, 28,32). The Fe-S clusters in nickel hydrogenases are postulatedto function in electron transport transferring reducing equiv-alents to or from Ni (17, 25). M. thermoautotrophicum AHhas two Ni-containing hydrogenases which also contain Fe-Sclusters. One reduces the hydride carrier deazaflavin F420coenzyme and is known as F420-reducing hydrogenase(FRH). This hydrogenase has been purified from severalmethanogens and extensively studied (7, 16, 22, 30). Theother hydrogenase readily reduces the one-electron acceptormethyl viologen (MV) and is known as methyl viologenhydrogenase (MVH). The natural electron acceptor may beferredoxin or polyferredoxin, but this is poorly defined atpresent (19, 31).FRH is a high-molecular-mass protein of 800 kDa, com-

posed of three subunits, a (47 kDa), ,B (31 kDa), and -y (26kDa), forming an (a1Y13Y1)8 complex, and a number of tightlybound cofactors, including a Ni atom, a flavin adeninedinucleotide, and several iron atoms arranged in Fe-S clus-ters (7). The size of this large protein was also estimated byunidirectional shadow casting (34). FRH is usually easy to

* Corresponding author.t Present address: Mikrobiologisches Institut, Zurich, Switzer-

land.

purify and characterize because it constitutes a larger por-tion of the total soluble protein than does MVH. The threegenes frihA, frhB, and frhG, encoding the three subunits,have been cloned and sequenced. The DNA sequence ofFRH also contains a fourth open reading frame, frhD (3).mvhDGAB, which encode the four polypeptides of MVH,

have been cloned and sequenced from M. thermoautotrophi-cum AH (25). On the basis of the deduced amino acidsequences, the subunit polypeptides were predicted to be15.8, 33, 53, and 44 kDa, respectively. The mvhB geneproduct (a polyferredoxin with six tandem repeats) has beenisolated and characterized from Methanothermus fervidus(31) and also from M. thermoautotrophicum Marburg (14).Four gene clusters encoding two [NiFeSe] hydrogenases

(FRH) and two [NiFe] hydrogenases (MVH) were recentlyidentified on the DNA level from Methanococcus voltae(13). In the present study, we found two MVH proteins fromM. thermoautotrophicum AH which were shown not to haveFRH activity. One of them, named MVH II (a new MVH), isreported here; the other, named MVH I, corresponded to theMVH which has been studied and characterized previously.In addition, some unique catalytic properties of MVH II,which is much less oxygen sensitive than MVH I, aredescribed and compared with those of MVH I.

MATERIALS AND METHODS

Materials. Chromatography resins and columns were ob-tained from Pharmacia LKB Biotechnology, Piscataway,N.J. Chemicals and reagents used were of the highest puritycommercially available.

Microorganism and culture conditions. M. thermoau-totrophicum AH (ATCC 29096) was cultivated on definedmedium as previously described (11) with the following

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modifications. The inoculum (10%) was transferred to a300-liter fermentor (B. Braun Biotech. Inc., Allentown, Pa.)containing 200 liters of medium. Cells were cultivated at60°C and pH 7.0 with a gas flow rate of 0.08 (H2) and 0.02(CO2) liter of gas per liter of culture per min and an agitationrate of 600 rpm. The flux of H2S was 1.63 ml/liter ofculture/min. The redox potential in the medium was main-tained below -400 mV throughout the cultivation. TheNH4C1 (100 g dissolved in 400 ml of H20) solution wasanaerobically added to the culture every 24 h. Cells wereharvested by continuous Sharples centrifugation at the earlystationary growth phase (87 h of growth).

Purification of MVH cell extract preparation (step 1). Forthe preparation of cell extract, harvested cells were sus-pended in an equal volume of 50 mM HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.5)containing 10 mM dithiothreitol (DTT) and 1 mM 3-[(3-cholamidopropyl) dimethylammonio] - 1 - propane sulfonicacid (CHAPS) in an anaerobic chamber (Coy ScientificProducts, Inc., Ann Arbor, Mich.). Cells were disrupted bytwo passages through a French pressure cell (AmericanInstrument Co. Inc., Silver Spring, Md.) at 20,000 lb/in2. Thebroken-cell suspension was ultracentrifuged for 1 h at150,000 x g and 4°C. The supematant containing hydroge-nase was stored frozen (-20°C) under H2 until used.

First ion-exchange chromatography (step 2). The purifica-tion and equilibration procedures mentioned below werecarried out at room temperature in an anaerobic chambercontaining an atmosphere of 95% N2 and 5% H2. Buffersolutions were filtered through a membrane filter (pore size,0.45 ,um; Millipore, Bedford, Mass.) before use. The cellextract was first passed through a surfactant-free celluloseacetate membrane filter (pore size, 0.2 ,um; Nalge Co.,Rochester, N.Y.). The filtered cell extract (1.42 g of totalprotein) was applied to a DEAE-Sephacel column (5.0 by20.0 cm gel bed height in an XK 50/30 column) which waspreequilibrated overnight with the 2.5 column volumes ofHEPES buffer containing 5 mM DTT and 1 mM CHAPS.The flow rate was 1.5 ml per min. After isocratic elution withbuffer (90 ml), the hydrogenase was eluted with a lineargradient of NaCl in the same buffer (0 to 1.0 M, 735 ml).Fractions of 8.0 ml each were collected. The active fractions(fractions 83 to 93) containing MVH were combined andconcentrated through a stirred cell (model 8400; AmiconInc., Beverly, Mass.) by using a cellulose membrane filter(YM30; molecular weight cutoff, 30,000). The concentratedpool was stored at -20°C in a 20% (vol/vol) aqueous solutionof glycerol.

First gel filtration chromatography (step 3). The concen-trated MVH fraction from the DEAE-Sephacel column wasloaded onto a Superose 6 Prep Grade (PG) HR 16/50 column(1.6 by 48.5 cm bed) in 400-,ul samples (8.9 mg of protein).The theoretical plate number per meter of Superose 6 PGcolumn was 10,890 m-1, which, being above 10,000 m-1,ensured satisfactory separation of proteins. The columnefficiency was tested by using acetone (5 mg/ml) as specifiedby the manufacturer. The column was equilibrated with 3column volumes of the buffer before the sample was applied.The elution was performed with 50 mM HEPES buffer (pH7.5) containing 0.3 M NaCl and 5 mM DTT at a flow rate of0.3 ml/min.

Second ion-exchange chromatography (step 4). The MVHpool from the Superose 6 PG column, desalted by ultrafil-tration on an Amicon YM-10 membrane, was loaded onto aMono Q HR 10/10 column. The proteins were applied in1.0-ml batches (6.9 mg of protein). Buffer A was 50 mM

HEPES buffer (pH 7.5) containing 5 mM DTT. Buffer B wasbuffer A with 1.0 M NaCl. MVH II and MVH I wereseparated and eluted at 0.5 M and 0.56 M NaCl, respec-tively, with a linear NaCl gradient from 0.45 M to 0.65 M(see Fig. 1B). The eluent flow rate was 4.0 ml/min.Second gel filtration chromatography (step 5). A Superose 6

prepacked HR 10/30 column was used for further purificationofMVH I and MVH II. The column had 30,000 plates per m.The same elution condition used for the first gel filtrationchromatography was used in this step, except that the flowrate in the Superose 6 column for MVH I was 0.2 ml/min.Enzyme and protein assays. The MVH activity of the

concentrated fractions at each purification step was deter-mined by using a reaction mixture that was constituted in ananaerobic chamber and contained 50 mM NaCl, 5 mM DTT,1 mM MV, and 50 mM HEPES (pH 7.5). The headspace gasof the assay vial was exchanged with hydrogen at 100 ml/minfor 5 min and incubated at 60°C for 7 min before addition ofenzyme. MV reduction (the increase in A601, e601 = 1.13 x104 M-1 cm-') was monitored after addition of enzymethrough a gastight syringe into the 1.5-ml semimicro poly-styrene cuvette which contained 0.7 ml of assay buffer. Oneunit of hydrogenase activity was defined as 1 ,umol ofhydrogen oxidized per min at 60°C (pH 7.5). Specific activityis expressed as units per milligram of protein. Proteinconcentration was determined by the Micro Protein Assay(Pierce Chemical Co., Rockford, Ill.) with bovine serumalbumin as a standard.

Gel electrophoresis. With the PhastSystem and PhastGelgradient (8 to 25%) media (Pharmacia LKB Biotechnology,Piscataway, N.J.), native polyacrylamide gel electrophoresis(PAGE) was carried out to check the purity of the enzyme ateach purification step. Anaerobic preparative native andsodium dodecyl sulfate (SDS)-PAGE slab gels (4% stackinggel and 10% resolving gel) were cast and polymerized in ananaerobic chamber. Prior to use, Tris-glycine electrophore-sis buffer (3.0 g of Tris base and 14.4 g of glycine dissolvedin 1.0 liter of H20) was sparged with O2-free nitrogen at 1.2liters/min for 1 h. For denaturing gel electrophoresis, SDS(0.1% [wt/vol]) was brought into the chamber and added tothe Tris-glycine buffer. Electrophoresis buffer, acrylamidestock solution (30% [wt/vol]), and lower and upper Trisstock solutions were filtered with a Sterifll-D GV filter unit(pore size, 0.22 ,um; Millipore) before they were brought intothe chamber. Standard proteins were obtained from Phar-macia LKB Biotechnology, and their sizes are indicated inthe figures.

Activity staining of native gels. At the completion ofelectrophoresis the gel was incubated in buffer containing 50mM NaCl, 5 mM DTT, 1 mM MV, and 50 mM HEPES (pH7.5) at room temperature until MV was reduced by MVHunder anaerobic conditions. After the reduction of MV, 3%(wt/vol) 2,3,5-triphenyltetrazolium chloride solution wasadded to the buffer until the colorless 2,3,5-triphenyltetra-zolium chloride had changed its color to a deep red on thehydrogenase bands.Amino acid composition. Homogeneous hydrogenase from

native gel electrophoresis was hydrolyzed in 6 N HCI at110°C for 16 h. The amino acid composition was analyzedwith an AminoQuant (Hewlett-Packard Co., Palo Alto,Calif.). The number of residues of each amino acid wascalculated as moles of amino acid per mole of protein.

N-terminal amino acid sequences. All sample preparationsexcept electroblotting and amino acid sequencing were per-formed under anaerobic conditions. The protein bands fromthe anaerobic preparative SDS-PAGE were transferred to a

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A

0.5

C D

.1-

'IH

1.0M 0.i[

(5-0

MVH0

MVHII1

MVH I

0 2 4 6 8 0 10 20 30

Time (hr)

laCI

1.5M

0

Time (min)

i II

0. MVH I

LVV

1 2 0 1 2 3

Time (hr) Time (hr)

FIG. 1. Purification of MVH by different chromatographic procedures. (A) Elution profile of the concentrated MVH fraction (8.92 mg)from DEAE-Sephacel column, which was applied to a Superose Prep Grade HR 16/50 column. Buffer solution (50 mM HEPES, 5 mM DTT,0.3 M NaCl [pH 7.5]) was used as the eluent. The flow rate was 0.3 mlmin. The protein concentration was measured by its A2.. (B)Separation of MVH I and MVH II on a Mono Q HR 10/10 column. An isocratic buffer solution (50 mM HEPES, 5 mM DTT [pH 7.5]) wasused as the eluent for the first 12 ml. A 6-ml linear gradient (0 to 0.45 M NaCl) was followed by the second step, a 100-ml 0.45 to 0.65 M NaClgradient. The flow rate was 4.0 ml/min. (C) Elution profile of MVH II on a Superose 6 HR 10/30 column. The same buffer solution and elutionconditions used in panel A were employed. (D) Elution profile ofMVH I on a Superose 6 HR 10/30 column. The same elution, except for theflow rate, was performed as described in Fig. 1A. The flow rate was 0.2 m/min.

polyvinylidine difluoride-type membrane (Applied Biosys-tems Inc., Richmond, Calif.) for 1 h in a Trans-Blot SDSemi-Dry Electrophoretic Transfer Cell (Bio-Rad Laborato-ries, Richmond, Calif.) as described by Matsudaria (21).Amino acids in the N-terminal region were sequenced with amodel ABI 477A or 475A protein sequencer (Applied Bio-systems) and identified by a model 120A on-line PTH ana-lyzer.Temperature. To study the effect of temperature, we

prepared the assay solution (690 ,ul) containing 50 mMHEPES, 50 mM NaCl, and 5 mM DTT (pH 7.5) in ananaerobic chamber. The headspace gas in the stopperedassay vial (a 1.5-ml polystyrene cuvette; Markson Sci. Inc.,

Phoenix, Ariz.) was flushed with 02-free hydrogen at 100ml/min for 5 min. The assay vial was then incubated in aThermo Circulator (model C 005-0405; Perkin-Elmer, OakBrook, Ill.) at 20, 30, 40, 50, 60, and 70°C for 15 min. Thereaction was started by the addition of 10 ,u1 of 70 mM MVfollowed by the enzyme sample through a microliter syringe(Hamilton Co., Reno, Nev.). Before use, each syringe wasflushed several times with oxygen-free water. For thesestudies as well as for studies on the effect of pH, heavymetals, and aerobic and anaerobic conditions, enzyme fromthe Mono Q column (step 4) was used, specific activities forMVH I and MVH II being 129.5 and 54.7 U/mg of protein,respectively.

TABLE 1. Purification of MVH II and MVH I from M. thermnoautotrophicumProtein Enzyme

Purification step Concn Total Concn Sp act Total amt Yield Purification(mg/ml) (amg) (U/ml) (U/mg) (U) (%) (fold)

MVH IICell extract 47.3 946.0 588.5 12.4 11,730 100 1.0DEAE-Sephacel 22.3 289.9 446.0 20.0 5,798 49 1.61Superose 6 PG 6.9 41.4 287.5 41.7 1,726 14.7 3.36Mono Q 0.34 0.51 18.6 54.7 27.9 0.24 4.41Superose 6 0.016 0.018 0.98 61.3 1.1 0.01 4.94

MVH ICell extract 47.3 946.0 588.5 12.4 11,730 100 1.0DEAE-Sephacel 22.3 289.9 446.0 20.0 5,798 49 1.61Superose 6 PG 6.9 41.4 287.5 41.7 1,726 14.7 3.36Mono Q 1.11 2.054 143.7 129.5 265.9 2.3 10.4Superose 6 0.092 0.120 17.0 184.8 22.1 0.19 14.9

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1 2

MVH 11- 1-| I MVH I

MVH II-2--s-

FIG. 2. Activity staining by dye reduction for MVH as shown on

a 10.0% native polyacrylamide gel under anaerobic conditions.Lanes: 1, Mono Q MVH II fraction (10.2 mg per well); 2, Mono QMVH I fraction (33.3 mg per well). Because of the thickness of thepreparative slab gel, a shadow was cast below the stained bandsduring photography.

pH. To study the effect of pH, we used the followingbuffers (final concentration, 50 mM, with 50 mM NaCl and 5mM DTT): MES [2-(N-morpholino)ethanesulfonic acid]buffer at pH 6.5, PIPES [piperazine-N,N'-bis(2-ethanesul-fonic acid)] buffer at pH 6.5, HEPES buffer at pH 7.0 and7.5, Tricine [N-tris(hydroxymethyl)methyl-glycine] buffer atpH 8.0 and 8.5, and CHES [2-(N-cyclohexylamino)ethanesul-fonic acid] buffer at pH 9.0 and 9.5. Buffers were storedunder anaerobic conditions, and the pH of the buffer was

determined before each use with a pH meter (model (110;Beckman Instruments Inc., Fullerton, Calif.) kept in theanaerobic chamber.

Effect of heavy metals on MVH activity. After the 1.0 ,ul ofMVH had been incubated with 1 mM metal chloride (60°Cfor 5 min), the residual activity of the enzyme was deter-mined as described above. The MVH activity in the absenceof a metal ion was assigned as the control activity (100%).

Stability of MVH under anaerobic and aerobic conditions.Enzyme solutions obtained from step 4 in the purificationwere kept in a serum vial for further purification withSuperose 6 HR 10/30. Part of the hydrogenase was trans-ferred to a serum vial and kept with 20% (vol/vol) glycerol at-20°C. Enzyme stability under anaerobic conditions was

observed every 24 h for 8 days by measuring the residualactivity of MVH. To study the oxygen sensitivity of MVH,each 10-,ul sample from the serum vial was placed aerobi-cally in a separate microcentrifuge tube. Each tube was

capped immediately and placed at -20°C. At each specifiedtime a centrifuge tube was removed from the freezer, and theenzyme solution was thawed quickly and added to thereaction mixture in a cuvette. The residual activity of MVHII was measured at 10, 20, 30, 60, 120, and 180 min. ResidualMVH I activity was monitored every 2 min and then at 15,20, and 30 min.

RESULTS

Separation of MVH I and MVH II by chromatography.MVH from M. thennoautotrophicum AH was purified as

described in Materials and Methods. The concentrated frac-tions containing MVH activity from a DEAE-Sephacel col-umn were dark brown. The elution profile of six proteinpeaks obtained after Superose 6 PG column chromatographyofMVH obtained from DEAE-Sephacel is shown in Fig. 1A.

MVH was eluted as the second peak in the purification ofstep 3, and most of an unknown yellow protein, a majorprotein during MVH purification, could be removed on theSuperose 6 PG HR 16/50 column. The third peak in Fig. 1Acontained most of the yellow protein. The yellow proteincould be effectively removed from MVH by the purificationprocedures used here. As shown in Fig. 1A, MVH I andMVH II were not separated by purification step 3 (Superose6 PG HR 16/50). When eluted from the Mono Q column (Fig.1B), MVH activity was found in two peaks. MVH II andMVH I were separated and eluted at 0.5 M and 0.56 M NaCl,respectively. Each MVH was further purified by Superose 6HR 10/30 (a prepacked column) (Fig. 1C and D).To simplify assays and because of limiting amounts of

enzymes, MVH I and MVH II activities were compared atpH 7.5, the optimum for MVH II but not for MVH I. Asshown in Table 1, the specific activity of MVH increased by3.4-fold through the first two steps of chromatography. Thespecific activity of the second hydrogenase peak (MVH I)eluted from Mono Q HR 10/10 column was more than twicethat of MVH II (the first hydrogenase peak) in Fig. 1B. Thespecific activity ofMVH I was three times higher than that ofMVH II after the last step of purification, Superose 6 (Fig.1C and D).Molecular properties of the enzymes. To ensure the isola-

tion of homogeneous MVH, we adopted the following strat-egy. Enzyme fractions of MVH I and MVH II from theMono Q column (step 4) were each subjected to a separatepreparative native-gel electrophoresis. The section whichcontained an isolated, reduced-dye hydrogenase band wasexcised from the gel slab with a razor blade, and each sectionwas loaded into a separate well of an anaerobic preparativeSDS-PAGE apparatus. Protein from appropriate bands insubsequent SDS-PAGE gels was pooled until a sufficientquantity of each protein subunit was obtained. Activitystaining for MVH in a 10.0% native polyacrylamide gel

A1 2

B1

kDa

-94

-67

-43

-30

p_a-o_-

-20.1

-14.4

kDa

-94

-67

-43

-30

-20.1

-14.4FIG. 3. (A) PAGE pattern (10.0% polyacrylamide) of homoge-

neous MVH II-1 (lane 1) and MVH II-2 (lane 2) under denaturingand anaerobic conditions. (B) PAGE pattern (10.0% polyacryl-amide) of homogeneous MVH I under denaturing and anaerobicconditions. Bands were visualized by Coomassie brilliant blue R-250staining. The running positions of the standard proteins (top tobottom), phosphorylase b, albumin, ovalbumin, carbonic anhy-drase, trypsin inhibitor, and a-lactalbumin are indicated in kilodal-tons.

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TABLE 2. Amino acid composition of subunits of MVH 11-1

No. of residues (% of total residues) in subunit:Amino acid

a 13 Sy 8

Asx 58.1 (11.34) 47.1 (11.29) 33.7 (8.94) 15.2 (10.12)Glx 56.6 (11.04) 47.0 (11.26) 44.9 (11.91) 15.9 (10.54)Ser 31.4 (6.14) 15.4 (3.70) 25.2 (6.69) 12.4 (8.25)His 9.9 (1.94) 10.2 (2.44) 5.2 (1.37) 3.4 (2.28)Gly 66.5 (12.99) 40.4 (9.68) 56.9 (15.09) 24.5 (16.30)Thr 25.3 (4.93) 24.2 (5.81) 14.3 (3.79) 5.9 (3.89)Ala 42.5 (8.29) 39.1 (9.37) 27.6 (7.31) 10.8 (7.20)Arg 22.2 (4.33) 22.4 (5.37) 14.4 (3.82) 9.8 (6.50)Tyr 17.9 (3.50) 17.7 (4.24) 9.2 (2.45) 4.8 (3.17)Val 24.4 (4.76) 22.0 (5.28) 17.8 (4.71) 7.3 (4.86)Met 14.8 (2.88) 10.1 (2.42) 20.3 (5.38) 2.7 (1.81)Phe 21.9 (4.27) 17.7 (4.25) 11.0 (2.92) 5.4 (3.56)Ile 27.6 (5.40) 24.1 (5.78) 25.1 (6.64) 8.2 (5.42)Leu 43.1 (8.42) 39.2 (9.40) 25.8 (6.84) 10.6 (7.05)Lys 24.8 (4.83) 22.7 (5.44) 25.5 (6.76) 8.6 (5.72)Pro 25.3 (4.94) 17.8 (4.27) 20.4 (5.40) 5.0 (3.32)Total 512.3 417.6 377.6 150.5

under anaerobic conditions is shown in Fig. 2. MVH II after identical with the deduced amino acid sequences of mvh-Mono Q HR 10/10 column chromatography (lane 1) showed DGAB from DNA analysis (25). The N-terminal amino acida major distinct band of hydrogenase activity (MVH II-1) sequence of each subunit indicated that the methionyl resi-and a smaller band of lower molecular weight (MVH II-2), due was excised at the N terminus of the subunit (15). Thewhereas MVH I showed only one stained band, none of the excised methionyl residue is shown in parentheses in Tablebands being related to the native FRH (7). The denatured 3. The sequences of a, -y, and 8 subunits of MVH II-1 andMVH II-1 (Fig. 3A, lane 1) showed four protein bands, these also those of a and y subunits of MVH II-2 were identicalsubunit patterns being very similar to those of MVH I (Fig. with those of corresponding subunits of MVH I. However,3B) which has been cloned and sequenced (25). The amino the N-terminal sequences of 1 subunits of MVH II-1 and II-2acid compositions of the four subunits of MVH II-1 are (shown in boldface type in Table 3) had no homology withpresented in Table 2. The three subunits (a, -y, and b) of those of the 1 subunit of MVH I. It was confirmed that theMVH 11-1 contain a relatively large number of glycine sequence of the 1 subunit of MVH II did not have anyresidues with polar uncharged groups as well as negatively homology with any other protein in the sequence data basescharged acidic amino acids; however, the acidic amino acids at the National Center for Biotechnology Information byappeared as the major amino acids in the composition of the using the BLAST network. The 1 subunit of MVH II-2 was1 subunit of MVH II-1. SDS-PAGE analysis of the 130-kDa identical with that of MVH II-1, indicating that MVH II-2native hydrogenase (MVH II-2) revealed it to be an a13y (a13y trimer) was derived from the tetrameric form of MVHtrimer (Fig. 3A, lane 2), suggesting that MVH II-2 is a II-1 (a13yb).derivative of MVH II-1. The N-terminal sequences of sub- Thermal stability of MVH. Since the two MVHs appearedunits from MVH I on the protein level (Table 3) were to be tightly associated in cell extracts, attempts were made

TABLE 3. Comparison of N-terminal amino acid sequences of MVH

Residue no.aSubunit

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

MVH II-Ia (M) V K L T M E P V T R I E G H A K I T Va3 (M) M K A ? A E K I A D G L Y ? T G V L D Dy (M) A E K I K I G T M ? L G G8 (M) A E D D I K I V M F

MVH II-2a (M) V K L T M E P V T R13 (M) M K A ? A E K I A D G L Y ? T G V L D Dly (M) A E K I K I G T M W L G G

MVH Ia (M) V K L T M E P V T R I E G H A K I T V R13 (M) I I V N K E D C I R C G A C Q G T C P Tly (M) A E K I K I G T M W L G G C S G C H L S8 (M) A E D D I K I V M F C C N W C S Y G G A

a Nonidentical amino acid residues are shown in boldface type; ?, amino acid not identified with certainty.

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100

-

0

0-

:L.._

(C)

C:

80

60

40

20

020 30 40 50 60 70

Temp (°C)FIG. 4. Dependence of MVH I and MVH II on temperature of

incubation. MVH activities were determined in assay solutionscontaining 50mM NaCl, 5 mM DTT, 1 mM MV, and 50 mM HEPES(pH 7.5) at the indicated temperatures under anaerobic conditionswith hydrogen as the headspace gas. MV reduction was monitoredat 601 nm. The headspace gas of each assay vial contained hydro-gen, and the reaction mixture was incubated at the indicatedtemperatures in a thermocirculator for 15 min before addition ofenzyme. The specific activities of MVH I and MVH II at 70°C were133.5 and 54.7 U/mg of protein, respectively.

to distinguish them on the basis of some biological andcatalytic properties. The assays were performed with MVHI and MVH II fractions obtained from the Mono Q HR 10/10column as described in Materials and Methods because ofinsufficient quantities of the most highly purified material.The relative activity of MVH I increased as incubationtemperatures increased from 20 to 70°C (Fig. 4). MVH IIactivity, however, was stimulated greatly, as seen by a 50%increase of relative activity at temperatures above 50°C (Fig.4). Both MVH I and MVH II showed the same optimal rangeof temperature between 60 and 70°C. The enzymes werestable in the reaction mixtures over the time course of theexperiment.

Effect of pH. The pH activity curve of MVH I was notcoincident with that ofMVH II (Fig. 5). The optimum pH ofMVH II was 7.5, with dramatic loss of activity to 32.5%when the pH of the reaction buffer was adjusted with Tricinebuffer at pH 8.5. MVH I activity had a pH optimum at 9.0but showed only 17.9% of relative activity at pH 7.5. The pHof the buffer used for the purification and the assay of MVHII was adjusted to 7.5 with HEPES buffer. When theactivities ofMVH I and MVH II at pH 7.5 are compared, theactivity of MVH I is underestimated by a factor of 5.6.MVH inhibition by divalent metal ions. Divalent metal ions

have been reported to be strong inhibitors of hydrogenase (9,23, 27, 29). The results in the present study show that theeight divalent metal ions are potent partial inhibitors ofMVH I and MVH II. To study the effect of various divalentmetals on the activity of the hydrogenases, each of thefollowing metal chloride salts at a final concentration of 1mM was added to a separate standard reaction mixture inwhich the specific activity of the hydrogenase for MVreduction without addition of metal salt was 129.5 U/mg ofprotein for MVH I and 54.7 U/mg of protein forMVH II. Thespecific activity of each enzyme in the presence of the metalsalt is presented as a percentage of the control for MVH Iand MVH II, respectively: Zn2+, 46 and 96%; Hg2+, 23 and

85%; MV+, 91 and 78%; Sr", 36 and 78%; Ba2", 27 and72%; Cd +, 7 and 70%; Mn2", 9 and 56%; Ca2+, 36 and 50%.MVH II was less sensitive to the metal salts except forMg2+; MVH I was dramatically more sensitive to Cd" andMn2+. All metal ions except Zn2+ and Mg2+ showed dra-matic inhibition of MVH I activity. Partial inhibition ofhydrogenase activity by Mg2+ was observed in both MVH Iand MVH II. No dramatic or complete inhibition of MVreduction was observed in MVH II.

Stability of hydrogenase activity. Under anaerobic condi-tions MVH I and MVH II showed excellent storage stabilityat -20°C with a maximum loss of enzyme activity of only12.0 and 7.0%, respectively, over 8 days. On exposure to air(Fig. 6A), decay of MVH II activity was a second-orderreaction (y = 97.713 - 0.41947x + 0.0008752x2; r2 = 0.964),with the residual activity being 50% (27.4 U/mg of protein)after 180 min. MVH I, however, lost 90% of its activitywithin 2 min and was completely inactivated within 30 min,although the original specific activity ofMVH I (129.5 U/mgof protein) (Fig. 6B) was 2.4 times higher than that of MVHII (54.7 U/mg of protein) (Fig. 6A).

DISCUSSION

Our purpose in this communication has been to presentevidence for the presence of a second MV-reducing hydro-genase in M. thermoautotrophicum AH. By extensive testingof chromatography resins and columns, we found that thepurification steps presented here yielded the best results forpurification of MVH, which was resolved into MVH I andMVH II by the combination of DEAE-Sephacel, Superose 6PG, and Mono Q column chromatography. Two MVHactivities (one major MVH and one minor MVH) were alsoobserved in extracts of Methanobacterium formicicum (17)during the purification of MVH and FRH. However, thecharacteristics of the minor MVH compared with the majorMVH were not identified. On the basis of the specificactivity, MVH I appeared to be the major MVH in M.thermoautotrophicum AH.The strong sequence similarity between the two gene

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carried out at 60°C with the pH adjusted by the following biologicalbuffers (final concentration, 50 mM): MES (pH 6.0), PIPES (pH6.5), HEPES (pH 7.0 to 7.5), Tricine (pH 8.0 to 8.5), and CHES (pH9.0 to 9.5). The specific activity ofMVH II at pH 7.5 was 54.7 U/mgof protein, and the specific activity of MVH I at pH 9.0 was 723.5U/mg of protein.

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clusters encoding two [NiFe] hydrogenases in Methanococ-cus voltae is further evidence for the presence of two MVHsinvolved in methanogenesis (13). The presence of two meth-yl-coenzyme M reductases (MCR) designated as MCR I andMCR II in M. thennoautotrophicum AH and Marburg hasbeen reported (26). The concentration of each depends onthe age of the culture (26). In the present study we made noattempt to monitor the relative amounts of MVH I and MVHII during growth. The duplication of gene products encodingtwo proteins with the same or similar physiological andbiological functions is a common phenomenon (12). Study ofthe control of duplicate enzymes involved in methanogenesisshould be fruitful.MVH II-2 and MVH II-1 were not completely separated

by chromatography. We suggest that MVH II-2 is a degra-dation product of MVH II-1. Although the ,B subunit differsin MVH I and II and the 8 subunit is absent in MVH II-2, thea and -y subunits are conserved, indicating that they serve anessential function in MVH catalysis.Heavy metals such as mercury and copper have been

reported to inhibit hydrogenase activity to a great extent (9,27, 29). One possible explanation for this phenomenon is thatthe heavy-metal ions result in a loss of the extractable ironatoms from the Fe-S clusters of the enzyme (24). Nakos andMortenson (23) observed that treatments with 8.15- and24.4-fold excesses of sodium merasyl over the molar con-centration of Clostridium pasteurianum W5 hydrogenasecauses, respectively, 40 and 83% loss of activity. However,no inhibition of hydrogenase activity was observed when themercury of the merasyl was first complexed with 2-mercap-toethanol and then added to the assay vial. This resultimplies that mercury reacts only with the acid-labile SHand/or S2- groups and also that the interaction shouldrelease iron, resulting in the loss of hydrogenase activity. Itis probable that the strong interaction of Cd2 , Mn2 , Ba2 ,

and Hg2e (73 to 93% inhibition) occurred with the Fe-Sclusters of the a subunit ofMVH I, whereas less labile sulfurwas available in the ,B subunit of MVH II.

Hydrogenase purified from the anaerobic bacterium De-

sulfovibrio vulgaris had only 10% of its original activity afterbeing exposed to air for 13 min (33). The bidirectionalhydrogenase from C pasteurianum showed a very similarinactivation rate with MVH I, with 50% inactivation occur-ring in 5 min. A newly purified unidirectional hydrogenasefrom the same organism was less oxygen sensitive than theclassical bidirectional hydrogenase. The half-life of the uni-directional hydrogenase under aerobic conditions was ap-proximately 30 min (4), resembling the inactivation patternof MVH II. Coremans et al. (5) observed that the hydrogenuptake activity and the hydrogen evolution activity in cellextracts of M. thermoautotrophicum Marburg were 6.8 and2.2 U/mg of protein, respectively. They also reported thatthe hydrogenase isolated in air did not show any hydrogenuptake activity. This result suggests a possible inactivationof MVH I under aerobic conditions on the basis of datapresented here. Lappi et al. (20) also observed the biphasicinactivation of hydrogenase from C. pasteurianum and notedthe presence of two forms of hydrogenase with differentoxygen sensitivities. The results obtained in this study showthat MVH II is responsible for the major MVH activity afterexposure of cell extract to oxygen.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutesof Health (AI 12277) and Department of Energy (DE-FG02-87ER13651).We thank Larry Daniels for assistance in purification of the

enzymes and Joe Marretta for help with amino acid sequenceanalysis.

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