Vol. 174, No. 3JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 1007-10120021-9193/92/031007-06$02.00/0Copyright © 1992, American Society for Microbiology

Structural Modifications and Kinetic Studies of the SubstratesInvolved in the Final Step of Methane Formation in

Methanobacterium thermoautotrophicumKARL D. OLSON,t LIDIA CHMURKOWSKA-CICHOWLAS, CHRISTOPHER W. McMAHON,

AND RALPH S. WOLFE*

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

Received 20 September 1991/Accepted 18 November 1991

The 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase catalyzes the final methane-yielding reactionin fastidiously anaerobic methanogenic archaebacteria. This step involves the reductive demethylation ofCH3-S-CoM with reducing equivalents from N-7-(mercaptoheptanoyl)-L-threonine 03-phosphate (HS-HTP) toyield methane and the nonsymmetrical disulfide of 2-mercaptoethanesulfonic acid and HS-HTP. We chemicallysynthesized modified analogs of CH3-S-CoM (which has two carbons in the ethylene bridge) and of HS-HTP(which has seven carbons in the side chain); analog pairs possessed an overall correct number of side chaincarbons (i.e., a total of nine in combination). They were simultaneously added to anaerobic cell extracts ofMethanobacterium therinoautotrophicum AH. The ability of the extracts to reductively demethylate the modifiedsubstrates was tested by gas chromatography. We also describe here previously unknown inhibitors ofmethanogenesis, 6-(methylthio)hexanoyl-L-threonine 03-phosphate (a structural analog of HS-HTP) andsodium bromomethanesulfonic acid (a structural analog of CH3-S-CoM). Both analogs were found to beeffective competitive inhibitors with respect to HS-HTP. These substrate analogs were also found to inhibit a

recently described photoactivation of homogeneous inactive reductase (K. D. Olson, C. W. McMahon, andR. S. Wolfe, Proc. Natl. Acad. Sci. USA 88:4099-4103, 1991). In addition, we probed the mechanism of actionof a potent inhibitor of the enzyme, 2-bromoethanesulfonic acid, a structural analog of CH3-S-CoM.

The final step in methane formation in methanogenicarchaebacteria (19) is the reductive cleavage of the terminalcarbon-sulfur bond of 2-(methylthio)ethanesulfonic acid(CH3-S-CoM) by the enzyme CH3-S-CoM reductase. Theelectron donor for this reaction is N-7-(mercaptoheptanoyl)-L-threonine 03-phosphate (HS-HTP). The balanced equationis as follows: CH3-S-CoM + HS-HTP -* CH4( T) + CoM-S-S-HTP (disulfide of 2-mercaptoethanesulfonic acid [HS-CoM] and HS-HTP). Two moles each of a nickel-containingtetrapyrrole (referred to as coenzyme F430) and HS-CoM aswell as an unknown number of moles of HS-HTP are boundper mol of CH3-S-CoM reductase (3, 10, 12). The role ofF430 in this reaction has not yet been elucidated. One way tobetter understand this complex reaction is through substratemodifications. Several chemical analogs of the substrateshave been tested. Replacement of the methyl moiety ofCH3-S-CoM by an ethyl group yielded an analog whichserved as a precursor for ethane biosynthesis (8). CH3-S-CoM reductase was also found to catalyze the reductivecleavage of CF2H-S-CoM to yield difluoromethane as aproduct (18). In addition, 2-(methylseleno)ethanesulfonicacid was a substrate for the reductase reaction (18). Someother substrate analogs which have been found to be inhib-itors of the final step include allyl-S-CoM, cyano-S-CoM,and CF3-S-CoM (18), CH3CH2CH2-S-CoM, 3-(methylthio)propanesulfonic acid, 2-bromoethanesulfonic acid (BES) (8),and 3-bromopropanesulfonic acid (6).

Since the structure of HS-HTP was elucidated only re-cently, few analogs have been tested. N-6-(Mercaptohex-

* Corresponding author.t Present address: Department of Microbiology and Molecular

Genetics, The University of Texas Medical School, Houston, TX77030.

anoyl)-L-threonine 03-phosphate (apparent K,, 0.1 ,uM) andN-8-(mercaptooctanoyl)-L-threonine 03-phosphate (appar-ent K,, 15 puM) were shown by Ellermann and coworkers tobe inhibitors of an active preparation of homogeneous re-ductase (4). In addition, the N-methylated analog of HS-HTP, N-7-(methylthio)heptanoyl-L-threonine 03-phosphate,was found to be a competitive inhibitor of the reductasereaction (5, 13). These data indicate that modified substratescan bind to the active site and that CH3-S-CoM reductase ishighly specific with regard to its tolerance of modifications ofits substrates. The enzyme is particularly specific withregard to a change in the number of methylene carbons in thesubstrates. CH3-S-CoM has two methylene carbons, andHS-HTP has seven carbons in the side chain. There aretherefore a combined total of nine carbons in the carbonchains of both coenzymes (see Fig. la). It seemed conceiv-able that the enzyme might accept a change in the number ofmethylene carbons in both substrates simultaneously whilemaintaining a total of nine carbons (see Fig. lb and c).Another approach to gain insights into the reductase

reaction involves understanding the mechanism of action ofinhibitors. BES, a structural analog of CH3-S-CoM, isknown to be a potent inhibitor of methanogenesis, causing50% inhibition at a 7.9 ,uM concentration (8). The mecha-nism of inhibition has not yet been elucidated, however. Apossible mechanism of BES inhibition is the chemical reac-tion with either HS-HTP or HS-CoM to form a thioether(i.e., CoM-S-HTP or CoM-S-CoM, respectively). As previ-ously noted, both HS-HTP and HS-CoM are known to bebound to CH3-S-CoM reductase.

In previous studies of the CH3-S-CoM reductase reaction,several different enzyme preparation types were used for themethane assay. (i) Gunsalus et al. (8) used a crude cellextract prepared by breakage of the cells and centrifugation

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1008 OLSON ET AL.

of the cell extract at 33,000 x g for 30 min. The supernatantsolution was used in the enzyme assay. (ii) Wackett et al.(18) used the supernatant of a cell extract prepared bycentrifugation of the cell extract for 1 h at 35,000 x g. (iii)Ellermann et al. (4) used highly purified CH3-S-CoM reduc-tase preparations. For many of the experiments described inthis communication, we have chosen to use Sephadex G-25-treated extract preparations. A Sephadex G-25-treated ex-tract preparation is defined as a cell extract depleted ofsoluble low-molecular-weight cofactors by passage througha Sephadex G-25 superfine-resin gel filtration column (seeMaterials and Methods). Photoactivation experiments, how-ever, were performed with highly homogeneous inactivereductase preparations as described in Materials and Meth-ods.

MATERIALS AND METHODS

Analytical methods. Reverse-phase high-pressure liquidchromatography (HPLC) (RP-HPLC) was performed with aWaters HPLC system. Eluates were monitored with aVarian 2050 variable-wavelength detector. The thiol concen-tration was determined with Ellman's reagent (7), and thephosphate concentration was determined as described pre-viously (1). Methane was measured by gas chromatographyas previously described (16). 'H nuclear magnetic resonancespectroscopy was performed on an XL 200-MHz, Nicolet360-MHz, or GN 500-MHz spectrometer. Mass spectra wereobtained with a ZAB-SE 10-kV mass spectrometer.

Light source. The lamp and power supply (Research Arcmodel 66024 lamp) were from Oriel, Stratford, Conn. Filterswere purchased from Oriel. The light source was a 1,000-WHg-Xe bulb (977BOO10; Hanovia, Newark, N.J.). Each sam-ple was placed approximately 25 cm from the bulb.

Preparation of analogs. We synthesized structural analogsof each substrate, the methyl group being added to either theHS-HTP or the CH3-S-CoM analog. Figure 1 shows thecombinations of structural analogs tested.

N-6-(Mercaptohexanoyl)-L-threonine 03-phosphate (4, 14),N-8-(mercaptooctanoyl)-L-threonine 03-phosphate (4, 14),ammonium 3-(mercaptopropane)sulfonate (8), ammonium3-(methylthio)propanesulfonate (8), and CoM-S-CoM (8)were synthesized and purified as previously described.

N-6-(Methylthio)hexanoyl-L-threonine 03-phosphate wassynthesized by alkylation of the N-6 analog (the reducedspecies) with CH3I. The N-6 analog was resuspended in 2 mlof concentrated NH40H, and 20 ,u (an excess) of CH3I wasadded. The solution was stirred overnight at an ambienttemperature and lyophilized, and the methylated productwas purified by RP-HPLC (Waters C18 ,uBondapak column;7.8 mm by 30 cm) (14). The conditions were as follows: a10-min isocratic application of 20 mM NH4 acetate (pH 5.0)and then a 20-min linear gradient to 20 mM NH4 acetate (pH5.0) in 80% methanol; the flow rate of 2 ml/min was moni-tored at 215 nm. The product eluted at a retention time (QR)of 19.8 min. The product was confirmed by high-resolutionfast atom bombardment (FAB) positive-ion mass spectros-copy (MS) (matrix, "magic bullet"; m/z = 344.09380). The Amillimers unit was -0.5. The composition was determined tobe C11H23N07SP. The symmetrical disulfide of N-6-(mer-captohexanoyl)-L-threonine 03-phosphate, which could bereduced in cell extracts by a thiol-disulfide interchange andcould therefore interfere with the assay, eluted at tR = 23.7min (confirmed by MS). The final methylated product testednegative for thiol and was therefore also free of N-6-(mercaptohexanoyl)-L-threonine 03-phosphate.

(a) 03S s -CH3 H - S R0

0(b) 003S ./ S-H H3C-SN . R

0(C) "03S N S-CH3 HS-VS R

(d)

2-7

3-6

3-6

0H - S R R

0(e) H3C-S R

H H 0o2e

02C H CH3FIG. 1. Chemical structures of some of the substrate analogs

tested. The numbers shown to the right of a, b, and c indicate thenumbers of carbon atoms in the (side) chains of CH3-S-CoM (oranalogs) and HS-HTP (or analogs), respectively. (a) Native sub-strates CH3-S-CoM and HS-HTP. (b) Structural modifications ofCH3-S-CoM and HS-HTP: 3-mercaptopropanesulfonic acid andN-6-(methylthio)hexanoyl-L-threonine 03-phosphate, respectively.(c) 3-(Methylthio)propanesulfonic acid and N-6-(mercaptohex-anoyl)-L-threonine 03-phosphate. The analogs shown in b and cwere added simultaneously to a Sephadex G-25-treated enzymeextract (cell extract depleted of low-molecular-weight cofactors) asdescribed in the text. (d) Chemical structure of N-8-(mercaptooc-tanoyl)-L-threonine 03-phosphate. (e) Chemical structure of N-8-(methylthio)octanoyl-L-threonine 03-phosphate. Each HS-HTP an-alog was the L-enantiomer of phosphothreonine.

N-8-(Methylthio)octanoyl-L-threonine 03-phosphate wassynthesized by alkylation of the N-8 analog (the reducedspecies) in a fashion analogous to that for the methylated N-6analog. The product, which eluted at tR = 21.0 min, waspurified by RP-HPLC as described above. A peak at m/z =372.1 was observed by FAB positive-ion MS (matrix, glyc-erol). The symmetrical disulfide of N-8-(mercaptooctanoyl)-L-threonine 03-phosphate eluted at tR = 30.6 min (confirmedby MS). Our product was therefore free of the N-8 symmet-rical disulfide, which could also be reduced in cell extractsby a thiol-disulfide interchange. The final product also testednegative for free thiol.CoM-S-HTP (the thioether analog of CoM-S-S-HTP) was

synthesized from BES and HS-HTP. HS-HTP (25 ,umol) wasadded anaerobically to 80 ,umol of BES in anoxic H20 (625,ul). The pH was adjusted to 9.0 with NaOH. The mixturewas slowly stirred for 3 days at room temperature. Theproduct was purified by injection into a Waters Cl8 ,uBon-dapak RP-HPLC column (3.9 mm by 30 cm). Application ofa linear gradient (2) eluted the thioether product at tR = 17min (HS-HTP eluted at tR = 22 min). The structure and thepurity of the sodium salt were confirmed by high-resolutionpositive-ion FAB MS (M + 1; observed mass, 474.0640;elemental composition, C13H26010PS2NaN; A millimersunit, -0.7). 'H nuclear magnetic resonance spectrum (360MHz, D20); b/ppm (relative to TSP, 0.0 ppm) = 1.31 (3H);1.37 (2H); 1.44 (2H); 1.64 (2H); 1.70 (2H); 2.35 (2H); 2.78(2H); 2.88 (2H); 3.18 (2H); 4.24 (1H); 4.69 (1H).

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KINETIC STUDIES OF CH3-S-CoM REDUCTASE 1009

c

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I I63 125 188[Br-CH2-SO3] (ILM)

[Br-CH2-SO:j (IM)

FIG. 2. CH3-S-CoM reductase inhibition by sodium bromomethanesulfonic acid (Br-CH2-SO3-). Shown are Lineweaver-Burk plots forthe CH3-S-CoM reductase reaction determined at several concentrations of the substrates CH3-S-CoM (top left) and HS-HTP (bottom left).In addition to the assay components listed in Materials and Methods, each cuvette contained 4.4mM HS-HTP (or the concentration indicated)and 10 mM CH3-S-CoM (or the concentration indicated). The concentration of Br-CH2-SO3- used in each assay is indicated. Br-CH2-SO3-was found to be noncompetitive with respect to CH3-S-CoM and competitive with respect to HS-HTP. The slopes of the lines of thedouble-reciprocal plots versus the inhibitor concentration are shown next to the respective substrate type: CH3-S-CoM (upper right) andHS-HTP (lower right).

Sodium bromomethanesulfonic acid (BrCH2SO3-Na')was synthesized as follows. A mixture of dibromomethane(21 g, 0.1208 mol), sodium sulfite (15.22 g, 0.1208 mol), anda transfer catalyst, tetrabutylammonium hydrogen sulfate(0.6 g, 0.18 mmol), was refluxed for 26 h (with stirring) in 48ml of H20. The solution was flash evaporated under vac-uum. Ethanol (108 ml) and H20 (19 ml) were added to theresidue. The mixture was heated and filtered through aBuchner funnel. The filtrate was cooled to -20°C, and thecrystals were collected by filtration. The yield of sodiumbromometharesulfonate was 17 g (72.0%). The product wasrecrystallized two times with ethanol (108 ml)-water (19 ml).Analytically calculated: C, 6.09; H, 1.02; S, 16.25; Br, 40.60.Found: C, 6.09; H, 1.06; S, 16.25; Br, 40.59. The meltingpoint was determined to be 285 to 290°C. Infrared spectrum(KBr): v = 2,953 cm-' (C-H, medium), 1,219 cm-' (S-O,strong), 1,060 cm-' (S=O, strong). Two peaks at m/z = 173and 175 (due to bromine isotopes) were found by negative-ion MS (M-; matrix, glycerol). The high-resolution positive-ion FAB MS data were as follows: m/z = 218.8703(M-Na2+); A millimers unit = 0.0; composition, CH2BrSO3

Na2. 'H nuclear magnetic resonance spectrum (500 MHz,D20): b/ppm (relative to HDO, 4.7 ppm) = 4.41 (s, 2H, 1-H).

Methanogenesis assay. Cells of M. thermoautotrophicumAH (ATCC 29096) were cultivated to an A660 of about 3.0and harvested anaerobically as previously described (11).Cells were resuspended 1:1 in 50 mM KPi (pH 7.0) contain-ing 10mM ,-mercaptoethanol. The cell extract was preparedas previously described (9). The Sephadex G-25-treatedextract preparation consisted of a cell extract which wasdepleted of soluble low-molecular-weight cofactors by pas-sage through a Sephadex G-25 superfine-resin gel filtrationcolumn (2.5 by 80 cm). The buffer consisted of 50 mM KPi(pH 7.0), 10 mM P-mercaptoethanol, and 1 mM dithiothreitol(DTT). The Sephadex G-25-treated extracts were concen-trated to approximately 70 mg ml-' by ultrafiltration. Allpreparations were stored at -20°C under N2 at 300 kPa.Each methane assay was performed in a calibrated vial aspreviously described (17). Each reaction mixture consistedof 1.5 mM ATP, 1.5 mM MgCl2, -5 mM DTT, 17 ,uMcyanocobalamin, 15 mM KP; (pH 7.0), and 2.2 mg of protein.The amount of protein used in each kinetics assay was within

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the range in which activity varied with protein concentrationin a linear fashion (18). Unless indicated otherwise, theconcentrations of CH3-S-CoM and HS-HTP were 10 and 4.4mM, respectively; these concentrations were saturating butnot inhibitory. All rates were determined from the maximalrate of methanogenesis after the lag as measured by gaschromatography. The lag was defined as the length of timebefore a maximum linear rate of methanogenesis wasachieved.

Purification of CH3-S-CoM reductase. The reductase fromM. thermoautotrophicum AH was purified aerobically aspreviously described (14). The solution containing the reduc-tase (in 20 mM KPi [pH 7.0]) was made anaerobic by flushingunder H2 for 30 min. The reductase stock was stored at 5°C.CH3-S-CoM reductase was determined to be MCR-I (15).

RESULTS AND DISCUSSION

Effect of thioethers. The thioethers (CoM-S-HTP andCoM-S-CoM) were not inhibitory at the highest concentra-tions tested: CoM-S-HTP, 2 mM; CoM-S-CoM, 5 mM (eachvial contained 1.25 mM CH3-S-CoM and 2 mM HS-HTP inaddition to other assay components). However, there was a5-min lag in the assay vial containing CoM-S-CoM. Weattempted to isolate the thioether product of CoM-S-HTP byadding BES (1 ,umol) and HS-HTP (1 ,umol) to a completeenzyme reaction mixture (at 60°C). After 10 min of incuba-tion under an H2 atmosphere, the protein was precipitatedby the addition of HCl04 (15 ,u of 70% HC104 per 200 ,ul ofreaction mixture). The supernatant was applied to the RP-HPLC system described previously (2). No CoM-S-HTPcould be detected. These data suggest that BES does notchemically react with enzyme-bound HS-CoM or HS-HTPbut inhibits methanogenesis by some other mechanism.

Inhibition by sodium bromomethanesulfonic acid. Eller-mann and coworkers previously reported that bromo-methanesulfonate was not inhibitory (in the micromolarconcentration range) in an assay of purified CH3-S-CoMreductase (6). In our Sephadex G-25-treated enzyme assay,however, inhibition was observed. Interestingly, the modeof inhibition was determined to be noncompetitive withrespect to CH3-S-CoM and competitive with respect toHS-HTP (Fig. 2). An apparent K, of 1.5 puM was determinedfrom Lineweaver-Burk plots at several inhibitor concentra-tions. The only other analog of CH3-S-CoM known to exhibitthis type of inhibition is 2-(methoxy)ethanesulfonic acid, asdescribed by Wackett et al. (18). The function of HS-HTPhad not yet been determined at that time, so the inhibitiontype (if any) of 2-(methoxy)ethanesulfonic acid with respectto HS-HTP is not known. Since sodium bromomethanesulfo-nic acid and 2-(methoxy)ethanesulfonic acid are structurallymore similar to CH3-S-CoM than to HS-HTP, they may beexpected to be competitive with respect to CH3-S-CoM.

Recently, we reported that an inactive CH3-S-CoM reduc-tase could be activated by light (14). The photoactivationwas inhibited by known inhibitors of methanogenesis, in-cluding N-6-(mercaptohexanoyl)-L-threonine 03-phosphate,N-8-(mercaptooctanoyl)-L-threonine 03-phosphate, BES,and sodium dithionite. We wanted to know whether thephotoactivation of the reductase could be inhibited by so-dium bromomethanesulfonic acid. The addition of bro-momethanesulfonic acid (1 mM) to an otherwise completereaction mixture completely inhibited methanogenesis viaphotoactivation (Fig. 3A; see the legend for reagent concen-trations).

Simultaneous addition of modified substrates. The first

80

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2 30

20

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Time (h)

80

0E 60

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FIG. 3. Inhibition of the photoactivation of homogeneous CH3-S-CoM reductase by sodium bromomethanesulfonic acid (A) andN-6-(methylthio)hexanoyl-L-threonine 03-phosphate (B). (A) Eachcuvette contained 37 ,uM HS-HTP, 370 ,uM DTT, 8 mM Ti(III)citrate, 1.4 mM CH3-S-CoM, and 12 mg of protein (in 20 mM KPi[pH 7.0]). Only the cuvette labeled + contained sodium bro-momethanesulfonic acid (1 mM) during photoactivation. (B) Eachcuvette contained 121 ,uM HS-HTP, 1.2 mM DTT, 8 mM Ti(III)citrate, 1.4 mM CH3-S-CoM, and 12 mg of protein (in 20 mM KPi[pH 7.0]). Only the cuvette labeled + contained N-6-(methylthio)-hexanoyl-L-threonine 03-phosphate 343 p.M during photoactivation.A cutoff filter eliminated light with wavelengths shorter than 385 nm.

combination of substrate analogs tested was CH3-S-CH2CH2CH2-SO3- [3-(methylthio)propanesulfonic acid] and N-6-(mercaptohexanoyl)-L-threonine 03-phosphate (Fig. 1). Eachreaction vial contained the concentrations of ATP, MgCl2,DTT, cyanocobalamin, Sephadex G-25-treated protein, andKPi listed in Materials and Methods. No CH3-S-CoM orHS-HTP was added. The highest concentrations of theanalogs tested simultaneously were 5 and 0.75 mM, respec-tively. Upon incubation at 60°C under an H2 atmosphere, nomethane could be detected by gas chromatography.We then tested HS-CH2CH2CH2-SO3- (3-mercaptopro-

panesulfonic acid) and N-6-(methylthio)hexanoyl-L-threo-nine 03-phosphate (Fig. 1). Each reaction vial contained theconcentrations of cyanocobalamin, ATP, MgCl2, protein,and KPi listed in Materials and Methods. CH3-S-CoM orHS-HTP was not added. The highest concentrations ofanalogs tested simultaneously were 3 and 5 mM, respec-tively. Once again, no methane could be detected by gaschromatography. We did, however, observe the inhibition ofmethanogenesis upon the addition of the methylated N-6analog to the Sephadex G-25-treated enzyme preparation.

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KINETIC STUDIES OF CH3-S-CoM REDUCTASE 1011

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FIG. 4. CH3-S-CoM reductase inhibition by 6-(methylthio)hexanoyl-L-threonine 03-phosphate (CH3-N-6). Shown are Lineweaver-Burkplots for the CH3-S-CoM reductase reaction determined at several concentrations of the substrates CH3-S-CoM (top left) and HS-HTP(bottom left). The analog was found to be competitive with respect to HS-HTP and noncompetitive with respect to CH3-S-CoM. An apparentKi of 8.9 ,uM was determined. The slopes of the lines of the double-reciprocal plots versus the inhibitor concentration are shown next tothe respective substrate type: CH3-S-CoM (upper right) and HS-HTP (lower right). In addition to the assay components listed in Materialsand Methods, each cuvette contained 4.4 mM HS-HTP (or the concentration indicated) and 10 mM CH3-S-CoM (or the concentrationindicated).

The methylated N-6 analog was competitive with respect toHS-HTP, with an apparent Ki of 8.9 ,uM (Fig. 4; see thelegend for details). The methylated N-6 analog was found tobe noncompetitive with respect to CH3-S-CoM (Fig. 4).Whereas the methylated N-6 analog was an effective inhib-itor of the reductase reaction, the methylated N-8 analog wasnot (within the concentration ranges tested). The highestconcentration of N-8-(methylthio)octanoyl-L-threonine 03-phosphate tested (1 mM) was not inhibitory.The methylated N-6 analog was also an inhibitor of the

photoactivated enzyme. The addition of 343 ,uM methylatedN-6 analog inhibited an otherwise complete reaction mixturedramatically (Fig. 3B).Of all the reactions leading to the biosynthesis of methane

in methanogens, the terminal reductive step is probably the

most complex, as reflected in the fact that there are threenovel coenzymes involved in this step alone. To betterunderstand this chemically complex and unique reaction, wehave synthesized structurally modified analogs of HS-HTPand CH3-S-CoM. It has been suggested that HS-HTP acts asan "arm" to donate its reducing equivalents in the enzyme(2). We tested this possibility by synthesizing structuralanalogs of both substrates with an overall correct number ofmethylene carbons. They were added simultaneously to acell extract to determine whether they could be processed bythe enzyme, with unsuccessful results. One analog of HS-HTP, 6-(methylthio)hexanoyl-L-threonine 03-phosphate,however, was found to be an effective competitive inhibitorof the reductase reaction (apparent Ki, 8.9 ,uM). The meth-ylated N-8 analog (1 mM) was found not to be an inhibitor.

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Under the conditions described here, bromomethanesulfonicacid was found to be an inhibitor of the reductase reaction(apparent Ki, 1.5 ,uM).

In addition, we have presented evidence that a potentinhibitor of the reductase reaction, BES, may not react withenzyme-bound HS-CoM or HS-HTP. The exact mechanismof BES inhibition has yet to be determined.

Finally, we have shown that the methylated N-6 analog ofHS-HTP and bromomethanesulfonic acid are inhibitors of arecently described photoactivation of aerobically purifiedCH3-S-CoM reductase. These data are consistent with theenzyme inhibition studies described here and are intended toadd to the data base to further the understanding of themechanism of methane formation and substrate processingin methanogenic archaebacteria.

ACKNOWLEDGMENTS

We thank Robert Coates and Thomas A. Bobik for helpfuldiscussions and Gary Schuster and Ingrid Wofle for the generoususe of their lamp. We thank Richard Milberg and associates for themass spectral analysis and Victor Gabriel for helpful technicalassistance.

This work was supported by National Science Foundation grantDMB 86-13679, National Institutes of Health grant Al 12277, andDepartment of Energy grant DE-FG02-87ER13651.

REFERENCES1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in

the neutralization of bacteriophage deoxyribonucleic acid. J.Biol. Chem. 235:769-775.

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. EHlefson, W. L., W. B. Whitman, and R. S. Wolfe. 1982.Nickel-containing factor F430: chromophore of the methylre-ductase of Methanobacterium. Proc. Natl. Acad. Sci. USA79:3707-3710.

4. Eilermann, J., R. Hedderich, R. B6cher, 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.

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