Crude vesicle preparations of the methano8enic strain Go1 were able to couple the reduction of the heterodisulfide of 2-mercaptoetbanesulfonate and 7-mercaptoheptanoylthreonine phosphate (CoM-99HTP) by H, with ATP formation. The rate of ATP synthesis was 1 mol/min mg protein. ATP synthesis and disulfide reduction were only observed with CoM-S-S-HTP, but not with CoM-4S-CoM or HTP-S-S-HTP. The methylreductase inhibitor 2-brorn~~~~ulfo~cacid had no effect on ATP synthesis induced by CoM-S-4HTP reduction with Hr. ATP synthesis was completely inhibited by the uncoupler SF 6847 whereas the con~mi~t CoM-S-S-HTP reduction was stimulated. The ATP synthase inhibitors DCCD and DES also inhibited ATP fo~ation completely and decreased the CoM-S-S-HTP reduction rate to 35% of the control. These ~bito~ effects were abolished by addition of the uncoupler. From these results it is concluded that energy couphng between the electron transfer from % to the heterodisultide and ATP synthesis occurs via a transmembrane proton gradient.

Methanogenesis from methyl-CoM and Ha, as catalyzed by inside-out vesicle preparations of the methanogenenic bacterium strain Go1, was associated with ATP synthesis. That this ATP synthesis proceeded via an uncoupler-sensitive transmembrane proton gradient was concluded from the following results :1. Various inhibitors that affected methane formation (e. g. 2-bromomethanesulfonate) also prcventcd ATP synthesis.2. The protonophore 3,5-di-tert-butyl-4-hydroxyben~ylidenemalononitrile, in combination with the K + ionophore valinomycin, inhibited ATP synthesis completely without affecting methanogenesis.3. The ATP synthase inhibitor diethylstilbestrol inhibited ATP synthesis. 4. Addition of the detergent sulfobetaine inhibited both methane formation and ATP synthesis; the former but not the latter could be restored by adding titanium(II1) citrate as electron donor.In addition it was shown that ATP synthesis could also be driven by transmembrane proton gradients artificially imposed on the vesicles. Furthermore net methanogenesis-dependent ATP formation was shown by measuring [32P]phosphate incorporation.The universal methanogenic reaction from all substrates is the reductive demethylation of methyl-CoM (CH3-S-CoM) by the methyl-coenzyme-M methylreductase system. This system catalyzes methanogenesis from methyl-CoM and molecular hydrogen [I] according to the following reaction:This reaction is highly exergonic and has always been considered as the reaction coupled with ATP synthesis. Investigations performed with whole cells of Methuna.surcirza barkepi led to the conclusion that methanogenesis from methanol plus H2 gives rise to an electrochemical proton gradient, which is subsequently used to synthesize ATP [2]. Taking advantage of the same whole cell system, it was indeed possible to nicasure directly the extrusion of 3 -4 H ' /methane formed from methanol and HZ [3]. Recently the structure of factor B, an obligatory cofactor of the methylreductase system, was unravelled [4] and thc reduction of the methylreductase system could be formulated as the sum of two reactions [5, 61: Correspondence to G. Gottschalk, Institut fur Mikrobiologic der Univcrsitlt Gottingen, Griscbachstrasse 8, D-3400 Gottingen, Federal Republic of GermanyAbbreviations. SF6847, 3,S-di-t~rt-butyl-4-hydroxybenzylidcncmalononi trile ; 3,5,4,5'-tetrachlorosalicylanilidc, 3,5-dichloro-N-(4,5-dichlorophenyl)-2-hydroxybcnzamide: (cHxN)*C, N,N'-dicyclohexylcarbodiimidc; sulfobetaine, N-tetradecyt-N./V-dimethylammonio-3-propanesulfonate; octyl glucoside; octyl-/h-glucopyranosidc; ApH', electrochemical gradient of H i ; ApH, (pHi-pH,), transmembrane chetnical gradient o f H'; CH3-S-CoM or methyl-CoM, 2-(methylthio)ethanesulfonate.Enzyme. li+-Transporling ATPasc, F,F,-ATP synthase (EC 3.6.1.34).CH,-S-CoM + HS-HTP + CH4 + HTP-S-S-COM HTP-S-S-COM + H2 + HS-HTP + HS-COM.Therefore, it can bc expected that electron transport from H2 via unknown clcctron carriers to the heterodisulfide gives rise to the proton extrusion observed.Based on the results obtained...

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient, however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+o05/Na+1n) was established.Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.Recently, it was shown that methanogenesis from H2 plus methanol, H2 plus trimethylamine, and H2 plus formaldehyde by resting cells of Methanosarcina barkeri is coupled to ATP formation by a chemiosmotic mechanism (2, 3, 19). This conclusion was based on the following findings. (i) The addition of the protonophore tetrachlorosalicylanilide (TCS) to resting cells of M. barkeri forming methane from methanol plus H2, formaldehyde plus H2, or trimethylamine plus H2 caused a dissipation of the proton motive force (Ap) and a decrease in the intracellular ATP content, whereas mnethane formation was stimulated. (ii) The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) inhibited methane formation and ATP synthesis but left Ap intact. (iii) Addition of TCS to cell suspensions incubated with DCCD restored their ability to form methane. We have extended these studies now to cells of M. barkeri grown on acetate. Important differences have been encountered in comparison to the substrate combination methanol plus H2; they are reported in this publication.It has been known for some time now that the growth of methanogenic bacteria and methane formation depend on sodium ions (20); an involvement of Na+ in the process of ATP synthesis was envisaged. However, we have shown that ATP synthesis during methanogenesis from methanol plus H2 does not depend on the presence of Na+ (4). Recently, one site of Na+ involvement was identified. Na+ is required for methyl group oxidation during dismutation of methanol to methane and carbon dioxide (4). It was of interest to study the effect of Na+ on methanogenesis from acetate. MATERIALS AND METHODSM. barkeri Fusaro (DSM 804) was grown on 100 mM sodium acetate as the sole carbon and energy source. The medium described by Hippe et al. (8) anaerobic techniques of...

Everted vesicles of the methanogenic strain Gö1 synthesized ATP in response to methanogenesis from methyl‐coenzyme M and H2. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl‐CoM and H2. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane‐bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Gö1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O2, K3[Fe(CN)6]) or SH reagents (Ag+, p‐chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl‐CoM and H2.

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