ATP synthesis coupled to methane formation from methyl‐CoM and H₂ catalyzed by vesicles of the methanogenic bacterial strain Gö1

Abstract

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...

Electron transport phosphorylation driven by glyoxylate respiration with hydrogen as electron donor in membrane vesicles of a glyoxylate-fermenting bacterium

“…With the exception of TCS at concentrations higher than 10 nmol (mg protein) -1, glyoxylate reduction was not affected by these protonophores. Interference of TCS with targets other than the proton-motive force has only recently been reported for luciferase (Peinemann et al 1989). We conclude that ATP was formed from ADP and Pi by a proton-translocating ATPase, probably of the F1F0-type; this reaction was driven by a proton-motive force generated by the electron transport from glyoxylate to glycolate with H2 as electron donor.…”

ATP synthesis coupled to electron transfer from H2 to the heterodisulfide of 2‐mercaptoethanesulfonate and 7‐mercaptoheptanoylthreonine phosphate in vesicle preparations of the methanogenic bacterium strain Gö1

“…Addition of the uncoupler SF 6847 abolished the inhibitory effect of DCCD on CoM-S-S-HTP reduction in~cating a stringent coupling between the electron transport from HZ to CoM-S-S-HTP and ATP synthesis. Such a stringent coupling was not observed with methyl-CoM as electron acceptor [6].…”

“…2-Bromoethanesulfonic acid inhibits methanogenesis and ATP formation in the latter system completely [6). CoM-S-S-HTP reduction (Fig.…”

“…Vesicles of strain Go1 were prepared from protoplasts as described previously [6]. The vesicle buffer had the same composition (20 mM potassium phosphate, 20 mM MgS04, 0.4 M sucrose, 1 mg/l resazurin, pH 6.8) except it was not reduced with 10 mM dithioerythritol but by stepwise addition of a few ~1 of 100 mM Ti(II1) citrate [8] until the resazurin turned colorless.…”

“…This was possible by taking advantage of a crude vesicle preparation of the methanogenic bacterium strain Gii I, which consists to 90% of vesicles with an inside-out orientation [5]. These vesicles are able to synthesize ATP in response to methanogenesis from Hz/methyl-CoM [6]. Here we report on a CoM-S-SHTP reduction-dependent ATP synthesis by this crude vesicle preparation.…”