On the role of <i>N</i>‐7‐mercaptoheptanoyl‐<i>O</i>‐phospho‐L‐threonine (component B) in the enzymatic reduction of methyl‐coenzyme M to methane

Ellermann, Joachim; Kobelt, André; Pfaltz, Andreas; Thauer, Rudolf K.

doi:10.1016/0014-5793(87)80846-3

Cited by 41 publications

(27 citation statements)

References 25 publications

(44 reference statements)

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“…One unit of activity was defined as 1 mol electrons transported per min. CoM-S-S-CoB (synthesized as described previously [9,23] Alternatively, H 2 :heterodisulfide oxidoreductase activity was determined by replacing CO, CO dehydrogenase/acetyl-CoA synthase, and ferredoxin with H 2 . In this analysis, only 50 g membrane preparation was used, and 1 U of activity was defined as the reduction of 1 mol CoM-S-S-CoB per min.…”

Section: Methodsmentioning

confidence: 99%

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

et al. 2010

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Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei ⌬ech mutant which shows decreased ferredoxin-dependent membranebound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C 1 compounds.The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO 2 and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C 1 compounds (methanol and methylamines), onequarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO 2 (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fd red ) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fd red :CoM-S-S-CoB oxidoreductase system (7, 24). The direct membranebound electron acceptor for Fd red is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H 2 -evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H 2 :CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20,21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fd red (8,10,17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).To investigate whether the Ech hydrogenase is the only means by which M. mazei channels electrons from Fd red into the respiratory chain, a mutant lacking the Ech hydrogenase (M. mazei ⌬ech mutant) was constructed. Electron transport experiments using Fd red as the el...

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Section: Methodsmentioning

confidence: 99%

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

et al. 2010

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“…CH3-S-EITP was synthesized as in [35]. Br-HTP was formed from 7-bromoheptanoic acid, which was directly activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide and then coupled with 0-phospho-DL-threonine.…”

Section: Synthesis Of Ch3-s-htp and Of Br-htpmentioning

confidence: 99%

The final step in methane formation

Ellermann¹,

Hedderich²,

Böcher³

et al. 1988

European Journal of Biochemistry

242

145

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Methyl‐coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3‐S‐CoM) with N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP = component B) to CH4. The mixed disulfide of H‐S‐CoM and H‐S‐HTP (CoM‐S‐S‐HTP) was the other major product formed. The specific activity was up to 75 nmol min−1 mg protein−1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3‐S‐CoM reduction to CH4 with H‐S‐HTP increased to 0.5–2 μmol min−1 mg protein−1. Under these conditions the CoM‐S‐S‐HTP formed from CH3‐S‐CoM and H‐S‐HTP was completely reduced to H‐S‐CoM and H‐S‐HTP. Methyl‐CoM reductase was specific for H‐S‐HTP as electron donor. Neither N‐6‐mercaptohexanoylthreonine phosphate (H‐S‐HxoTP) nor N‐8‐mercaptooctanoylthreonine phosphate (H‐S‐OcoTP) nor any other thiol compound could substitute for H‐S‐HTP. On the contrary, H‐S‐HxoTP (apparent Ki=0.1 μM) and H‐S‐OcoTP (apparent Ki= 15 μM) were found to be effectives inhibitors of methyl‐CoM reductase, inhibition being non‐competitive with CH3‐S‐CoM and competitive with H‐S‐HTP.

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“…Approximately 1 billion tons of methane are produced by these anaerobic microorganisms per year (1), which generates significant amounts of a valuable fuel but poses a potential environmental problem because methane is a potent greenhouse gas. Methyl-coenzyme M reductase (MCR) 3 catalyzes the final step in biological methane formation: the reaction of methyl-coenzyme M (methyl-SCoM) with N-7-mercaptoheptanoyl-threonine phosphate (HSCoB) to generate methane and the mixed disulfide CoBS-SCoM (Reaction 1) (1,2). HSCoB serves as the two-electron donor (3).…”

mentioning

confidence: 99%

Spectroscopic and Kinetic Studies of the Reaction of Bromopropanesulfonate with Methyl-coenzyme M Reductase

Kunz

Horng

Ragsdale

2006

Journal of Biological Chemistry

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On the role of N‐7‐mercaptoheptanoyl‐O‐phospho‐L‐threonine (component B) in the enzymatic reduction of methyl‐coenzyme M to methane

Cited by 41 publications

References 25 publications

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

The final step in methane formation

Spectroscopic and Kinetic Studies of the Reaction of Bromopropanesulfonate with Methyl-coenzyme M Reductase

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