Evidence for the involvement and role of a corrinoid enzyme in methane formation from acetate in Methanosarcina barkeri

Methane formation from acetate in cell suspensions of Methanosarcina barkeri was inhibited by low concentrations (5 M) of propyl iodide. Inhibition was abolished by short exposure of the suspension to light which strongly indicates that a corrinoid enzyme is involved in methanogenesis from acetate. Propyl iodide (5M) had no effect on the exchange reaction between the carboxyl group of acetate and ¹⁴CO₂, and on methane formation from methanol, from H₂ and methanol, or from H₂ and CO₂. These findings indicate that the proposed corrinoid enzyme has a role in methyl group transfer to coenzyme M after C-C cleavage of acetate.Dedicated to Professor N. Pfennig on the occasion of his 60th birthday     

Coenzyme M derivatives and their effects on methane formation from carbon dioxide and methanol by cell extracts of Methanosarcina barkeri.

Extracts of Methanosarcina barkeri reduced methanol and CO2 to CH4 in the presence of H2 and converted methanol stoichiometrically into CH4 and CO2 in the absence of H2. In dialyzed cell-free extracts these reactions were stimulated by 2-mercaptoethanesulfonic acid (coenzyme M) and some derivatives (acetyl and formylcoenzyme M and the oxidized form of coenzyme M), which could be converted to coenzyme M by enzyme systems present in the extracts. Methylcoenzyme M could not be used in these systems.     

Acetate catabolism by Methanosarcina barkeri: Hydrogen-dependent methane production from acetate by a soluble cell protein fraction

Abstract Cell extracts prepared from Methanosarcina barkeri converted acetate into methane and carbon dioxide under a hydrogen atmosphere. Methanogenesis by cell extracts required acetate and ATP and, the in vitro rate was 5 to 10% of the rate of methanogenesis observed during exponential growth of cells on acetate. Methane and carbon dioxide produced by cell extracts originated predominantly from the methyl and carboxyl groups of acetate, respectively, in a manner consistent with that observed in whole cells. Acetate degradation activity was detected in the soluble (150000 × g supernatant) fraction and not in the membrane fraction. These results are discussed in relation to a proposed model for ATP generation from acetate that involves both membrane-bound and soluble enzymatic components such as CO dehydrogenase.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

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+ out/Na+ in) 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.     

Hydrogen metabolism during methanogenesis from acetate by Methanosarcina barkeri

A tritium exchange assay and a sensitive gas chromatographic technique were used to demonstrate that hydrogenase was active and that hydrogen was produced by Methanosarcina barkeri strain MS grown on acetate. Both methane and hydrogen production rates were dependent on the concentration of acetate in the medium. H₂ was produced at 0.5–2% of the rate of CH₄ formation. Chloroform and potassium cyanide, inhibitors of methanogenesis from acetate, inhibited H₂ production but not hydrogenase activity. The addition of hydrogen gas to cell suspensions did not inhibit CH₄ or carbon dioxide production from the methyl group of acetate. H₂ production appears to be linked to several intracellular redox processes which follow the cleavage of acetate.     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.     

Structure of a methanofuran derivative found in cell extracts of Methanosarcina barkeri

Cell extracts prepared from cells of Methanosarcina barkeri grown on hydrogen and carbon dioxide, acetate, or methanol contain a coenzyme structurally related to methanofuran. This modified coenzyme was highly purified and its structure assigned as 4-[N-(gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamyl-gamma-L-glutamy l)-p- (beta-amino-ethyl)phenoxymethyl]-2-(aminomethyl)furan. The key structural evidence was obtained by high-resolution fast atom bombardment-mass spectrometry and 1H NMR spectroscopy. Quantitative analysis of the hydrolytic fragments of the coenzyme supported the assigned structure. We propose that this coenzyme be called methanofuran-b.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Formation of factor 390 by cell extracts of Methanosarcina barkeri.

Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Involvement of a corrinoid enzyme in methanogenesis from acetate in Methanosarcina barkeri

Abstract Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H₂. Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate.
An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Effect of trimethylamine on acetate utilization by Methanosarcina barkeri

During growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate, methane production and acetate consumption were biphasic. In the first phase trimethylamine (33 mmol x l^-1) was depleted and some acetate (11–14 from 50 mmol x l^-1) was metabolized simultaneously. In the second phase the remaining acetate was cleaved stoichiometrically into CH₄ and CO₂. Kinetic experiments with (2-¹⁴C)acetate revealed that only 2.5% of the methane produced in the first phase originated from acetate: 18% of the acetate metabolized was cleaved into CH₄ and CO₂, 23% of the acetate was oxidized, and 55% was assimilated. Methane produced from CD₃–COOH in the first phase consisted of CD₂H₂ and CD₃H in a ratio of 1:1.     

Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro)

The 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing and methyl-viologen-reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64-fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 mumol coenzyme F420 reduced.min-1.mg protein-1 and the yield was 4.8% of the initial deazaflavin-reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (alpha), 33 kDa (beta) and 30 kDa (gamma). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2-mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 microM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s-1 and 9226 s-1.     

Methanogenic cleavage of acetate by lysates of Methanosarcina barkeri.

Cell lysates of acetate-grown Methanosarcina barkeri 227 were found to cleave acetate to CH4 and CO2. The aceticlastic reaction was identified by using radioactive methyl-labeled acetate. Cell lysates decarboxylated acetate in a nitrogen atmosphere, conserving the methyl group in methane. The rate of methanogenesis from acetate in the cell lysates was comparable to that observed with whole cells. Aceticlastic activity was found in the particulate fraction seperate from methylcoenzyme M methylreductase activity, which occurs in the soluble fraction. Pronase treatment eliminated methylcoenzyme M methylreductase activity in lysates and stimulated aceticlastic activity, indicating the aceticlastic activity was not derived from unbroken cells, which are unaffected by proteolytic treatment.     

Protonmotive force-driven synthesis of ATP during methane formation from molecular hydrogen and formaldehyde or carbon dioxide in Methanosarcina barkeri

Abstract Methane formation from formaldehyde and H₂ or from carbon dioxide and H₂, as performed by cell suspensions of Methanosarcina barkeri , was coupled to ATP synthesis. In correspondence with this, methane formation was inhibited by N , N '-dicyclohexylcarbodiimide (DCCD), which at the same time, caused a decrease of the intracellular ATP concentration but only a slow decrease of the membrane potential. Addition of the uncoupler tetrachlorosalicylanilide (TCS) led to a relief of the inhibition of methane formation from CH₂O + H₂, but not from CO₂+ H₂.     

Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri.

Corrinoid proteins have been implicated as methyl carriers in methane formation from acetate, yet specific corrinoid proteins methylated by acetate-derived intermediates have not been identified. In the presence of ATP, H2, and bromoethanesulfonic acid, label from 3H- or 2-14C-labeled acetate was incorporated into the protein fraction of cell extracts of Methanosarcina barkeri. Incorporated label was susceptible to photolysis, yielding labeled methane as the anaerobic photolysis product. Size exclusion high-pressure liquid chromatography (HPLC) demonstrated the presence of at least three labeled proteins with native molecular sizes of 480, 200, and 29 kDa, while electrophoresis indicated that four major labeled proteins were present. Dual-label experiments demonstrated that these four proteins were methylated rather than acetylated. Two of the proteins (480 and 29 kDa) contained the majority of radiolabel and were stably methylated. After labeling with [2-14C]acetate, the stable 14CH3-proteins were partially purified, and 14CH3-cofactors were isolated from each protein. UV-visible spectroscopy and HPLC demonstrated these to be methylated corrinoids. When the 480-kDa corrinoid protein was purified to 70% homogeneity, the preparation was found to have subunits of 40 and 30 kDa. The 480-kDa protein but not the 29-kDa protein was methylated during in vitro methanogenesis from acetate and demethylated as methanogenesis ceased, consistent with the involvement of this protein in methane formation.     

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.     

Lack of peptidoglycan in the cell walls of Methanosarcina barkeri

Neither muramic acid and glucosamine nor d-glutamic acid or other amino acids typical of peptidoglycan were found in cell walls of two strains of Methanosarcina barkeri. The main components are galactosamine, neutral sugars and uronic acids. Therefore, the structural component of the cell wall most likely consists of an acid heteropolysaccharide, resembling that of Halococcus morrhuae. It is, however, not sulfated.