Carbon monoxide-driven electron transport in Clostridium thermoautotrophicum membranes.

Membrane vesicles of Clostridium thermoautotrophicum prepared by osmotic lysis after lysozyme treatment contained carbon monoxide dehydrogenase and methylenetetrahydrofolate dehydrogenase with specific activities three- to fourfold higher than the specific activity of the cytoplasm. The membrane-associated carbon monoxide dehydrogenase mediated the reduction with CO or the oxidation with CO2 of b-type cytochromes and other electron carriers in the membrane.     

One-carbon metabolism in methanogenic bacteria: analysis of short-term fixation products of 14CO2 and 14CH3OH incorporated into whole cells.

Methanobacterium thermoautotrophicum, M. ruminantium, and Methanosarcina barkeri were labeled with 14CO2 (14CO2 + H14CO3- + 14CO32-) for from 2 to 45 s. Radioactivity was recovered in coenzyme M derivatives, alanine, aspartate, glutamate, and several unidentified compounds. The properties of one important structurally unidentified intermediate (yellow fluorescent compound) displayed UV absorbance maxima at pH 1 of 290 and 335 nm, no absorbance in the visible region, and a fluorescence maximum at 460 nm. Label did not appear in organic phosphates until after 1 min. 14CH3OH was converted by M. barkeri primarily into coenzyme M derivatives at 25 s. [2-14C]acetate was assimilated by M. thermoautotrophicum mainly into alanine and succinate during 2 to 240 s, but not into coenzyme M derivatives or yellow fluorescent compound. Cell-free extracts of M. thermoautotrophicum lacked ribulose 1,5-bisphosphate carboxylase activity. The data indicated the absence of the Calvin, serine, and hexulose phosphate paths of C1 assimilation in the methanogens examined and indicated that pyruvate was an early intermediate product of net CO2 fixation. The in vivo importance of coenzyme M derivatives in methanogenesis was demonstrated.     

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide.

NaCN was tested for its inhibitory effects on growth of and metabolism by Methanosarcina barkeri 227. NaCN (10 microM) inhibited catabolism of acetate methyl groups to CH4 and CO2 but did not inhibit methanogenesis from methanol, CO2, methylamine, or trimethylamine. NaCN also inhibited the assimilation of methanol or CO2 (as the sole carbon source) into cell carbon and stimulated the assimilation of acetate. These results suggest that inhibition by NaCN was a result of its action as an inhibitor of in vivo CO dehydrogenase. The results also implicate CO dehydrogenase in the oxidation of acetate but not methanol methyl groups to CO2.     

Formation of carbon monoxide from CO2 and H2 by Methanobacterium thermoautotrophicum

Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol X min-1 X mg protein-1. Half-maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy-driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers. CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme. CO production by growing cells was 5-10-times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.     

Formylmethanofuran dehydrogenase from methanogenic bacteria, a molybdoenzyme

Formylmethanofuran dehydrogenase, a key enzyme of methanogenesis, was purified 100-fold from methanol grown Methanosarcina barkeri to apparent homogeneity and a specific activity of 34 mumol.min-1.mg protein-1. Molybdenum was found to co-migrate with the enzyme activity. The molybdenum content of purified preparations was 3-4 nmol per mg protein equal to 0.6-0.8 mol molybdenum per mol enzyme of apparent molecular mass 200 kDa. Evidence is presented that also formylmethanofuran dehydrogenase from H2/CO2 grown Methanobacterium thermoautotrophicum (strain Marburg) is a molybdoenzyme.     

Carbon Monoxide Oxidation by Methanogenic Bacteria

Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.     

Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum

Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.     

Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.     

Dissimilatory sulphate reduction with acetate as electron donor

Acetate oxidation by sulphate was studied with desulfobacter postgatei. Cell extracts of the organism were found to contain high activities of the following enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and pyruvate synthase. It is concluded that acetate oxidation with sulphate in D. postgatei proceeds via the citric acid cycle with the synthesis of pyruvate from acetyl CoA and CO2 as an anaplerotic reaction. The apparent Ks for acetate oxidation by D. postgatei as determined in vivo was near 0.2 mM. The apparent Ks for acetate fermentation to methane and CO2 by methanosarcina barkeri was 3 mM. The significantly lower ks for acetate of the sulphate reducer explains why methane formation from acetate in natural habitats is apparently inhibited by sulphate.     

Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase.

The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.     

Enzymology of one-carbon metabolism in methanogenic pathways

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.     

Effect of nitrate on the autotrophic metabolism of the acetogens Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H2-CO2-dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H2-CO2 did not replace CO. These results indicated that nitrate blocked the reduction of CO2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.     

N-furfurylformamide as a pseudo-substrate for formylmethanofuran converting enzymes from methanogenic bacteria

Methanofuran (4-[N-(4,5,7-tricarboxyheptanoyl-gamma-L-glutamyl)-gamma-L- glutamyl)-p-(beta-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan is a coenzyme involved in methanogenesis. The N-formyl derivative is an intermediate in the reduction of CO2 to CH4 and the disproportionation of methanol to CO2 and CH4. Formylmethanofuran dehydrogenase and formylmethanofuran:tetrahydromethanopterin formyltransferase are the enzymes catalyzing its conversions. We report here that the two enzymes from Methanosarcina barkeri and the formyltransferase from Methanobacterium thermoautotrophicum can also use N-furfurylformamide as a pseudo-substrate albeit with higher apparent Km and lower apparent Vmax values. N-Methylformamide, formamide, and formate were not converted indicating that the furfurylamine moiety of methanofuran is the minimum structure required for the correct binding of the coenzyme.     

Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria

The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [(13)C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate. Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2). Most likely, this organism uses the beta-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2). Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.     

Electron transport and electrochemical proton gradient in membrane vesicles of Clostridium thermoautotrophicum.

Membrane vesicles of Clostridium thermoautotrophicum containing carbon monoxide dehydrogenase generated a proton motive force when exposed to CO. This proton motive force, with a value of -140 mV, consisted of only an electrical potential at pH 7.5 and above and of an electrical potential and pH gradient at a lower pH. The proton motive force drove the uptake of L-alanine by the vesicles to a concentration of 300 times that of the medium.     

Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.     

Influence of corrinoid antagonists on methanogen metabolism.

Iodopropane inhibited cell growth and methane production when Methanobacterium thermoautotrophicum, Methanobacterium formicicum, and Methanosarcina barkeri were cultured on H2-CO2. Iodopropane (40 microM) inhibited methanogenesis (30%) and growth (80%) when M. barkeri was cultured mixotrophically on H2-CO2-methanol. The addition of acetate to the medium prevented the observed iodopropane-dependent inhibition of growth. The concentrations of iodopropane that caused 50% inhibition of growth of M. barkeri on either H2-CO2, H2-CO2-methanol, methanol, and acetate were 112 +/- 6, 24 +/- 2, 63 +/- 11, and 4 +/- 1 microM, respectively. Acetate prevented the iodopropane-dependent inhibition of one-carbon metabolism. Cultivation of M. barkeri on H2-CO2-methanol in bright light also inhibited growth and methanogenesis to a greater extent in the absence than in the presence of acetate in the medium. Acetate was the only organic compound examined that prevented iodopropane-dependent inhibition of one-carbon metabolism in M. barkeri. The effect of iodopropane and acetate on the metabolic fates of methanol and carbon dioxide was determined with 14C tracers when M. barkeri was grown mixotrophically on H2-CO2-methanol. The addition of iodopropane decreased the contribution of methanol to methane and cell carbon while increasing the contribution of CO2 to cell carbon. Regardless of iodopropane, acetate addition decreased the contribution of methanol and CO2 to cell carbon without decreasing their contribution to methane. The corrinoid antagonists, light and iodopropane, appeared most specific for methanogen metabolic reactions involved in acetate synthesis from one-carbon compounds and acetate catabolism.