Biochemistry of methanogenesis.

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH3-S-CoM) to CH4. Seminal studies on the CO2-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO2 to the methyl level (CH3-S-CoM) with electrons from H2 or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH3-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO2 by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH3-S-CoM; electrons for reduction of CH3-S-CoM to CH4 are provided by the oxidation of methyl groups to CO2.     

Biochemistry of Methanogenesis

Abstract

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH₃-S-CoM) to CH₄. Seminal studies on the CO₂-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO₂ to the methyl level (CH₃-S-CoM) with electrons from H₂ or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH₃-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO₂ by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH₃-S-CoM; electrons for reduction of CH₃-S-CoM to CH₄ are provided by the oxidation of methyl groups to CO₂.     

Methane formation and methane oxidation by methanogenic bacteria.

Methanogenic bacteria were found to form and oxidize methane at the same time. As compared to the quantity of methane formed, the amount of methane simultaneously oxidized varied between 0.3 and 0.001%, depending on the strain used. All the nine tested strains of methane producers (Methanobacterium ruminantium, Methanobacterium strain M.o.H., M. formicicum, M. thermoautotrophicum, M. arbophilicum, Methanobacterium strain AZ, Methanosarcina barkeri, Methanospirillum hungatii, and the "acetate organism") reoxidized methane to carbon dioxide. In addition, they assimilated a small part of the methane supplied into cell material. Methanol and acetate also occurred as oxidation products in M. barkeri cultures. Acetate was also formed by the "acetate organism," a methane bacterium unable to use methanogenic substrates other than acetate. Methane was the precursor of the methyl group of the acetate synthesized in the course of methane oxidation. Methane formation and its oxidation were inhibited equally by 2-bromoethanesulfonic acid. Short-term labeling experiments with M. thermoautotrophicum and M. hungatii clearly suggest that the pathway of methane oxidation is not identical with a simple back reaction of the methane formation process.     

The biosynthesis of methylated amino acids in the active site region of methyl-coenzyme M reductase

The global production of the greenhouse gas methane by methanogenic archaea reaches 1 billion tons per annum. The final reaction releasing methane is catalyzed by the enzyme methyl-coenzyme M reductase. The crystal structure of methyl-coenzyme M reductase from Methanobacterium thermoautotrophicum revealed the presence of five modified amino acids within the alpha-subunit and near the active site region. Four of these modifications were C-, N-, and S-methylations, two of which, 2-(S)-methylglutamine and 5-(S)-methylarginine, have never been encountered before. We have now confirmed these modifications by mass spectrometry of chymotryptic peptides. With methyl-coenzyme M reductase purified from cells grown in the presence of L-[methyl-D(3)]methionine, it was shown that the methyl groups of the modified amino acids are derived from the methyl group of methionine rather than from methyl-coenzyme M, an intermediate in methane formation. The D(3) labeling pattern was found to be qualitatively and quantitatively the same as in the two methyl groups of the methanogenic coenzyme F(430), which are known to be introduced via S-adenosylmethionine. From the results, it is concluded that the methyl groups of the modified amino acids in methyl-coenzyme M reductase are biosynthetically introduced by an S-adenosylmethionine-dependent post-translational modification. A mechanism for the methylation of glutamine at C-2 and of arginine at C-5 is discussed.     

Life with CO or CO2 and H2 as a source of carbon and energy

An account is presented of the recent discovery of a pathway of growth by bacteria in which CO or CO2 and H2 are sources of carbon and energy. The Calvin cycle and subsequently other cycles were discovered in the 1950s, and in each the initial reaction of CO2 involved adding CO2 to an organic compound formed during the cyclic pathway (for example, CO2 and ribulose diphosphate). Studies were initiated in the 1950s with the thermophylic anaerobic organism Clostridium thermoaceticum, which Barker and Kamen had found fixed CO2 in both carbons of acetate during fermentation of glucose. The pathway of acetyl-CoA biosynthesis differs from all others in that two CO2 are combined with coenzyme A (CoASH) forming acetyl CoA, which then serves as the source of carbon for growth. This mechanism is designated the acetyl CoA pathway and some have called it the Wood pathway. A unique feature is the role of the enzyme carbon monoxide dehydrogenase (CODH), which catalyzes the conversion of CoASH, CO, and a methyl group to acetyl CoA, the final step of the pathway. The pathway involves the reduction of CO2 to formate, which then combines with tetrahydrofolate (THF) to form formyl THF. It in turn is reduced to CH3-THF. The methyl is then transferred to the cobalt on a corrinoid-containing enzyme. From there the methyl is transferred to CODH, and CO and CoASH bind with the enzyme at separate sites. Acetyl CoA is then synthesized. CODH would more properly be called carbon monoxide dehydrogenase-acetyl CoA synthase as it catalyzes oxidation of CO to CO2 and the synthesis of acetyl CoA. The solution of the mechanism of this pathway required more than 30 years, in part because the intermediate compounds are bound to enzymes, the enzymes are extremely sensitive to O2 and must be isolated under strictly anerobic conditions, and the role of a corrinoid and CODH was unprecedented. It is now apparent that this pathway occurs (perhaps with some modification) in many bacteria including the methane and sulfur bacteria. In some humans this pathway is catalyzed by the bacteria of the gut and acetate is produced rather than methane; it is calculated that 2.3 x 10(6) metric tons of acetate are formed daily from CO2. A similar synthesis occurs in the hind gut of termites. It is becoming apparent that the acetyl CoA pathway plays a significant role in the carbon cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Metabolism of methanogens

Methanogenic archaea convert a few simple compounds such as H₂ + CO₂, formate, methanol, methylamines, and acetate to methane. Methanogenesis from all these substrates requires a number of unique coenzymes, some of which are exclusively found in methanogens. H₂-dependent CO₂ reduction proceeds via carrier-bound C₁ intermediates which become stepwise reduced to methane. Methane formation from methanol and methylamines involves the disproportionation of the methyl groups. Part of the methyl groups are oxidized to CO₂, and the reducing equivalents thereby gained are subsequently used to reduce other methyl groups to methane. This process involves the same C₁ intermediates that are formed during methanogenesis from CO₂. Conversion of acetate to methane and carbon dioxide is preceeded by its activation to acetyl-CoA. Cleavage of the latter compound yields a coenzyme-bound methyl moiety and an enzyme-bound carbonyl group. The reducing equivalents gained by oxidation of the carbonyl group to carbon dioxide are subsequently used to reduce the methyl moiety to methane. All these processes lead to the generation of transmembrane ion gradients which fuel ATP synthesis via one or two types of ATP synthases. The synthesis of cellular building blocks starts with the central anabolic intermediate acetyl-CoA which, in autotrophic methanogens, is synthesized from two molecules of CO₂ in a linear pathway.     

Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum.

A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.     

The nickel enzyme methyl-coenzyme M reductase from methanogenic archaea: in vitro interconversions among the EPR detectable MCR-red1 and MCR-red2 states

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme contains tightly bound the nickel porphinoid F430. The nickel enzyme has been shown to be active only when its prosthetic group is in the Ni(I) reduced state. In this state MCR exhibits the nickel-based EPR signal red1. We report here for the MCR from Methanothermobacter marburgensis that the EPR spectrum of the active enzyme changed upon addition or removal of coenzyme M, methyl coenzyme M and/or coenzyme B. In the presence of methyl-coenzyme M the red1 signal showed a more resolved 14N-superhyperfine splitting than in the presence of coenzyme M indicating a possible axial ligation of the substrate to the Ni(I). In the presence of methyl-coenzyme M and coenzyme B the red1 signal was the same as in the presence of methyl-coenzyme M alone. However, in the presence of coenzyme M and coenzyme B a highly rhombic EPR signal, MCR-red2, was induced, which was found to be light sensitive and appeared to be formed at the expense of the MCR-red1 signal. Upon addition of methyl-coenzyme M, the red2 signal disappeared and the red1 signal increased again. The red2 signal of MCR with 61Ni-labeled cofactor was significantly broadened indicating that the signal is nickel or nickel-ligand based.     

Methane production by the membranous fraction of Methanobacterium thermoautotrophicum.

Intact membrane vesicles are required to synthesize methane from CO2 and H2 by disrupted preparations of Methanobacterium thermoautotrophicum cells. When membrane vesicles were removed by high-speed centrifugation at 226 600 g, the remaining supernatant fraction no longer synthesized methane. Alternatively, if vesicle structure was disrupted by passage through a Ribi cell fractionator at very high pressures (345 MPa), the bacterial cell extract, with all the particulate fraction in it, did not synthesize methane. Methyl-coenzyme M, a new coenzyme first described by McBride & Wolfe [(1971) Biochemistry 10, 2317--2324], was shown to stimulate methane production from CO2 and H2, as previously reported, but the methyl group of the coenzyme did not appear to be a precursor of methane in this reaction. No methyl-coenzyme M reductase activity was detected in the cytoplasmic fraction of M. thermoautotrophicum cells.     

Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B. The electron density at 1.6 A resolution of the M. barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations.     

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.     

Enzymes involved in the anoxic utilization of phenyl methyl ethers by <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> DCB2 and <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> PCE-S

Phenyl methyl ethers are utilized by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S; the methyl group derived from the O-demethylation of these substrates can be used as electron donor for anaerobic fumarate respiration or dehalorespiration. The activity of all enzymes involved in the oxidation of the methyl group to carbon dioxide via the acetyl-CoA pathway was detected in cell extracts of both strains. In addition, a carbon monoxide dehydrogenase activity could be detected. Activity staining of this enzyme indicated that the enzyme is a bifunctional CO dehydrogenase/acetyl-CoA synthase.     

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.     

Methanopterin and methanogenic bacteria

Methanogenic bacteria comprise a selected group of microorganisms that derive their energy for growth from the hydrogen-dependent reduction of CO2 to methane or the disproportionation of reduced one-carbon compounds and acetate to CO2 and methane. In the reduction and oxidation steps at the formyl, hydroxymethyl and methyl level the one-carbon unit remains bound to the reduced form of methanopterin, a pterin derivative typical of methanogenic bacteria. In addition, the reduced methanopterin, 5,6,7,8-tetrahydromethanopterin, is involved in a number of anabolic reactions. Methanopterin is structurally and functionally the counterpart of folic acid found in other organisms. In this review the occurrence and properties of methanopterin and its derivatives, as well as the biosynthesis and the role in the different catabolic and anabolic reactions are discussed against the background of folic acid biochemistry.     

Methyl-coenzyme M formation in methanogenic archaea. Involvement of zinc in coenzyme M activation.

Methyl-coenzyme M (2-methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mM EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M approximately 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl-coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn2+ of MtaA was re-determined and found to be > 15 and not 9.6 as previously reported by us.     

Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS

The synthesis of methylmercury by Desulfovibrio desulfuricans LS was investigated on the basis of ¹⁴C incorporation from precursors and the measurement of relevant enzyme activities in cell extracts. The previously observed incorporation of C-3 from serine into methylmercury was confirmed by measurement of relatively high activities of serine hydroxymethyltransferase and other enzymes of this pathway. High rates of label incorporation into methylmercury from H¹⁴COO^- and H¹⁴CO₃^- prompted the assay of enzymes of the acetyl coenzyme A (CoA) synthase pathway. These enzymes were found to be present but at activity levels much lower than those reported for acetogens. Propyl iodide inhibited methylmercury and acetyl-CoA syntheses to similar extents, and methylmercury synthesis was found to compete with acetyl-CoA synthesis for methyl groups. On the basis of these findings, we propose that in methylmercury synthesis by D. desulfuricans LS the methyl group is transferred from CH₃-tetrahydrofolate via methylcobalamin. The methyl group may originate from C-3 of serine or from formate via the acetyl-CoA synthase pathway. These pathways are not unique to D. desulfuricans LS, and thus the ability of this bacterium to methylate mercury is most likely associated with the substrate specificity of its enzymes.     

Methanogenic Decomposition of Ferulic Acid, a Model Lignin Derivative

Ferulic acid, a model lignin derivative, was observed to be biodegradable to methane and carbon dioxide under strict anaerobic conditions. This conversion appears to be carried out by a consortium of bacteria similar to that previously described for the methanogenic degradation of benzoic acid. A temporary buildup of acetate in these cultures indicates that it is a likely intermediate and precursor for methane formation. An analog of coenzyme M, 2-bromoethanesulfonic acid (BESA), inhibited gas production and enhanced the buildup of propionate, butyrate, isobutyrate, and isovalerate. Phenylacetate, cinnamate, 3-phenylpropionate, benzoate, cyclohexane carboxylate, adipate, and pimelate were also detected in BESA-inhibited cultures. A pathway is proposed which includes these various acids as possible intermediates in the methanogenic degradation of ferulic acid. This model overlaps previously described benzoic acid degradation pathways, suggesting that this type of anaerobic degradation may be common for aromatic compounds.     

The bioenergetics of methanogenesis

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)