Biological Formation Of Methane Research Articles

A Cobalamin‐Dependent Radical SAM Enzyme Catalyzes the Unique Cα‐Methylation of Glutamine in Methyl‐Coenzyme M Reductase

AbstractMethyl‐coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2‐(S)‐methylglutamine. The enzyme responsible for the Cα‐methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin‐dependent radical SAM enzyme as the glutamine C‐methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base‐off, His‐off conformation and contains a single [4Fe‐4S] cluster. The cobalamin cofactor cycles between the methyl‐cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5′‐deoxyadenosine and S‐adenosyl‐l‐homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C‐methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3‐hybridized carbon atoms.

A Cobalamin-Dependent Radical SAM Enzyme Catalyzes the Unique Cα -Methylation of Glutamine in Methyl-Coenzyme M Reductase.

Methyl‐coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2‐(S)‐methylglutamine. The enzyme responsible for the Cα‐methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin‐dependent radical SAM enzyme as the glutamine C‐methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base‐off, His‐off conformation and contains a single [4Fe‐4S] cluster. The cobalamin cofactor cycles between the methyl‐cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5′‐deoxyadenosine and S‐adenosyl‐l‐homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C‐methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3‐hybridized carbon atoms.

Stimulation of methanogenesis in bituminous coal from the upper Silesian coal basin

Biological methane production from coal seams through their biological conversion is one of the methods that have been postulated to utilize unmineable resources. Furthermore, energy production from methane is considered less environmentally-harmful than the combustion of coal. In the current study, meta-bituminous coal extracted from the Upper Silesian Coal Basin (USCB) was investigated in terms of its potential for biological methane formation. Both natural and stimulated (by bioaugmentation) methanogenic activity was assayed. Consortia of microorganisms selected from highly methanogenic peat (Moszne peat bog) and sediments (Szczecin subsidence reservoir) were enriched on various media and used for coal bioaugmentation. It was found that both peat- and sediment-derived microbiota are capable of coal degradation, with the latter being more effective. FT-IR revealed changes in the utilization patterns of organic matter by each community that were linked to differences in the stable carbon isotope indices of the gases produced. The ability to cleave aromatic rings was recognized as the most important feature of the microbial community used in the production of methane from the tested coal.

Methane Production and Bioactivity-A Link to Oxido-Reductive Stress.

Biological methane formation is associated with anoxic environments and the activity of anaerobic prokaryotes (Archaea). However, recent studies have confirmed methane release from eukaryotes, including plants, fungi, and animals, even in the absence of microbes and in the presence of oxygen. Furthermore, it was found that aerobic methane emission in plants is stimulated by a variety of environmental stress factors, leading to reactive oxygen species (ROS) generation. Further research presented evidence that molecules with sulfur and nitrogen bonded methyl groups such as methionine or choline are carbon precursors of aerobic methane formation. Once generated, methane is widely considered to be physiologically inert in eukaryotes, but several studies have found association between mammalian methanogenesis and gastrointestinal (GI) motility changes. In addition, a number of recent reports demonstrated anti-inflammatory potential for exogenous methane-based approaches in model anoxia-reoxygenation experiments. It has also been convincingly demonstrated that methane can influence the downstream effectors of transiently increased ROS levels, including mitochondria-related pro-apoptotic pathways during ischemia-reperfusion (IR) conditions. Besides, exogenous methane can modify the outcome of gasotransmitter-mediated events in plants, and it appears that similar mechanism might be active in mammals as well. This review summarizes the relevant literature on methane-producing processes in eukaryotes, and the available results that underscore its bioactivity. The current evidences suggest that methane liberation and biological effectiveness are both linked to cellular redox regulation. The data collectively imply that exogenous methane influences the regulatory mechanisms and signaling pathways involved in oxidative and nitrosative stress responses, which suggests a modulator role for methane in hypoxia-linked pathologies.

Identification of a unique Radical SAM methyltransferase required for the sp3-C-methylation of an arginine residue of methyl-coenzyme M reductase

The biological formation of methane (methanogenesis) is a globally important process, which is exploited in biogas technology, but also contributes to global warming through the release of a potent greenhouse gas into the atmosphere. The last and methane-releasing step of methanogenesis is catalysed by the enzyme methyl-coenzyme M reductase (MCR), which carries several exceptional posttranslational amino acid modifications. Among these, a 5-C-(S)-methylarginine is located close to the active site of the enzyme. Here, we show that a unique Radical S-adenosyl-L-methionine (SAM) methyltransferase is required for the methylation of the arginine residue. The gene encoding the methyltransferase is currently annotated as “methanogenesis marker 10” whose function was unknown until now. The deletion of the methyltransferase gene ma4551 in Methanosarcina acetivorans WWM1 leads to the production of an active MCR lacking the C-5-methylation of the respective arginine residue. The growth behaviour of the corresponding M. acetivorans mutant strain and the biophysical characterization of the isolated MCR indicate that the methylated arginine is important for MCR stability under stress conditions.

The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.

Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

Structural Analysis of a Ni-Methyl Species in Methyl-Coenzyme M Reductase from Methanothermobacter marburgensis

We present the 1.2 Å resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR), the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 Å proximal of the Ni atom of the MCR coenzyme F(430). A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.

Detection of Organometallic and Radical Intermediates in the Catalytic Mechanism of Methyl-Coenzyme M Reductase Using the Natural Substrate Methyl-Coenzyme M and a Coenzyme B Substrate Analogue

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.

Methane formation in aerobic environments

Environmental context. Methane is an important greenhouse gas and its atmospheric concentration has drastically increased since pre-industrial times. Until recently biological methane formation has been associated exclusively with anoxic environments and microbial activity. In this article we discuss several alternative formation pathways of methane in aerobic environments and suggest that non-microbial methane formation may be ubiquitous in terrestrial and marine ecosystems. Abstract. Methane (CH4), the second principal anthropogenic greenhouse gas after CO2, is the most abundant reduced organic compound in the atmosphere and plays a central role in atmospheric chemistry. Therefore a comprehensive understanding of its sources and sinks and the parameters that control emissions is prerequisite to simulate past, present and future atmospheric conditions. Until recently biological CH4 formation has been associated exclusively with anoxic environments and methanogenic activity. However, there is growing and convincing evidence of alternative pathways in the aerobic biosphere including terrestrial plants, soils, marine algae and animals. Identifying and describing these sources is essential to complete our understanding of the biogeochemical cycles that control CH4 in the atmospheric environment and its influence as a greenhouse gas.

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

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F 430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 Å and 1.8 Å resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F 430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg 2+ and 9 Na + ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 Å resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.

Methane formation from long-chain alkanes by anaerobic microorganisms.

Biological formation of methane is the terminal process of biomass degradation in aquatic habitats where oxygen, nitrate, ferric iron and sulphate have been depleted as electron acceptors. The pathway leading from dead biomass to methane through the metabolism of anaerobic bacteria and archaea is well understood for easily degradable biomolecules such as carbohydrates, proteins and lipids. However, little is known about the organic compounds that lead to methane in old anoxic sediments where easily degradable biomolecules are no longer available. One class of naturally formed long-lived compounds in such sediments is the saturated hydrocarbons (alkanes). Alkanes are usually considered to be inert in the absence of oxygen, nitrate or sulphate, and the analysis of alkane patterns is often used for biogeochemical characterization of sediments. However, alkanes might be consumed in anoxic sediments below the zone of sulphate reduction, but the underlying process has not been elucidated. Here we used enrichment cultures to show that the biological conversion of long-chain alkanes to the simplest hydrocarbon, methane, is possible under strictly anoxic conditions.

Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation.

Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha2beta2gamma2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F430 are embedded between the subunits alpha, alpha', beta, and gamma and alpha', alpha, beta', and gamma', forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.

Environmental impact of biomethanogenesis

The environmental impact of biomethanogenesis is related to its ecological role, accumulation and effect as a greenhouse gas, and application in anaerobic digestion for conversion of biomass and wastes to methane and compost. Biological formation of methane is the process by which bacteria decompose organic matter using carbon dioxide as an electron acceptor in the absence of dioxygen or other electron acceptors. This microbial activity is responsible for carbon recycling in anaerobic environments, including wetlands, rice fields, intestines of animals sediments, and manures. The mixed consortium of microorganisms involved includes a unique group of bacteria, the methanogens, which may be considered to be in a separate kingdom based on genetic and phylogenetic variance from all other life forms. Because methane is a significant and increasing greenhouse gas, its source fluxes and their potential reduction are of concern. Biomethanogenesis may be harnessed for reduction of wastes and conversion of renewable resources to significant quantities of substitute natural gas which could mitigate carbon dioxide and other pollutants related to use of fossil fuels.

Coenzym F430 aus Methan-Bakterien: Zusammenhänge zwischen der Struktur des hydroporphinoiden Liganden und der Redoxchemie des Nickelzentrums

The hydroporphinoid nickel complex coenzyme F430 ist the prosthetic group of methyl-coenzyme M reductase, the enzyme catalyzing the last step of biological methane formation. Although the enzyme mechanism is still unknown, it has been shown that the isolated cofactor can be reduced to the NiI-form at surprisingly positive potentials. Recent in vivo EPR studies indicate that the NiI form of F430 is indeed formed in whole cells and in highly active enzyme preparations. A comparative study of the redox chemistry of partially synthetic derivatives of F430 helped to identify the structural elements that allow coenzyme F430 to be reduced at the metal rather than at the ligand and at a physiologically accessible potential.

Elemental metals as electron sources for biological methane formation from CO2

Several elemental metals were examined as potential electron donors for methanogenic bacteria, using both a single tube system where the metal was in direct contact with the cells, and a two-flask system, where metal and cells were not in direct contact, but had contact via the gas phase. With all organisms examined in the direct contact system, Fe degree, Al degree and Zn degree served as electron donors for methanogenesis; some organisms used Ni degree or Sn degree as low-level electron donors. Of the metals tested, methanogenesis from H2 + CO2 was inhibited by direct contact with Zn degree or Cu degree, but not by Fe degree or Al degree. Ni degree and Co degree were inhibitory to some methanogens, with Ni degree being particularly inhibitory to the thermophilic strains tested. With all organisms examined in the two-flask system, Fe degree and Zn degree served as good electron sources for both methanogenesis and growth; Co degree generated a very low level of methane and Cu degree did not work at all. In either system V degree, Ti degree or Cd degree did not serve as electron donors. The results suggest that some elemental metals (notably Fe degree, Al degree and Zn degree) produce gaseous H2 by cathodic depolarization which is then consumed by the methanogen, thus accelerating oxidation of the metal by its metabolic activity. All of these reactions are thermodynamically favorable; however, some other metals that are clearly favorable for such a reaction on thermodynamic grounds (Ti degree and V degree) are very stable and do not serve as electron donors.

Role of vitamin B12 in methyl transfer for methane biosynthesis by Methanosarcina barkeri.

When Methanosarcina barkeri is grown on methanol as the sole carbon source, a B12-containing protein is synthesized by this organism. This B12 protein contains bound aquocobalamin, and when this cofactor is reduced and methylated with [14C]methyl iodide, the resultant [14C]methyl B12 protein is extremely active in the biosynthesis of 14C-labeled methane. These findings indicate that a B12-dependent system is operative in the biological formation of methane in addition to other systems that are B12-independent.

Microbial Formation of Methane

This chapter discusses the microbial formation of methane. The formation of methane is a unique biological event, which is confined to a small group of bacteria. The biological formation of methane is common in nature and methane bacteria are readily found in anaerobic environments where organic matter is being vigorously decomposed. Organic polymers such as cellulose are hydrolyzed by extracellular cellulases to sugars, which are fermented to a variety of compounds: fatty acids, alcohols, carbon dioxide, and hydrogen. Some of these compounds, such as acetate, methanol, or carbon dioxide and hydrogen, are the preferred substrates for methane bacteria. Methane, the simplest member of the hydrocarbon family, is poorly soluble, is essentially inert under anaerobic conditions and is volatile. Thus, the final reduced product has ideal non-toxic properties and readily escapes from the anaerobic environment. Methane found in coal mines is believed to have been trapped when the coal has been formed, and the carboniferous swamps must have been an ideal environrment for methane bacteria. Methane is commonly produced in the digestive tract of animals (for example cows), where similar anaerobic food chains are important in the degradation of food. Man has designed an anaerobic environment in which the sludge from the aerobic portion of sewage treatment is allowed to digest under anaerobic conditions. The chapter also discusses the biochemistry of methane formation.

ATP-dependent formation of methane from methylcobalamin by extracts of [formula omitted][formula omitted

The significant observation by Blaylock and Stadtman (1) that the methyl moiety of methylcobalamin serves as a precursor of methane using extracts of Methanosarcina barkeri has prompted us to extend our previous observations on cell-free methane formation (2). At the present time, it is not known whether methylcobalamin is a natural intermediate in the biological formation of methane or whether it only serves as an active methyl donor. This communication presents evidence that extracts of Methanobacillus omelianskii form methane stoichiometrically from methylcobalamin, that this formation is dependent upon ATP, and that the formation apparently does not require an added source of electrons.

Biological Formation of Methane

Biological Formation of Methane