Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as “Methanoplasmatales”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “Candidatus Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “Ca. Methanoplasma termitum” lacks the entire pathway for CO₂ reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F₄₂₀:methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F₄₂₀-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

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

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Δech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C₁ compounds.The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO₂ and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C₁ compounds (methanol and methylamines), one-quarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO₂ (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fd_red) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fd_red:CoM-S-S-CoB oxidoreductase system (7, 24). The direct membrane-bound electron acceptor for Fd_red is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H₂-evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H₂:CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20, 21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought to be involved in the aceticlastic pathway of methanogenesis as an acceptor for Fd_red (8, 10, 17). The Methanosarcina mazei genome, however, contains genes coding for the Ech hydrogenase, but this species lacks the Rnf complex (5).To investigate whether the Ech hydrogenase is the only means by which M. mazei channels electrons from Fd_red into the respiratory chain, a mutant lacking the Ech hydrogenase (M. mazei Δech mutant) was constructed. Electron transport experiments using Fd_red as the electron donor and CoM-S-S-CoB as the electron acceptor were conducted with wild-type and mutant membranes to gain deeper insight into the actual membrane-bound protein complexes that accept electrons from Fd_red. Furthermore, an in-depth characterization of the growth and trimethylamine (TMA) consumption of the Δech mutant was performed, which provided insight into the in vivo role of Ech hydrogenase.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

Membrane-bound F420H2-dependent heterodisulfide reduction in Methanococcus voltae

Washed membranes prepared from H₂+CO₂- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F₄₂₀H₂ and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F₄₂₀H₂ + COB–S–S–CoM → F₄₂₀ + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F₄₂₀H₂-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F₄₂₀H₂-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F₄₂₀H₂ oxidation is catalyzed by a membrane-associated F₄₂₀-reducing hydrogenase. The purified protein catalyzed the H₂-dependent reduction of methyl viologen and F₄₂₀. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F₄₂₀-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F₄₂₀-reducing hydrogenase from Mc. voltae. A distinct F₄₂₀H₂ dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism. Received: 18 September 1998 / Accepted: 2 November 1998     

Anaerobic treatment of protein-containing waste waters: correlation between coenzyme F420 and methane production

Summary Anaerobic treatment of gelatine-containing model waste water and baker's yeast manufacturing effluent was investigated in upflow anaerobic sludge blanket (UASB) reactors. During start up a correlation between coenzyme F ₄₂₀ content and methane production in the reactor was observed. By monitoring coenzyme F ₄₂₀ concentrations a certain prediction of methanogenic activities was possible.     

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V_max and K_M values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO₂. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

The F420-Reducing [NiFe]-Hydrogenase Complex from Methanothermobacter marburgensis, the First X-ray Structure of a Group 3 Family Member

The reversible redox reaction between coenzyme F₄₂₀ and H₂ to F₄₂₀H₂ is catalyzed by an F₄₂₀-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7 Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H₂ and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F₄₂₀-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°′ ≈ − 400 mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges

In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F₄₂₀−2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F₄₂₀−4 and F₄₂₀−5 in sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

VhuD Facilitates Electron Flow from H2 or Formate to Heterodisulfide Reductase in Methanococcus maripaludis

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H₂ via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F₄₂₀-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth.     

Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport of Methanosarcina mazei Gö1

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F₄₂₀H₂ dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F₄₂₀H₂ was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The K_m values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F₄₂₀-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.     

Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO₂ assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the ΔehbO::pac strain. The membrane-spanning ion translocator (ΔehbF) and the large hydrogenase subunit (ΔehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The ΔehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.Hydrogenotrophic methanococci specialize in utilizing H₂ as an electron donor, and these organisms possess six different Ni-Fe hydrogenases. These enzymes include two F_420--reducing hydrogenases, two non-F₄₂₀-reducing hydrogenases, and two membrane-bound hydrogenases (Eha and Ehb [5]). The F₄₂₀-reducing hydrogenases reduce coenzyme F₄₂₀, which subsequently reduces methenyltetrahydromethanopterin and methylenetetrahydromethanopterin, intermediates in the pathway of methanogenesis. In Methanococcus voltae, the F₄₂₀-reducing hydrogenase is also reported to reduce the 2-mercaptoethanesulfonate:7-mercaptoheptanoylthreonine phosphate heterodisulfide formed in the final step of methanogenesis (2). In contrast, Methanothermobacter marburgensis utilizes the non-F₄₂₀-reducing hydrogenase to reduce the heterodisulfide (22, 25).The two membrane-bound hydrogenases couple the chemiosmotic energy of ion gradients to H₂ oxidation and ferredoxin reduction. In the aceticlastic methanogen Methanosarcina barkeri, the homologous enzyme is called energy conserving hydrogenase or Ech and performs a variety of physiological functions, including the generation of a proton motive force during CO oxidation and concomitant proton reduction in aceticlastic methanogenesis and the generation of low potential electron donors for CO₂ reduction to formylmethanofuran in the first step of methanogenesis and the reductive carboxylation of acetyl coenzyme A (acetyl-CoA) to pyruvate in carbon assimilation (11, 12). In the hydrogenotrophic methanogens, it is predicted that the two energy-conserving hydrogenases (Eha and Ehb) have distinct roles (26). The Ehb appears to reduce low potential electron carriers utilized in autotrophic CO₂ fixation (16). Anabolic enzymes likely to be coupled to Ehb in this manner include (i) the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and the pyruvate oxidoreductase (Por), which catalyze the first two steps of carbon assimilation; (ii) the α-ketoglutarate oxidoreductase (Kor), which catalyzes the final step in the incomplete reductive tricarboxylic acid cycle; and (iii) the indolepyruvate oxidoreductase (Ior) and the 2-oxoisovalerate oxidoreductase (Vor), which are involved in amino acid biosynthesis from aryl and branched-chain acids, respectively. Support for these conclusions comes in large part from the phenotype of an M. maripaludis ehb gene replacement mutant S40, which was only capable of limited growth in the absence of acetate and amino acids (16). Furthermore, expression of CODH/ACS, Por, and Vor were significantly upregulated in the mutant, providing further evidence for a role of Ehb in these processes (16). In contrast, there is no direct evidence for the role of Eha. By analogy with the Methanosarcina Ech, it could be involved in generating reducing equivalents for the reduction of CO₂ to formylmethanofuran. Alternatively, hydrogenotrophic methanogens may have an alternative method of CO₂ reduction (27), and Eha could have another function entirely.In spite of some functional similarities between the Ech of the aceticlastic methanogens and Eha or Ehb of hydrogenotrophs, the structures of their operons are very different (Fig. ​(Fig.1).1). Based upon sequence comparisons, all of these membrane-bound hydrogenases possess conserved large and small hydrogenase subunits, a 2[4Fe-4S] ferredoxin, and an integral membrane ion translocator (3, 8, 26). Otherwise, the structures are very different. The purified Ech from Methanosarcina barkeri contains six polypeptides encoded by the six genes of the ech operon (8, 11). The Eha and Ehb hydrogenases have never been purified. The eha and ehb operons from the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus comprise 20 and 17 genes, respectively (23, 26). Most of these genes are predicted to encode transmembrane proteins, although there are also several polyferredoxins and hydrophilic proteins (26). Many of these genes are not homologous to the M. barkeri ech genes. The Methanococcus maripaludis genome contains homologs to the M. thermautotrophicus eha and ehb genes, although only nine of the ehb genes are contiguous on the genome (Fig. ​(Fig.1).1). In the present study, the Ehb from the hydrogenotrophic methanogen Methanococcus maripaludis was analyzed. M. maripaludis is a model organism that can be easily genetically modified. Furthermore, its genome has been sequenced, and many of its biochemical pathways have been characterized.Open in a separate windowFIG. 1.Genetic map of Methanosarcina barkeri ech (A), Methanothermobacter marburgensis ehb (MTH1235-1251) (B), and Methanococcus maripaludis ehb (MMP1631-1629) (C) operons. Genes encoding integral membrane proteins found only in Ehb are indicated in blue, integral membrane proteins conserved in both Ech and Ehb are blue with diagonal stripes, hydrogenase small subunits are yellow, hydrogenase large subunits are red, 4Fe-4S motif-containing proteins are brown, and other hydrophilic proteins present in Ehb but absent from Ech are gray. Notably, M. maripaludis contains homologs to all of the M. marburgensis ehb genes, but many are unlinked to the major gene cluster and not shown. Based upon references 5, 8, 11, and 26.     

Methanosarcina spp. Drive Vinyl Chloride Dechlorination via Interspecies Hydrogen Transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-¹⁴C]acetate to ¹⁴CO₂ when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H₂) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H₂ levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H₂ levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H₂ as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO₂ plus H₂, driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Relation of coenzyme F420 to the activity of methanogenic microorganisms

Summary The relationship between the coenzyme F₄₂₀ content and the activity of methanogenic microorganisms was investigated under different cultivation conditions in anaerobic reactors. The coenzyme F₄₂₀ concentration depends on the substrate used and the cultivation conditions. Coenzyme F₄₂₀ appears not to be a measure of the total methanogenic activity but rather a measure of the amount of methanogenic microorganisms in mixed anaerobic cultures.