Volker Müller

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2+CO2 and formate are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H2:heterodisulfide oxidoreductase, the F420H2:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H+ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na+ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.

Bioenergetics of Methanogenesis

The bioenergetics of methanogens could be expected to have special features, given that they are members of the Archaea and utilize a number of unique reactions and coenzymes in the pathways of methanogenesis. This turns out to be so; however, it was not until 1980 that the elucidation of the biochemistry of methanogenesis attained a stage where the bioenergetics could be studied. Since then, progress has been made, reactions involved in energy conservation have been identified, and it became clear that at least some of these reactions proceed in or at the cytoplasmic membrane. For example, it now makes sense that only methylotrophic methanogens contain cytochromes that were first discovered in methanogens in 1979; likewise, the general sodium ion dependence of methanogenesis is now understood. Indeed, the bioenergetics of methanogenesis has unique features: ATP synthesis takes advantage of proton as well as of sodium gradients, both of which are generated by primary pumps.

Energetics of methanogenesis studied in vesicular systems.

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain. Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism. Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake. Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction. Their role and importance for the methanogenic metabolism are described.

Subunit Structure and Organization of the Genes of the A1A0 ATPase from the Archaeon Methanosarcina mazei Gö1

The proton-translocating A1A0 ATP synthase/hydrolase of Methanosarcina mazei Gö1 was purified and shown to consist of six subunits of molecular masses of 65, 49, 40, 36, 25, and 7 kDa. Electron microscopy revealed that this enzyme is organized in two domains, the hydrophilic A1 and the hydrophobic A0 domain, which are connected by a stalk. Genes coding for seven hydrophilic subunits were cloned and sequenced. From these data it is evident that the 65-, 49-, 40- and 25-kDa subunits are encoded by ahaA, ahaB, ahaC, and ahaD, respectively; they are part of the A1 domain or the stalk. In addition there are three more genes, ahaE, ahaF, and ahaG, encoding hydrophilic subunits, which were apparently lost during the purification of the protein. The A0 domain consists of at least the 7-kDa proteolipid and the 36-kDa subunit for which the genes have not yet been found. In summary, it is proposed that the A1A0 ATPase of Methanosarcina mazei Gö1 contains at least nine subunits, of which seven are located in A1 and/or the stalk and two in A0.

The Sodium Ion Cycle in Acetogenic and Methanogenic Bacteria: Generation and Utilization of a Primary Electrochemical Sodium Ion Gradient

Acetogenic bacteria are strictly anaerobic bacteria which use a wide variety of organic substrates for growth and acetate formation. Glucose, for example, is metabolized via the Embden-Meyerhoff pathway to pyruvate which is then split in the phosphoroclastic reaction to CO2 and acetyl CoA; the latter is converted to acetate via acetyl phosphate. The term “acetogen” is used for a variety of different organisms which produce acetate as a major fermentation end product. Homoacetogenic bacteria (which are referred to in the following as “acetogens”) differ from other organisms in that the CO2 formed is not an end product; the reducing equivalents obtained during glycolysis are used to reduce the 2 mol of CO2 produced in the phosphoroclastic reaction to acetate via the acetyl-CoA pathway. Therefore, homoacetogenic bacteria convert 1 mol of glucose to 3 mol of acetate via glycolysis and acetyl-CoA pathway. As the pathway is well established, one can calculate a net formation of 4 mol of ATP per mole of glucose fermented by the mechanism of substrate-level phosphorylation (SLP) (Fuchs, 1986; Ljungdahl, 1986; Wood et al., 1986), which is sufficient to ensure the energy supply of the cells during heterotrophic acetogenesis.

Proton translocation coupled to methanogenesis from methanol + hydrogen in methanosarcina barkeri

Addition of methanol to resting cells of Methanosarcina barkeri incubated under an atmosphere of molecular hydrogen resulted in an acidification of the medium. This acidification was not observed when H2 was replaced by N2 or air, or when the uncoupler tetrachlorosalicylanilide was present. 2‐Bromoethanesulfonate completely inhibited both methanogenesis and proton extrusion. N,N′‐Dicyclohexylcarbodiimide, an inhibitor of the proton‐translocating ATPase in M. barkeri, did not affect proton extrusion. Therefore, it could be concluded that proton translocation was coupled to the terminal methylcoenzyme M methylreductase reaction and that it was not due to an H+‐translocating ATPase. A maximum value of 4 H+ translocated per CH4 formed was calculated.

Differential effects of sodium ions on motility in the homoacetogenic bacteria Acetobacterium woodii and Sporomusa sphaeroides

AbstractThe strictly anaerobic homoacetogenic bacteriaAcetobacterium woodii andSporomusa sphaeroides differ with respect to their energy metabolism. Since growth as well as acetate and ATP formation ofA. woodii is strictly dependent on Na+, but that ofS. sphaeroides is not, the question arose whether these organisms also use different coupling ions for mechanical work, i.e. flagellar rotation. During growth on fructose in the presence of Na+ (50 mM), cells ofA. woodii were vigorously motile, as judged by light microscopy. At low Na+ concentrations (0.3 mM), the growth rate decreased by only 15%, but the cells were completely non-motile. Addition of Na+ to such cultures restored motility instantaneously. Motility, as determined in swarm agar tubes, was strictly dependent on Na+; Li+, but not K+ partly substituted for Na+. Of the amilorides tested, phenamil proved to be a specific inhibitor of the flagellar motor ofA. woodii. Growth and motility ofS. sphaeroides was neither dependent on Na+ nor inhibited by amiloride derivatives. These results indicate that flagellar rotation is driven by % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9vq-xc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0Firpepe0le9vr0-vr% 0-vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaiabgs5aeHGaai% qb-X7aTzaaiaWaaSbaaKqaGgaaieaacaGFobGaa4xyamaaCaaajiaO% beqaaiadacLHRaWkaaaaleqaaaaa!3D99!

The molecular structure of the Na+-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H+-translocating ATPases

\Delta \tilde \mu _{Na^ + }