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Microbial life under extreme energy limitation and the evolution of living matter

 

Life on earth evolved in the absence of oxygen and still many natural environments like the intestinum of animals, soils of terrestric origin, of ponds and oceans as well as sewage and biogas plants do not contain oxygen. Nevertheless these environments are heavily populated by microbes that grow in the absence of oxygen, like the typical fermenting bacteria such as lactic acid bacteria. However, there are groups of strictly anaerobic microbes that evolved very early in life and have an autotrophic life style: CO₂ is reduced to biomass with electrons coming from molecular hydrogen or carbon monoxide. Typical examples for this are methanogenic archaea and acetogenic bacteria that use the same pathway, the Wood-Ljungdahl pathway (WLP) for CO₂ reduction. The Wood-Ljungdahl pathway is the only pathway known to couple CO₂ fixation with the synthesis of ATP. Therefore, both prerequisites to sustain life are fulfilled and, thus, the WLP is considered as one of the first or even the first biochemical pathway on earth. Although the pathway allows growth, the energy that can be gained from it is very, very little. Our group is interested in how the pathway is coupled to energy conservation (ATP synthesis). The question can be made more general: what is the minimal biological energy quantum to sustain life? What are the enzymes involved, what are their reaction mechanisms and how can we make use of the pathway in industrial applications?

 

 

1. The Wood-Ljungdahl pathway in acetogenic bacteria

Acetogenic bacteria are a specialized group of strictly anaerobic bacteria that produce acetate from hydrogen and carbon dioxide according to:

 

4 H₂ + 2 CO₂ -> CH₃COOH + 2 H₂O       ΔG^0’ = -95 kJ/mol

 

Acetogens all employ the Wood-Ljungdahl pathway for CO₂ reduction, but they differ in how they couple it to the synthesis of ATP. The figure below shows the recently established biochemistry and bioenergetics for the model bacterium Acetobacterium woodii.

 

The WLP module reduces two mol of CO₂ to acetate. Electrons are gained from the oxidation of H₂ by an electron-bifurcating hydrogenase (Hyd A-D) that reduces ferredoxin and NAD simultaneously. CO₂ reduction to formate is catalyzed by a novel type of enzyme, a hydrogen-dependent CO₂ reductase. The redox pool (ferredoxin and NAD) is balanced by the Rnf complex, a novel type of respiratory enzymes that couples the exergonic electron transfer from reduced ferredoxin to NAD with export of Na⁺. The electrochemical Na⁺ gradient across the cytoplasmic membrane then drives the synthesis of ATP via a unique Na⁺ -F₁F_O ATP synthase. All together, only 0.3 mol of ATP is synthesized via the formation of one mol of acetate. We are currently in the process of analyzing structure and function of the novel enzymes (click on their names to learn more about them).

The Wood-Ljungdahl pathway (WLP) in A. woodii. Hydrogen is oxidized by an electron-bifurcating hydrogenase reducing NAD and ferredoxin (Fd) in equal amounts. The reduction of two CO₂ to acetate consumes more NADH then ferredoxin thus ferredoxin needs to be oxidized at the Rnf complex fueling sodium-ion translocation. The hydrogen-dependent CO₂ reductase is an adaptation to this mode of energy conservation by using hydrogen instead of ferredoxin for CO₂ reduction.

 

 

References

Schuchmann, K., Müller, V. (2013) Direct and reversible hydrogenation of CO₂ to formate by a bacterial carbon dioxide reductase. Science 342 : 1382-1385.

Mayer, F., Müller, V. (2013) Adaptations of anaerobic archaea to life under extreme energy limitation. FEMS Microbiol. Rev. 38 : 449-472.

Müller, V., Frerichs, J. (2013) Acetogenic bacteria. In: Encyclopedia of life sciences. www.els.net.

Poehlein, A., Schmidt, S., Kaster, A.-K., Goenrich, M., Vollmers, J., Thürmer, A., Bertsch, J., Schuchmann, K., Voigt, B., Hecker, M., Daniel, R., Thauer, R.K., Gottschalk, G., Müller, V. (2012) An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis. PLoS ONE 7 : e33439.

Biegel, E., Müller, V. (2010) Bacterial Na⁺ translocating ferredoxin:NAD-oxidoreductase. Proc. Natl. Acad. Sci. USA 107 : 18138-18142.

Schmidt, S., Biegel, E., Müller, V. (2009) The ins and outs of Na⁺ bioenergetics in Acetobacterium woodii. Biochim Biophys. Acta 1787 : 691-696.

Müller, V. (2009) Incredible anaerobes – more bioenergetic surprises to come. Environ. Microbiol. Reports 1 : 13-14.

Deppenmeier U., Müller, V. (2008) Life close to the thermodynamic limit: how methanogenic archaea conserve energy. Results Probl. Cell Differ. 45 : 123-152.

Lewalter, K.Y., Müller, V. (2006) Bioenergetics of archaea: ancient energy conserving mechanisms developed in the early history of life. Biochim. Biophys. Acta 1757 : 437-445.

 

 

2. Caffeate-Respiration in A. woodii

The WLP module catalyzing CO₂ reduction to acetate can be regarded to have a function solely to reoxidize NADH, the end product of the anaerobic respiration catalyzed by the Rnf complex. Indeed, acetogens can use other modules instead of the WLP for reoxidizing NADH. These modules catalyze reduction of nitrate, fumarate or phenylacrylates. Under these conditions, acetate is not produced and the lifestyle of the acetogenic bacteria becomes non-acetogenic!

 

Phenylacrylates are products of lignin degradation and the biochemistry and bioenergetics of caffeate metabolism has been studied in detail in A. woodii. Caffeate is reduced to hydrocaffeate that is not further metabolized but secreted into the medium. Since caffeate is only used as an electron acceptor, this type of metabolism was termed caffeate respiration, a novel type of anaerobic respiration. The first step in caffeate respiration in A. woodii is the activation of the electron acceptor caffeate to caffeyl-CoA. This is first done by a caffeyl-CoA synthethase at the expense of ATP hydrolysis. Caffeyl-CoA is then reduced to hydrocaffeyl-CoA by the caffeyl-CoA reductase. After the first molecule of hydrocaffeyl-CoA has been produced, the CoA is transferred by a CoA transferase from hydrocaffeyl-CoA to caffeate, thus saving the ATP for activation.

 

The caffeyl-CoA reductase is another electron bifurcating enzyme  that reduces ferredoxin. Reduced ferredoxin is then reoxidized at the Rnf complex. Caffeate respiration is another exciting example of a metabolic scenario that relies only on electron-bifurcation followed by Rnf-mediated ion export for ATP synthesis.

 

Model of caffeate reduction in Acetobacterium woodii. Electron transfer from molecular hydrogen to the acceptor caffeate is coupled to the translocation of Na+ across the cytoplasmic membrane via the Rnf complex. The CarC/EtfAB complex regenerates reduced ferredoxin that can again fuel the Rnf complex. Caffeate is either initially activated by an ATP-dependent caffeyl-CoA synthase (CarB) or, in the steady state of caffeate respiration, by the CoA transferase CarA.

 

 

 

References

Hess, V., González, J.M., Parthasarathy, A., Buckel, W., Müller, V. (2013) Caffeate respiration in the acetogenic bacterium Acetobaceterium woodii: a CoA loop saves energy for caffeate activation. Appl. Environ. Microbiol. 79 : 1942-1947.

Bertsch, J., Parthasarathy, A., Buckel, W., Müller, V. (2013) An electron-bifurcating caffeyl-CoA reductase. J. Biol. Chem. 288 : 11304-11311.

Hess, V., Vitt, S., Müller, V. (2011) A caffeyl-coenzyme A synthetase initiates caffeate activation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii. J. Bacteriol. 193 : 971-978.

Imkamp, F., Biegel, E., Jayamani, E., Buckel, W., Müller, V. (2007) Dissection of the caffeate respiratory chain in the acetogen Acetobacterium woodii: identification of a Rnf-type NADH dehydrogenase as potential coupling site. J. Bacteriol. 189 : 8145-8153.

Dilling, S., Imkamp, F., Schmidt, S., Müller, V. (2007) Regulation of caffeate respiration in the acetogenic bacterium Acetobacterium woodii. Appl. Environ. Microbiol. 73 : 3630-3636.

Imkamp, F., Müller V. (2002) Chemiosmotic energy conservation with Na⁺ as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii. J. Bacteriol. 184 : 1947-1951.

 

 

3. Formate oxidation to hydrogen and carbon dioxide

 

The thermophilic archaeon Thermococcus onnurineus isolated from a deep sea floor growth by the conversion of

 

HCOOH -> CO₂ + H₂

 

The free energy charge of this reaction is positive (ΔG⁰ = + 1.3 kJ/mol) at 25°C but becomes slightly exergonic at 80°C (ΔG⁰ = - 2.6 kJ/mol). An exciting example of a substrate that can only sustain life at high temperatures. However, the reaction is still only slightly exergonic and therefore had always been excluded to sustain life of pure cultures. How T. onnurineus makes a living from it, is illustrated in the figure below.

 

It has a membrane-bound formate hydrogen lyase system composed of a formate dehydrogenase, a membrane-bound hydrogenase and a Na⁺/H⁺ antiporter. Oxidation of formate first leads to H⁺ export followed by an exchange to Na⁺ via the Na⁺/H⁺ antiporter. The Na⁺ gradient drives synthesis of ATP via the Na⁺ ATP synthase. A coupling of the membrane-bound hydrogenase module to the antiporter module may result in ion/e^- stoichiometries lower than one. So far, the minimum biological energy quantum was seen at around -20 kJ/mol, the amount of energy required to pump out one ion into the periplasm. With the combined action of a transporter and an exchanger, any value below –20 kJ/mol would be sufficient for ion translocation. Since the exchanger has a stoichiometry of <1, less than one ion can be pumped out of the cell, which is sufficient to sustain life.   

 

Energy conservation in T. onnurineus. Fdh, formate dehydrogenase; Mfh, multisubunit membrane-bound hydrogenase; Mrp, multisubunit Na⁺/H⁺ antiporter. The A₁A_O uses Na⁺ as coupling ion.

 

 

 

 

References

Lim, J.K., Mayer, F., Kang, S.G., Müller, V. (2014) Energy conservation by oxidation of formate to carbon dioxide and hydrogen via sodium ion current in a hyperthermophilic archaeon. Proc. Natl. Acad. Sci. USA, doi:10.1073/pnas.1407056111.