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Electron-bifurcating enzymes in the energy metabolism of the model acetogen, Acetobacterium woodii

Subject Area Metabolism, Biochemistry and Genetics of Microorganisms
Term from 2012 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 221575835
 
Up to the year 2008, only two mechanisms to drive endergonic cellular reactions were known. Cytoplasmic reactions are usually driven by hydrolysis of ATP (or other nucleotides) and membrane-bound processes by the electrochemical ion potential across the membrane. In 2008, a third mechanism was discovered: soluble enzymes that drive endergonic redox reactions by coupling to an exergonic redox reaction. Usually one electron donor, but two different electron acceptors are used. The electrons coming from the donor are bifurcated: one goes energetically „downhill“ and this provides the energy for the other electron going energetically „uphill“. Bifurcation of electrons usually requires a flavin, and therefore this process is also referred to as flavin-based electron bifurcation (FBEB). The discovery of FBEB has changed our understanding of anaerobic metabolism fundamentally. It does not only increase the energetic efficiency of anaerobic metabolism in general but in particular allows autotrophic growth of acetogenic bacteria, an ecophysiological and evolutionary important group of strictly anaerobic bacteria, on H2+CO2. The pathway involved is considered „ancient“ and maybe one of the oldest on earth since it is the only one that combines carbon dioxide fixation with the synthesis of ATP. Therefore, FBEB may be an ancient mechanism to overcome energetic barriers in metabolism. The acetogenic bacterium Acetobacterium woodii conserves energy by carbonate respiration (acetogenesis) or caffeate respiration. The respiratory chain is identical in both cases and consists of a ferredoxin-NAD:oxidoreductase (Rnf) and an ATP synthase that are coupled via a transmembrane electrochemical Na+ gradient. Reduction of ferredoxin with H2 as electron donor is highly endergonic. In the first funding period we have isolated and characterized a tetrameric, electron bifurcating hydrogenase that overcomes the barrier by FBEB and reduces ferredoxin and NAD simultaneously; mutagenesis experiments revealed that this hydrogenase is essential for autotrophy. During caffeate respiration, there is an additional electron-bifurcating enzyme, the caffeyl-CoA reductase/Etf complex that couples caffeyl-CoA reduction with NADH as electron donor to simultaneous ferredoxin reduction. The caffeyl-CoA reductase/Etf complex was purified and characterized, its 3D structure was solved and enabled first steps into a structure-based analysis of electron flow and energy coupling by site-directed mutagenesis. The lactate dehydrogenase/Etf complex is the third electron-bifurcating enzyme discovered; it enables A. woodii to grow on lactate. Since the encoding genes are widespread in bacteria, this seems to be a common way of lactate oxidation in anaerobes. In the next funding period, we will built on these discoveries and unravel electron flow and energetic coupling in these enzymes as well as study their physiological importance by genetic means.
DFG Programme Research Grants
 
 

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