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The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na+-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70–120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg2+. In addition, activity was strictly dependent on Na+ with a Km of 1.1 mm. Hydrolysis of pyrophosphate was accompanied by 22Na+ transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that 22Na+ transport was primary and electrogenic. Next to the Na+-motive ferredoxin:NAD+ oxidoreductase (Fno or Rnf), the Na+-pyrophosphatase is the second primary Na+-translocating enzyme in A. woodii.
This thesis is based on the following publications (in chronological order): 1. Biegel, E., S. Schmidt & V. Müller (2009) Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodii. Environ. Microbiol. 11: 1438-1443. My contribution: Amplification, sequence determination and analysis of Rnf homologues, enrichment of the Rnf complex 2. Biegel, E. & V. Müller (2010) Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc. Nat. Acad. Sci. U. S. A. 107: 18138-18142. My contribution: I designed and performed all experiments shown and interpreted the data. 3. Biegel, E., S. Schmidt, J. Gonzáles & V. Müller (2010) Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell. Mol. Life Sci., in press. DOI: 10.1007/s00018-010-0555-8. My contribution: I was involved in writing all chapters except chapters: „phylogenetic analyses of rnf genes“ and „distribution of rnf genes“. 4. Biegel, E. & V. Müller (2010) A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii. J. Biol. Chem., in press. DOI: 10.1074/jbc.M110.192823. My contribution: I designed and performed all experiments shown and interpreted the data.