Energy conservation by a hydrogenase-dependent chemiosmotic mechanism in an ancient metabolic pathway

The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H-2 formation and the generation of a transmembrane electrochemical ion gradient (Delta(mu ion)). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H-2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across themembrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.