Functional production of an archaeal ATP synthase with a V-type c subunit in Escherichia coli.

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na+-dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min-1·mg protein-1. Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na+ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na+, indicating a functional coupling of the F1 and FO domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg530) in the FO motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.

The structural features of Acetobacterium woodii F-ATP synthase reveal the importance of the unique subunit γ-loop in Na+ translocation and ATP synthesis.

The Na+ translocating F1 FO ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α3 :ß3 :γ:δ:ε:a:b2 :(c2/3 )9 :c1 and reveals an evolutionary path between synthases and pumps involving adaptations in the rotor c-ring, which is composed of F- and vacuolar-type c subunits in a stoichiometry of 9 : 1. This hybrid turbine couples rotation with Na+ translocation in the FO part and rotation of the central stalk subunits γ-ε to drive ATP synthesis in the catalytic α3 :ß3 headpiece. Here, we isolated a highly pure recombinant A. woodii F-ATP synthase and present the first projected structure of this hybrid engine as determined by negative-stain electron microscopy and single-particle analysis. The uniqueness of the A. woodii F-ATP synthase is also reflected by an extra 17 amino acid residues loop (195 TSGKVKITEETKEEKSK211 ) in subunit γ. Deleting the loop-encoding DNA sequence (γΔ195-211 ) and purifying the recombinant F-ATP synthase γΔ195-211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F-ATP synthase γΔ195-211 mutant revealed the same subunit composition as the wild-type enzyme and a minor reduction in ATP hydrolysis. When reconstituted into proteoliposomes ATP synthesis and Na+ transport were diminished, demonstrating the importance of the γ195-211 loop in both enzymatic processes. Based on a structural model, a coupling mechanism for this enzyme is proposed, highlighting the role of the γ-loop. Finally, the γ195-211 loop of A. woodii is discussed in comparison with the extra γ-loops of mycobacterial and chloroplasts F-ATP synthases described to be involved in species-specific regulatory mechanisms.

The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii.

The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+ When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.