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 F₁F_O ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α₃:β₃:γ:δ:ε:a:b₂:(c_2/3)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 F_O part and rotation of the central stalk subunits γ-ε to drive ATP synthesis in the catalytic α₃:β₃ 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 (₁₉₅TSGKVKITEETKEEKSK₂₁₁) in subunit γ. Deleting the loop-encoding DNA sequence (γ_Δ195–211) and purifying the recombinant F-ATP synthase γ_Δ195–211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F-ATP synthase γ_Δ195–211 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.