The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus 0F4 is adapted to alkaliphilic lifestyle

Summary: In the c-ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c-ring from the alkaliphile Bacillus pseudofirmusOF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X-ray structures of the wild-type c₁₃ ring at pH 9.0 and a 'neutralophile-like' mutant (P51A) at pH 4.4, at 2.4 and 2.8 Å resolution, respectively, reveal a dependency of the conformation and protonation state of the proton-binding glutamate (E⁵⁴) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide (DCCD) demonstrate a greater flexibility of E⁵⁴ in the mutant due to reduced water occupancy within the H⁺ binding site. A second 'neutralophile-like' mutant (V21N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E⁵⁴ carboxylate. The study directly connects subtle structural adaptations of the c-ring ion binding site to in vivo effects of alkaliphile cell physiology.