Mutations in the β-subunit Thr¹⁵⁹ and Glu¹⁸⁴ of the Rhodospirillum rubrum F₀F₁ ATP synthase reveal differences in ligands for the coupled Mg²⁺- and decoupled Ca²⁺-dependent F₀F₁ activities

In the crystal structure of the mitochondrial F₁-ATPase, the β- Thr¹⁶³ residue was identified as a ligand to Mg²⁺ and the β-Glu¹⁸⁸ as directly involved in catalysis. We replaced the equivalent β-Thr¹⁵⁹ of the chromatophore F₀F₁ ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu¹⁸⁴ with Gln or Lys. The mutant β subunits were isolated and tested for their capacity to assemble into β-less chromatophore F₀F₁ and restore its lost activities. All of them were found to bind into the β-less enzyme with the same efficiency as the wild type β subunit, but only the β-Thr¹⁵⁹ → Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr¹⁵⁹ and Glu¹⁸⁴ are not required for assembly and that Glu¹⁸⁴ is indeed essential for all the membrane-bound chromatophore F₀F₁ activities. A detailed comparison between the wild type and the β. Thr¹⁵⁹ → Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F₀F₁ proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton- decoupled CaATPase activity. This clear difference between the ligands for Mg²⁺ and Mn²⁺, where threonine can he replaced by serine, and Ca²⁺, where only threonine is active, suggests that the β-subunit catalytic site has different conformational states when occupied by Ca²⁺ as compared with Mg²⁺. These different states might result in different interactions between the and γ subunits, which are involved in linking F₁ catalysis with F₀ proton-translocation and can thus explain the complete absence of Ca- dependent proton-coupled F₀F₁ catalytic activity.