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J Biol Chem, Vol. 275, Issue 2, 901-905, January 14, 2000

Mutations in the -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^*

Lubov Nathanson and Zippora Gromet-Elhanan^§

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

In the crystal structure of the mitochondrial F₁-ATPase, the -Thr¹⁶³ residue was identified as a ligand to Mg²⁺ and the -Glu¹⁸⁸ as directly involved in catalysis. We replaced the equivalent -Thr¹⁵⁹ of the chromatophore F₀F₁ ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu¹⁸⁴ with Gln or Lys. The mutant  subunits were isolated and tested for their capacity to assemble into a -less chromatophore F₀F₁ and restore its lost activities. All of them were found to bind into the -less enzyme with the same efficiency as the wild type  subunit, but only the -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 -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 be replaced by serine, and Ca²⁺, where only threonine is active, suggests that the -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  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.

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