Mutations in the beta-subunit Thr(159) and Glu(184) 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 F(1)ATPase, the beta-Thr(163) residue was identified as a ligand to Mg2+ and the beta-Glu(188) as direct ly involved in catalysis. We replaced the equivalent beta-Thr(159) Of the c hromatophore F0F1 ATP synthase of Rhodospirillum rubrum with Ser, Ala, or V al and the Glu(184) with Gin or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F 0F1 and restore its lost activities. All of them were found to bind into th e beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the asse mbled enzyme. These results indicate that both Thr(159) and Glu(184) not re quired for assembly and that Glu(184) is indeed essential for all the membr ane-bound chromatophore F0F1 activities. A detailed comparison between the wild type and the beta-Thr(159), Ser mutant revealed a rather surprising di fference. Although this mutant restored the wild type levels and all specif ic 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 threo nine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca2+ as compared with Mg2+ These di fferent states might result in different interactions between the beta and gamma subunits, which are involved in linking F-1 catalysis with F-0 proton -translocation and can thus explain the complete absence of Ca-dependent pr oton-coupled F0F1 catalytic activity.