Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F₁-ATPase and its role in divalent cation-dependent catalysis

We introduced mutations at the fully conserved residue Glu-195 in subunit β of Rhodospirillum rubrum F₁-ATPase. The activities of the expressed wild type (WT) and mutant β subunits were assayed by following their capacity to assemble into the earlier prepared β-depleted, membrane- bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, β-E195K, β-E195Q, and β-E195G, were found to bind as the WTβ into the β-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only β-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl₂ or MnCl₂ concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F₁β-E195 is equivalent to the mitochondrial F₁β-E199, which points into the tunnel leading to the F₁ catalytic nucleotide binding sites (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F₁ catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F₁ catalytic sites between different conformational states.