Mutations within the C-Terminus of the γ Subunit of the Photosynthetic F1-ATPase Activate MgATP Hydrolysis and Attenuate the Stimulatory Oxyanion Effect†

Two highly conserved amino acid residues near the C-terminus within the y subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three /5 subunits within the catalytic αβ hexamer of the F 1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to y subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F 1 y subunit were changed to leucine and alanine, respectively. Each mutant y was assembled together with a and /? subunits from Rhodospirillum rubrum F 1 into a hybrid photosynthetic F 1 that carries out both MgATPase and CaATPase activities and ATP-dependent y rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the k cat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the k cat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F 1 y subunit, like its mitochondrial counterpart, forms a catch with the a and β subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg 2+ ions, but is not essential for cooperative nucleotide exchange.