Studies of the C-Terminal Region of the Gamma Subunit of the Chloroplast Atp Synthase

Two highly conserved amino acid residues, an arginine and a glutamine, near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment. Forming the catch is hypothesized to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. A series of single and multiple amino acid substitutions of the two analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit, as well as the neighboring residue gammaArg302, were constructed utilizing site-directed mutagenesis. The mutations were introduced with the intention of eliminating any charge-charge or hydrogen bonding interactions between the catch residues and the anionic loop on the beta subunit, thereby testing the hypothesis. Each mutant gamma subunit was assembled together with the alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma subunit rotation. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat 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 >90% inhibited, whereas the CaATPase activities were inhibited by less than 50%. In a second study, the gamma catch mutants were assembled with the native CF1 alpha and beta subunits and, in some cases, with the epsilon and delta subunits followed by reconstitution with CF1 -free thylakoid membranes. The Mg2+-dependent and Ca 2+-dependent ATP hydrolysis activities of the native assembled alpha 3beta3gamma complex both increased by more than 3-fold as a result of the mutations; however, the sulfite-stimulated activity decreased by more than 60%. Proton-driven ATP synthesis of the thyloakoid-bound mutant enzyme also decreased by about 80% compared to the wild type enzyme. Nucleotide binding and exchange studies of CF1 using TNP-ADP revealed two nucleotide binding sites with completely different properties. One site binds and exchanges nucleotides rapidly and is likely to be a catalytic site while the other site displays slow nucleotide binding and exchange and appears to be non-catalytic. The triple mutant containing three alanines in place of Arg302, Arg304 and Gln305 within the CF1 gamma subunit displayed higher exchange rates in the fast filling, catalytic site but failed to exchange the majority of the TNP-ADP in the slow filling, non-catalytic site even after 30 minutes of incubation with ATP hydrolysis substrates. The results of these studies indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). However, contrary to the hypothetical role in orchestrating catalytic cooperativity among the nucleotide binding sites, the catch is not essential for ATP hydrolysis. Instead, the catch appears to be involved primarily in the process of oxyanion activation of MgATP hydrolysis and in coupling the proton gradient to ATP synthesis. The results have led to the new hypothesis that the catch residues perform the critical function of substrate (MgADP) recruitment during photophosphorylation.