Studies of the C-terminal Region of the Gamma Subunit of the Chloroplast ATP Synthase

Abstract

The C-terminal region of the γ subunit of the ATP synthase forms a "catch" with an anionic loop on one of the three β subunits within the catalytic αβ hexamer of the F1 segment. A series of single and multiple amino acid substitutions within this region of the chloroplast γ subunit were constructed utilizing site-directed mutagenesis. The mutations were reconstituted with α and β subunits into both hybrid and native enzymes. The kinetics analysis of the mutant enzymes suggests that the "catch" is not required for ATP hydrolysis but critical for proton coupling and ATP synthesis functions. Nucleotide binding and exchange studies using TNP-ADP showed that substituting three residues in the "catch" region simultaneously inhibited the nucleotide exchange at a non-catalytic site. The results have led to the new hypothesis that the catch residues perform the critical function of substrate (MgADP) recruitment and enzyme activation during photophosphorylation.