Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F-1 is a soluble part of F0F1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The gamma subunit, which is the rotary shaft of F-1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of gamma assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of alpha(3)beta(3) hexamer of the F-1 molecule. Cyanobacterial and chloroplast gamma subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial gamma. To understand the structure function relationship of the gamma subunit, we prepared a mutant F-1 ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the gamma subunit is split into N-terminal alpha-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type gamma. Contrary to our expectation, the complexes containing the split gamma subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the gamma subunit. We demonstrate that insertion of the nick between two helices of the gamma subunit imparts resistance to ADP inhibition, and the C-terminal alpha-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F-1-ATP synthase.