The N-terminal region of the E subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their E subunits. To clarify the function of the E subunit from phototrophs, here we analyzed the E subunit-mediated inhibition (E-inhibition) of cyanobacterial F-1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal alpha-helix null E-mutants; one lacked the C-terminal alpha-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two alpha-helices or an alpha-helix and a beta-sandwich structure. All of these E-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F-1-ATPase in the presence of these E-mutants. The stop angular position of the gamma subunit in the presence of the E-mutant was identical to that in the presence of the WT E. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80 degrees forcing of the gamma subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the E-mutants or by the WT E subunit. These results imply that the C-terminal alpha-helix domain of the E subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial E, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the E subunit-mediated regulation of ATP synthase.