Engineering of ATP synthase for enhancement of proton-to-ATP ratio

Summary F o F 1 -ATP synthase (F o F 1 ) interconverts the energy of the proton motive force ( pmf ) and that of ATP via mechanical rotation of the rotor complex. The H + /ATP ratio, one of the most crucial parameters in bioenergetics, varies among species due to the different number of H + -binding c -subunits, resulting in H + /ATP ratios ranging from 2.7 to 5. The present study attempted to enhance the H + /ATP ratio significantly by employing a novel approach that differs from that of nature. We engineered F o F 1 to form multiple peripheral stalks, each bound to a proton-conducting a -subunit. Engineered F o F 1 showed an H + /ATP ratio of 5.9, beyond the highest among naturally occurring F o F 1 s, enabling ATP synthesis at a low pmf , at which wild-type enzymes are unable to synthesize ATP. Structural analysis showed that the engineered F o F 1 formed up to three peripheral stalks and the a -subunits. This study not only provides important insights into the H + -transport mechanism of F o F 1 but also opens the possibility of engineering the foundation of cell bioenergetics.