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

FoF1-ATP synthase (FoF1) 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 FoF1 to form multiple peripheral stalks, each bound to a proton-conducting a-subunit. Engineered FoF1 showed an H+/ATP ratio of 5.9, beyond the highest among naturally occurring FoF1s, enabling ATP synthesis at a low pmf, at which wild-type enzymes are unable to synthesize ATP. Structural analysis showed that the engineered FoF1 formed up to three peripheral stalks and the a-subunits. This study not only provides important insights into the H+-transport mechanism of FoF1 but also opens the possibility of engineering the foundation of cell bioenergetics.