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

F_oF₁-ATP synthase (F_oF₁) interconverts the energy of the proton motive force (pmf) and that of ATP through the mechanical rotation. The H⁺/ATP ratio, one of the most crucial parameters in bioenergetics, varies among species due to differences in the number of H⁺-binding c-subunits, resulting in H⁺/ATP ratios ranging from 2.7 to 5. In this study, we seek to significantly enhance the H⁺/ATP ratio by employing an alternative approach that differs from that of nature. We engineer F_oF₁ to form multiple peripheral stalks, each bound to a proton-conducting a-subunit. The engineered F_oF₁ exhibits an H⁺/ATP ratio of 5.8, surpassing the highest ratios found in naturally occurring F_oF₁s, enabling ATP synthesis under low pmf conditions where wild-type enzymes cannot synthesize ATP. Structural analysis reveals that the engineered F_oF₁ forms up to three peripheral stalks and a-subunits. This study not only provides valuable insights into the H⁺-transport mechanism of F_oF₁ but also opens up possibilities for engineering the foundation of cellular bioenergetics.