Electrostatic Interaction Between Rotor and Stator Subunits in the F_o Motor of E. coliATP Synthase

Abstract

F1Fo ATP synthase is the ubiquitous biomolecular machine that catalyzes the final step of oxidative phosphorylation and is therefore the primary producer of ATP across all domains of life. The membrane‐embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which provides two aqueous half channels that facilitate proton translocation. The mechanism by which proton translocation is converted into torque on the c‐ring is not fully defined. Previous work has suggested that conserved residues aAsp92, aGlu196, and cArg50 in the proton exit pathway are important for proton transport and possibly for torque generation. To clarify the roles of these residues, we generated 22 mutants and assayed their growth on succinate and in vitro ATP synthesis, H+ pumping, and permeability activities. Mutations of aGlu196 had only a mild effect on proton pumping, while moderately inhibiting ATP synthesis. These results indicate that aGlu196 is likely not interacting with cArg50 but interestingly do suggest that it may have a greater role in ATP synthesis than proton pumping. In contrast, mutations of aAsp92 were not well tolerated, and mutations that reverse the charge of cArg50 caused a substantial defect. These results along with the structural proximity of these residues suggest that they may interact electrostatically across the rotor stator interface.