pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

Abstract

Most cellular ATP is made by rotary F1Fo ATP synthases using proton translocation-generated clockwise torque on the Fo c-ring rotor, while F1-ATP hydrolysis can force anticlockwise rotation and proton pumping. Although the interface of stator subunit-a containing the transmembrane half-channels and the c-ring is known from recent F1Fo structures, the torque generating mechanism remains elusive. Here, single-molecule studies reveal pH-dependent 11{degrees} rotational sub-steps in the ATP synthase direction of the E. coli c10-ring of F1Fo against the force of F1-ATPase-dependent rotation that result from H+ transfer events from Fo subunit-a groups with a low pKa to one c-subunit of the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. Mutations of subunit-a residues in the proton translocation channels alter these pKa values, and the ability of synthase substeps to occur. Alternating 11{degrees} and 25{degrees} sub-steps then result in sustained ATP synthase rotation of the c10-ring.