The F1Fo ATP synthase is a multisubunit, membrane-bound enzyme that catalyzes the synthesis of the majority of cellular ATP. Mutations in several subunits in the human mitochondrial enzyme are of important clinical relevance. The enzyme works as a molecular motor that drives the rotary motion of some of the subunits. The enzymes from mitochondria, chloroplasts and the plasma membranes of bacteria are closely similar. In E. coli, F1 is composed of an (alpha-beta)3 ring that surrounds the gamma subunit, and also contains delta and epsilon subunits. The membrane embedded Fo is composed of a c10 subunit ring to which is attached the a subunit and two b subunits. During ATP synthesis, the movement of protons through Fo drives the rotation of the c10 subunit ring to which the gamma and epsilon subunits are attached that forces sequential conformational changes in the (alpha-beta)3 ring and results in the synthesis of ATP from each of the three (alpha-beta)3 heterodimers. The hydrolysis of ATP can also drive the rotation of the gamma subunit in the opposite direction. When F1 molecules are attached to a microscope cover slip, and a probe that can be observed under a microscope is attached to the gamma subunit, single molecules can be observed to rotate upon addition of ATP. Our long term objective is to elucidate the mechanism of ATPase-driven rotation of the gamma subunit. We propose the induction mechanism as a working hypothesis for rotation that will be critically examined. This hypothesis posits that Coulombic potential originating from residues that form hydrogen bonds and salt bridges between the (alpha-beta)3 ring and the gamma subunit contribute to the generation of gamma subunit torque. We will test this hypothesis by accomplishing the following aims. (1) The contribution of beta Catch Loop-gamma Subunit interactions to gamma subunit rotation will be examined by measuring the effects of mutations that eliminate these interactions on the rate of gamma subunit rotation measured using an innovative single molecule assay that we developed for this purpose. (2) The contribution of other known (alpha-beta)3 ring-gamma subunit interactions to gamma subunit rotation will be determined that include contacts at the gamma subunit N and C termini. (3) The contribution to rotation of (alpha-beta)3 ring residues that do not interact directly with the gamma subunit will be examined including those that communicate between the gamma subunit and the catalytic sites. (4) The contribution to gamma subunit rotation of (alpha-beta)3 ring-gamma subunit interactions that have not been identified in known crystal structures of F1 but are identified by tracking the rotational path of charged and polar gamma subunit residues around the inside of the (alpha-beta)3 ring.
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