Across the tree of life, rotary molecular motors like the F1FO ATP synthase utilize a transmembrane nonequilibrium proton gradient to synthesize adenosine triphosphate (ATP), the biological energy currency. The catalytic portion of rotary motors, such as the F1 complex from E. coli and the V1 complex from S. cerevisiae, was purified and studied during ATP hydrolysis. Single-molecule assays utilized gold nanorods to investigate the kinetics of the F1-ATPase catalytic dwell, the biophysics of V1-ATPase, and the kinematics of the F1-ATPase power stroke. Observation of oscillatory rotor motion during the F1 catalytic dwell provided new insight as to how energy from ATP binding is stored during its three stages. That motion indicated a ratchet mechanism, in which F1 changed states according to first-order kinetics with a time constant τ = 0.182, showing that Stage-1 represents a pre-hydrolysis state and Stage-2 represents a post-hydrolysis state. F1 was then observed to return to 0° prior to its next power stroke (Stage-3), which explained why the three catalytic dwells remain 120° apart after many revolutions. Analysis of the 120° power stroke following Stage-3 was conducted in both V1 and F1, allowing comparative biology to elucidate defects in the ATPase mechanism, such as ADP inhibition and faltering rotation. It is noteworthy that the V1 rotary positions of ADP release and ATP binding are the opposite of F1, and that less elastic energy is stored in the V1 rotor due to differences in its catch loop. In both rotary ATPases, energy contributed by binding and hydrolysis can dissipate at multiple points. When the F1 catch loop contact between F1 βD305 and γQ269 was mutated, the elastic energy stored in the rotor dissipated dramatically. Dissipation was clearly shown by sustained Phase-1 decelerations, the distribution of ATP-binding dwells, and high-amplitude oscillations in γQ269L. These findings clarify evolutionary similarities and differences between eukaryotic V1, which is exclusively a hydrolase, and F1, which can both hydrolyze and synthesize ATP.

The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to the coiled-coil and globular foot domains of subunit-γ. The F1 complex can hydrolyze ATP in vitro in a manner that drives counterclockwise (CCW) rotation, in 120° power strokes, as viewed from the positive side of the membrane. The power strokes that occur in ≈ 300 μsec are separated by catalytic dwells that occur on a msec time scale. A single-molecule rotation assay that uses the intensity of polarized light, scattered from a 75 × 35 nm gold nanorod, determined the average rotational velocity of the power stroke (ω, in degrees/ms) as a function of the rotational position of the rotor (θ, in degrees, measured in reference to the catalytic dwell). The velocity is not constant but rather accelerates and decelerates in two Phases. Phase-1 (0° - 60°) is believed to derive power from elastic energy in the protein. At concentrations of ATP that limit the rate of ATP hydrolysis, the rotor can stop for an ATP-binding dwell during Phase-1. Although the most probable position that the ATP-binding dwell occurs is 40° after the catalytic dwell, the ATP-binding dwell can occur at any rotational position during Phase-1 of the power stroke. Phase-2 of the power stroke (60° - 120°) is believed to be powered by the ATP-binding induced closure of the lever domain of a β-subunit (as it acts as a cam shaft against the γ-subunit). Algorithms were written, to sort and analyze F1-ATPase power strokes, to determine the average rotational velocity profile of power strokes as a function of the rotational position at which the ATP-binding dwell occurs (θATP-bd), and when the ATP-binding dwell is absent. Sorting individual ω(θ) curves, as a function of θATP-bd, revealed that a dependence of ω on
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.