Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F o F 1 -ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F o has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F o subunits a and c by improving the time resolution to 10 ls at unprecedented S/N, and by using Escherichia coli F o F 1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 ls to form and 175 ls to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of B361 while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F o motor where c-ring rotational diffusion is limited to 361.

F 1 -ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120°power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35°from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85°to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85°of rotation, consistent with a transition from F 1 structures 2HLD1 and 1H8E (Protein Data Bank).

A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F(1)-ATPase gamma-subunit rotation was determined to be 7.62 +/- 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg(2+)-ATPase k(cat) of 130 s(-)(1) (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F(1) was 63.3 +/- 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F(1), resulting in direct implications for its rotational mechanism.

The complexes [VO(H(2)O)ada] (1), [VO(H(2)O)Hheida] (2), and [VO(H(2)O)aeida] (3) (H(2)ada, N-(carbamoylmethyl)iminodiacetic acid; H(3)heida, N-(2-hydroxyethyl)iminodiacetic acid; H(2)aeida, N-(2-aminoethyl)iminodiacetic acid) were synthesized and crystallographically characterized. Crystallographic parameters for 1.2H(2)O: monoclinic, space group P2(1)/c (No. 14), a = 7.327(2) Å, b = 23.386(7) Å, c = 7.258(3) Å, alpha = 90 degrees, beta = 110.95(2) degrees, gamma = 90 degrees, V = 1204.6(7) Å(3), Z = 4, R1 = 0.0353, and wR(2)() = 0.0848. Crystallographic parameters for 2.H(2)O: orthorhombic, space group Pbca (No. 61), a = 10.512(2) Å, b = 11.727(2) Å, c = 16.719(5) Å, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees, V = 2060.6(8) Å(3), Z = 8, R1 = 0.0297, and wR(2)() = 0.0758. Crystallographic parameters for 3: monoclinic, space group P2(1)/c (No. 14), a = 6.785(1) Å, b = 9.714(2) Å, c = 14.959(2) Å, alpha = 90 degrees, beta = 95.12(1) degrees, gamma = 90 degrees, V = 982.2(3) Å(3), Z = 4, R1 = 0.0298, and wR(2)() = 0.0762. In each structure, the tetradentate ligand is disposed so that the tertiary nitrogen is bound trans to the vanadyl oxo, and the rest of the donors occupy equatorial coordination positions. In solution, the structural integrity of these compounds is maintained as observed by UV/visible and EPR spectroscopies, and axial ligation by nitrogen is inferred on the basis of ESEEM spectroscopy. The implications of this study with respect to understanding the coordination environment of VO(2+) in the reduced, inactive form of vanadium bromoperoxidase (VBrPO) are discussed, and it is proposed that significant changes in the coordination environment of vanadium in VBrPO occur upon its reduction, which may provide a plausible explanation for its irreversible inactivation.