Wayne D. Frasch

Microsecond time scale rotation measurements of single F1-ATPase molecules

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.

Direct observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase

Although single‐molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the FoF1‐ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in Fo has been particularly hampered by signal‐to‐noise and time resolution. Here, we show the presence of a transient dwell between Fo subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli FoF1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs 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 ∼36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the Fo motor where c‐ring rotational diffusion is limited to 36°.

Anatomy of F1-ATPase powered rotation

Significance We present a description of the angular velocity of the power stroke as a function of rotational position for the F1-ATPase molecular motor. The angular velocity of this motor, which rotates in 120° power strokes separated by catalytic dwells, is part of the FoF1 ATP synthase in oxidative phosphorylation, and has been thought not to vary. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur, providing direct evidence that angular velocity depends on substrate binding affinity. The data support a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank). F1-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 F1 structures 2HLD1 and 1H8E (Protein Data Bank).

Hydrogen peroxide as an alternate substrate for the oxygen-evolving complex

Photosystem II reaction centers evolve O2 in the dark when H2O2 is added as a substrate. Although some of this activity can be attributed to catalase, as much as 75% of the activity was not affected by the addition of 1 mM KCN. Several lines of evidence demonstrate that this KCN-insensitive O2 evolution from H2O2 in the dark is catalyzed by the cycling of S states in the oxygen-evolving complex including: inactivation of H2O2-mediated O2 evolution by Ca/EDTA washing; susceptibility of the activity to inhibition by amines like ammonia and Tris; inhibition by CCCP which is known to accelerate the rate of deactivation of the S2 state and; a direct dependence of the rate of O2 evolution on the presence of calcium and chloride.

Single Molecule Measurements of F1-ATPase Reveal an Interdependence between the Power Stroke and the Dwell Duration

Increases in the power stroke and dwell durations of single molecules of Escherichia coli F(1)-ATPase were measured in response to viscous loads applied to the motor and inhibition of ATP hydrolysis. The load was varied using different sizes of gold nanorods attached to the rotating gamma subunit and/or by increasing the viscosity of the medium using PEG-400, a noncompetitive inhibitor of ATPase activity. Conditions that increase the duration of the power stroke were found to cause 20-fold increases in the length of the dwell. These results suggest that the order of hydrolysis, product release, and substrate binding may change as the result of external load on the motor or inhibition of hydrolysis.

The participation of metals in the mechanism of the F1-ATPase

The Mg(2+) cofactor of the F(1)F(0) ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V(IV)=O)(2+) is a functional surrogate for Mg(2+) in the F(1)-ATPase. The (51)V-hyperfine parameters derived from EPR spectra of VO(2+) bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the (51)V-hyperfine parameters of the bound VO(2+), thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to beta(E) in mitochondrial F(1), activation of the chloroplast F(1)-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the gamma subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the beta(DP) site of mitochondrial F(1), the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from betaE197 in Chlamydomonas CF(1). Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.

The involvement of photosystem II-generated H2O2 in photoinhibition

The involvement of H2O2 generated by photosystem II (PSII) in the process of photoinhibition of thylakoids with a functional oxygen‐evolving complex (OEC) was investigated. The rate of photoinhibition was decreased to the rate of loss of activity in the dark when bovine Fe‐catalase was present during the photoinhibitory illumination. Photoinhibition was accelerated for both Cl−‐depleted and Cl−‐sufficient thylakoids when KCN was present to inhibit the thylakoid‐bound Fe‐catalase. We propose that these preparations become photoinhibed by reactions with H2O2 produced via oxidation of water by the Cl−‐depleted OEC and by reduction of O2 at the QB site when PSII is illuminated without an electron acceptor.

Interactions among γR268, γQ269, and the β Subunit Catch Loop of Escherichia coli F1-ATPase Are Important for Catalytic Activity

Removal of the ability to form a salt bridge or hydrogen bonds between the β subunit catch loop (βY297-D305) and the γ subunit of Escherichia coli F1Fo-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues βT304, βD305, βD302, γQ269, and γR268 were found to be very important for ATP hydrolysis catalyzed by soluble F1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue βD305 would not tolerate substitution with Val or Ser and had extremely low activity as βD305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the γ subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which αβγ intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven γ subunit rotation.

Interactions between Gamma R268, Gamma Q269 and the Beta subunit catch-Loop of E. coli F1 ATPase are Important for catalytic activity

Removal of the ability to form a salt bridge or hydrogen bonds between the β subunit catch loop (βY297-D305) and the γ subunit of Escherichia coli F1Fo-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues βT304, βD305, βD302, γQ269, and γR268 were found to be very important for ATP hydrolysis catalyzed by soluble F1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue βD305 would not tolerate substitution with Val or Ser and had extremely low activity as βD305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the γ subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which αβγ intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven γ subunit rotation.

A quantitative estimation of chloroplast thylakoid-bound coupling factor 1 by rocket immunoelectrophoresis

Chloroplast coupling factor 1 (CF,) is the extrinsic membrane protein fraction of the chloroplast energytransducing compIex [ 1,2]. It contains the active site(s) for both ATP synthesis and ATP hydrolysis, as well as other binding sites for adenine nucleotides [3,4]. Many experiments performed with washed thylakoid membranes designed to either alter the activity of CFt or measure the binding of ligands to the protein are often expressed on a chlorophyll basis because qu~titative estimations of the amount of CFr are difficult and time consuming. However, in [ 51 it is clearly shown that the amount of thylakoidbound CF, is directly related to the amount of surface area of the thylakoid membrane exposed to the stroma phase. The fraction of exposed lamellae is dramatically influenced by the environment in which the plants are grown, and hence, the amount of CFi can vary substantially from different chloroplast preparations. It would, therefore, be desirable to have a fairly rapid, quantitative method to estimate the amount of thylakoid-bound CFr. Two methods that have been used to estimate the amount of thylakoid-bound CF, are electron microscopy [6] and the amount of trypsin-induced ATPase activity [7]. Here we describe a rocket immunoelectrophoresis system for the quantitative analysis of CF, and show how the system can be used to estimate the amount of thylakoid-bound CF1.