The two opposed rotary molecular motors of the FF-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120° power strokes separated by dwells when F synthesizes or hydrolyzes ATP. F and F complexes connect via a central rotor stalk and a peripheral stator stalk. A major unresolved question is the mechanism in which the interaction between subunit-a and rotating subunit-c-ring in the F motor uses the flux of H across the membrane to induce clockwise rotation against the force of counterclockwise rotation driven by the F-ATPase. In single-molecule measurements of FF embedded in lipid bilayer nanodiscs, we observed that the ability of the F motor to form transient dwells increases with decreasing pH. Transient dwells can halt counterclockwise rotation powered by the F-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of F, and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Because the F proton half-channels that access the periplasm and the cytoplasm are exposed to the same pH, these data are consistent with the conclusion that the periplasmic half-channel is more easily protonated in a manner that halts ATPase-driven rotation by blocking ATPase-dependent proton pumping. The fit of transient dwell occurrence to the sum of three Gaussian curves suggests that the asymmetry of the three ATPase-dependent 120° power strokes imposed by the relative positions of the central and peripheral stalks affects c-subunit stepping efficiency.
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 counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.
F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.
The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa’s. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a.
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° 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° and 25° sub-steps then result in sustained ATP synthase rotation of the c10-ring.
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