Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocation-generated clockwise torque on the F O c-ring rotor, while F 1 -ATP hydrolysis can force counterclockwise rotation and proton pumping. The F O torque-generating mechanism remains elusive even though the F O interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F 1 F O structures. Here, single-molecule F 1 F O 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 F O to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through F O 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 Escherichia coli c 10 -ring of F 1 F O against the torque of F 1 -ATPase-dependent rotation that result from H + transfer events from F O 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 c 10 -ring occur to sustain F 1 F O ATP synthesis.
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F1FO ATP synthase molecular motor mechanisms
The F-ATP synthase, consisting of F 1 and F O motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F 1 (αβ) 3 ring stator contains three catalytic sites. Single-molecule F 1 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 (β E , empty; β D , ADP bound; β T , ATP-bound). During a power stroke, β E binds ATP (0°–60°) and β D 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 β E upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from β E to β D , which changes catalytic site conformations. In F 1 F O , the membrane-bound F O 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 F 1 . Single-molecule studies of F 1 F O embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F 1 -ATPase-driven rotation every 36° (at each c-subunit in the c 10 -ring of E. coli F 1 F O ) 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.
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- Award ID(s):
- 2119963
- NSF-PAR ID:
- 10418022
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 13
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Biological molecular motors transform chemical energy into mechanical work by coupling cyclic catalytic reactions to large-scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the F 1 F O ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in a surrounding stator orchestrated by mechanical deformation. We design the mechanism using DNA origami, characterize its structure via cryo-electron microscopy, and examine its dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. While the camshaft can rotate inside the stator by diffusion, the stator’s mechanics makes the camshaft pause at preferred orientations. By changing the stator’s mechanical stiffness, we accelerate or suppress the Brownian rotation, demonstrating an allosteric coupling between the camshaft and the stator. Our mechanism provides a framework for manufacturing artificial nanomachines that function because of coordinated movements of their components.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmicb.2022.965620
