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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pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism
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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- Award ID(s):
- 2119963
- NSF-PAR ID:
- 10331015
- Date Published:
- Journal Name:
- eLife
- Volume:
- 10
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.70016
