PH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism.

Seiga Yanagisawa,Wayne D Frasch

doi:10.7554/elife.70016

Abstract

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 Escherichia 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.

Highlights

The F1FO ATP synthase (Figure 1) that is found in all animals, plants, and eubacteria is comprised of two molecular motors that are attached by their rotors, and by their stators [1,2]
Contributions of subunit-a residues putatively involved in the ATP synthase H+ half-channels were assessed by the effects on transient dwell formation caused by mutations that converted charged or polar groups in subunit-a to hydrophobic leucine
Coli FOF1 molecules embedded in lipid bilayer nanodiscs [31], hereafter F1FO (Figure 1C)

Summary

Introduction

The F1FO ATP synthase (Figure 1) that is found in all animals, plants, and eubacteria is comprised of two molecular motors that are attached by their rotors, and by their stators [1,2]. Pmf-powered CW rotation of subunit- forces conformational changes to all catalytic sites in the ( )3-ring, which releases ATP from one catalytic site with each 120° rotational step [1,2]. In this manner, F1FO converts the energy from the pmf ( H+) into energy in the form of a non-equilibrium chemical gradient ( ATP) where the ATP/ADPPi concentration ratio is far in excess of that found at equilibrium

Methods

Results

Conclusion