Rotational Mechanism of FO Motor in the F-Type ATP Synthase Driven by the Proton Motive Force

Abstract

In F_OF₁ ATP synthase, driven by the proton motive force across the membrane, the F_O motor rotates the central rotor and induces conformational changes in the F₁ motor, resulting in ATP synthesis. Recently, many near-atomic resolution structural models have been obtained using cryo-electron microscopy. Despite high resolution, however, static information alone cannot elucidate how and where the protons pass through the F_O and how proton passage is coupled to F_O rotation. Here, we review theoretical and computational studies based on F_O structure models. All-atom molecular dynamics (MD) simulations elucidated changes in the protonation/deprotonation of glutamate-the protein-carrier residue-during rotation and revealed the protonation states that form the "water wire" required for long-range proton hopping. Coarse-grained MD simulations unveiled a free energy surface based on the protonation state and rotational angle of the rotor. Hybrid Monte Carlo and MD simulations showed how proton transfer is coupled to rotation.

Keywords: FO motor; FOF1 ATP synthases; Monte Carlo simulations; coarse-grained model; molecular dynamics simulations.

Figure 1

Overview of the F_O motor. (A) Cartoon showing the geometry of the mitochondrial F_O motor. The rotational direction of the c-ring and movement of protons required for ATP synthesis are indicated. (B) Cartoons showing the mechanism of proton transfer between the a-subunit and c-ring. Green and orange spheres represent protonated (EH) and deprotonated (E⁻) glutamate, respectively. Black arrows indicate the net proton flow during ATP synthesis. R⁺ (blue) is a highly conserved arginine residue of the a-subunit. (C) Top (left) and side (middle) views of the mitochondrial F_O motor a-subunit (gray), and c-ring (cyan) (PDB ID: 6F36). Close-up (right) views of the square region in the middle cartoon. Representative residues with acidic, basic, and neutral side chains are indicated in red, blue, and green, respectively. The proton-carrier residue of c-subunit is cE111, the conserved arginine of a-subunit is aR239, and the representative residues of two half-channels are aE288 for the IMS channel and aE225 for the matrix channel. Water-accessible regions in the two half-channels, namely the IMS and matrix channels, are shown in yellow. The funnel-like space in the matrix channel is shown in pink.

Figure 2

Side-chain conformation coupled with the protonation states. (A) A 2.7 map of the V_O motor a-subunit (green) and c-ring (pink and yellow) (right panel). Water molecules near the interface between the α-subunit and c-ring (left panel). Red beads are defined by cryo-EM map and Coot function. Blue dots are defined by all-atom MD simulation (Roh et al., 2020) (B) Representative snapshot of all-atom MD simulation with protonated (left) and deprotonated (right) cE137. The left panel shows a water-wire (Roh et al., 2020) (C) Free energy surfaces of the closed Pogoryelov et al. (2010) and open conformations of cE62 in S. platensis. (D) Proton movement with changes in glutamate conformation. Black and red side chains indicate the closed and open glutamate conformation, respectively. Blue side chain indicates the highly conserved arginine of the a-subunit.

Figure 3

Free energy surface based on the rotational angle. (A,B) Free energy surfaces of individual protonation states obtained from coarse-grained MD simulations (Kubo et al., 2020). “Path 1” and “Path 2” represent the deprotonation-first and the protonation-first pathways, respectively. (C) Free energy surface of the c-ring rotation in yeast V_O motor. Five local minima (A–E) and transition states (TS) (Roh et al., 2020). (D) Representative snapshot of the TS state in (C). The a-subunit and c-ring interface is focused, with some polar residue side chains indicated with sticks. Blue dashed lines indicate hydrogen bonds (Roh et al., 2020).

Figure 4

Hybrid MC/MD simulation protocol and results. (A) Our hybrid MC/MD simulation protocol. We performed alternative short MC and MD simulations. (B) Representative trajectory of the cumulative c-ring rotational angle (top) and protonation state of each c-subunits (bottom). The bottom time courses are obtained from the red trajectory in the top panels. Protonated and deprotonated states are indicated in green and orange, respectively. (C) Protonation state distribution depends on the c-ring rotational angle. The rotational angle is indicated in red, and the proton transfer event at each angle is indicated in green (Kubo et al., 2020).

Figure 5

Coupling among c-subunits for F_O rotation. (A) Schematic cartoon of the a-subunit and c-ring of the F_O motor. The 10 c-subunits are labeled a–j. Circles represent 10 glutamates of the c-ring and 2 glutamates of the IMS and matrix channels. Green and orange represent protonated and deprotonated states, respectively. Purple represent cE56D mutant c-subunit. (B) ATP synthesis driven by NADH oxidation. c10 is the wild-type (WT); e to ej are the results of cE56D mutants, and E56Q are the results of c10(E56Q)- F_OF₁. Bars represent standard error. (C) Average rotational velocities for WT and mutants in coarse-grained MC/MD simulations. Bars represent standard error. (D) Cartoon showing the four phases of proton transfer, namely the resting time (gray), proton release duration (red), deprotonated rotation (green), and proton uptake duration (blue). (E,F) Representative time course of durations for the double mutant “ef” in (E) and “ej” in (F) (Mitome et al., 2022).