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

In FOF1 ATP synthase, driven by the proton motive force across the membrane, the FO 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 FO and how proton passage is coupled to FO rotation. Here, we review theoretical and computational studies based on FO 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.

FOF1 ATP synthase, a ubiquitous enzyme that synthesizes most ATP in living cells, comprises two rotary motors: the membrane-embedded proton-driven FO motor and the catalytic F1 motor; these motors share the central rotor and peripheral stalk. The FO motor harbors a ring-shaped c-subunit oligomer, which serves as the rotor, and an a-subunit (stator), which mediates proton transfer between the c-ring and outer membrane aqueous environment [matrix and intermembrane space (IMS) of mitochondrial ATP synthase]. The a-subunit possesses two half-channels [confirmed for bacterial, mitochondrial (Srivastava et al., 2018; Spikes et al., 2020), and chloroplast (Hahn et al., 2018) FO motors]. One exchanges protons with IMS or outside the cell, and the other shuttles protons between the matrix and cytoplasm. Since these two half-channels are not directly connected, protons can only pass through the membrane via rotation of the c-ring. Therefore, the passage of protons through the membrane is coupled to the rotational motion of the c-ring (Figure 1A).

Recent cryo-electron microscopic (EM) studies have elucidated details of FO rotary mechanisms. For instance, Klusch et al. (2017) confirmed that the two half-channels of the a-subunit harbor continuous spaces through which protons can pass from the matrix/IMS region to the key residues responsible for proton transport in the c-subunit. In the c-ring, the key residues for proton transport receive and pass protons via protonation and deprotonation, respectively. Pogoryelov et al. (2010) elucidated the c-ring structure of FO in Spirulina platensis and reported that the key residues are in a proton-locked conformation in the lipid membrane environment and in a proton-unlocked conformation in the hydrophilic environment, assuming an interface with the a-subunit. In addition, molecular dynamics (MD) simulations confirmed that these two states could move back and forth depending on the surrounding environment. Thus, high-resolution structures, together with MD simulations, have offered important insights into the proton pathway.

In the present review, we describe theoretical and computational studies aimed at clarifying the rotational mechanisms of the FO motor in FOF1 ATP synthase. First, we present a chronological overview of the structural and theoretical studies on FO. Next, we introduce a recent all-atom MD simulation study that examines how the conformation of side chains of the key residues changes depending on the protonation state and environment. All-atom MD simulations have provided substantial information on water molecule accessibility. The free energy profile of a transferring proton, including its pKa value, is briefly discussed. Then, we describe coarse-grained MD simulations, which allow for modeling large-scale dynamics. Coarse-grained MD simulations have revealed protonation state-dependent free energy surfaces. We explain a computational methodology to simulate proton transfer-coupled FO rotation through a hybrid Monte Carlo/MD approach, which identified a preferred pathway in which two or three c-subunit key residues are deprotonated during a single rotational step. A recent biochemical experiment, together with the corresponding simulations, supports this view. Finally, we discuss the effects of a mutation of the conserved arginine of the a-subunit, the symmetry mismatch between FO and F1, and the coupling between side-chain reorientation of the c-subunit key residues and proton transfer-coupled FO rotation.

As the fundamental mechanism of proton transfer-driven FO rotation, the model first proposed by Vik and Antonio (1994) has been widely accepted (Figure 1A). The model assumes the presence of two half-channels in FO: one that shuttles protons from the IMS aqueous environment to the middle of the membrane, and the other that connects the middle of the membrane to the matrix-side aqueous environment. However, the two half-channels are not directly connected to the membrane. Instead, the end of each half-channel is close to a proton carrier amino acid in the middle of the FO c-subunit such that the proton can be transferred between each half channel and the proton-carrier residue in the c-subunit. Importantly, the c-subunits connected to the two half-channels are not the same in the c-ring. Thus, for the proton to move across the membrane, c-ring rotation is obligatory. Vik and Antonio introduced various mutations in the half-channel residues on the cytoplasmic side of the a-subunit (aE219 in Escherichia coli) adjacent to the proton carrier acidic residue of the c-ring (cD61 in E. coli) and measured ATP hydrolysis-driven proton pump activity; the authors noted that the proton-accepting residue at the aE219 position is critical for proton transfer. Subsequently, Oster et al. extended the Vik and Antonio model to a quantitative mathematical model (Elston et al., 1998). In this model, the authors focused on switches in electrostatic attractions between protonated and deprotonated acidic residues of the c-ring (cD61 in E. coli) and the conserved arginine of the a-subunit (aR210 in E. coli), which serves key regulatory roles in enzyme function (Figure 1B, left). This model was also applied to sodium-transporting FOF1-ATPase, suggesting that it captures the universal nature of the FO motor (Dimroth et al., 1999; Xing et al., 2004).

Together, the results successfully explained the experimental data (Vik and Antonio, 1994), and many subsequent theoretical studies were based on these models. For example, Aksimentiev et al. used a low-resolution structure (Girvin et al., 1998; Rastogi and Girvin, 1999) which allowed for speculating the positional association between c-ring and a-subunit for their simulations. Consequently, in addition to distance, the angle of the c-ring relative to the conserved arginine of the a-subunit garnered attention, and their kinetic model was constructed to explain the experimental results (Aksimentiev et al., 2004). In summary, it was theoretically confirmed that the FO motor achieves rotational motion by the kinetics of proton transfer and the associated effects of electrostatic interactions changing.

Then, cryo-EM revolutionized the resolution of structural information. In 2015, cryo-EM structural models of the FO part were obtained at a resolution of ~7 Å (Allegretti et al., 2015; Zhou et al., 2015). Interestingly, the a-subunit of FO harbors two long helices within the membrane, which are highly tilted from the normal to the membrane surface, and the conserved arginine residue is located in the middle of these helices (Figure 1C). This structural model clearly support the half-channel model of Vik and Antonio (1994). Based on the structural information at that time, Warshel group performed coarse-grained MD simulations to evaluate the free energy surface depending on the rotation angle of the c-ring under fixed protonated/deprotonated conditions (Mukherjee and Warshel, 2011). In addition, more recent advances in cryo-EM allowed for obtaining the structure of FO in mitochondrial, chloroplast, and bacterial ATP synthases at a resolution of ~3–4 Å (Guo et al., 2017; Klusch et al., 2017; Hahn et al., 2018; Srivastava et al., 2018; Gu et al., 2019). With the higher resolution models, Warshel group re-performed similar coarse-grained MD simulations to more reliably evaluate the free energy surface (Bai and Warshel, 2019) because their earlier work assumed a different orientation of the a-subunit with respect to the c-ring. Moreover, Roh et al. (2020) and Marciniak et al. (2022) performed all-atom MD simulations to evaluate the free energy surface of FO and VO motors, respectively. We also performed hybrid Metropolis Monte Carlo (MC)/MD simulations of proton transfer-coupled FO rotation, where proton transfer was mimicked by MC moves and the rotational motion of the FO motor was simulated by MD (Kubo et al., 2020).

This method is based on the constant-pH MD (Baptista et al., 2002; Mongan et al., 2004) and empirical valence bond (EVB) approaches (Kato et al., 2006; Olsson and Warshel, 2006), and similar approach, as called MCCE approach, used to study conformationally-dependent protonation states in Complex I (Khaniya et al., 2020). Note that as shown in Figure 1B (left), the proton-carrier residue in only one or two c-subunits can be deprotonated under rotation in the conventional model. For instance, Leone and Faraldo-Gómez (2016) predicted the possible c-ring rotational mechanism as follows: (1) only the ith c-subunit is deprotonated [denoted as DEP(ci)]; (2) the protonated jth c-subunit releases protons to the matrix region; (3) c-ring rotates; (4) the ith c-subunit receives the proton from the IMS region; and (5) c-ring rotates and completes the 36° step rotation (Leone and Faraldo-Gómez, 2016). This mechanism can be represented as follows:

In contrast, our MC/MD simulations (Kubo et al., 2020) and recent all-atom MD simulations (Marciniak et al., 2022) based on recent high-resolution structures suggested a new pathway, in which two or three c-subunits are simultaneously deprotonated under rotation, as shown in Figure 1B (right). This pathway can be described as follows:

The results of our MC/MD simulations are described in greater detail below. Discrepancies between the two pathways may be due to the different species (the former was for FO in Polytomella sp. and the latter for FO in yeast). If two half-channels are close to each other, there is no space for an additional deprotonated c-subunit. Alternatively, these differences may arise from the resolution of the original cryo-EM maps (~6.2 vs. 3.6 Å).

The hybrid MC/MD approach has many benefits for understanding mechanisms of molecular motors, in general. It can track conformational dynamics together with some elementary reactions, such as proton movements in the FO motor. We have succeeded in theoretically reproducing the rotational motion of the FO motor corresponding to the proton motion by combining MD and MC. Remarkably, the model that used the hybrid MC/MD approach rotated with a force equivalent to the torque confirmed in the experiment. However, it should be noted that these methods were based on many assumptions and have many limitations. Coarse-grained MD does not take into account the side chains orientation, and the simulated time correspondence with the real time is not perfect. All-atom MD can solve these problems, but it is hard to handle the entire motion of a huge system such as a FOF1 motor at once because of the computational cost. The MC methods were able to treat the proton movement only empirically; nothing that is not designed happens. In principle, it is possible to handle the proton transfer by the hybrid quantum mechanics/molecular mechanics simulation, but the computational cost is way beyond the current reach. The hybrid MC/MD simulation we performed is a sort of compromise. Along this line, the constant-pH MD simulation is a closely related approach, in which protonation states in relevant amino acids are updated via MC processes. However, the commonly used constant-pH MD simulation allows appearance and disappearance of protons at any protonatable amino acids instead of considering the proton transfer from one site to the other. Thus, this cannot directly be used in the proton-transport study. This can be modified in a similar manner to the introduced hybrid MC/MD approach.

Despite much room of improvement, the hybrid MC/MD approach has the advantage of being able to track proton and conformational motions with a high spatiotemporal resolution than experiments.

Several high-resolution structural models have been obtained using cryo-EM, allowing unambiguous discussions on proton transfer pathways. Klusch et al. (2017) identified water-accessible channels in their mitochondrial ATP synthase structure model used the program Hollow (Ho and Gruswitz, 2008). Based on the water-accessible region and molecular model constructed from a map at 3.7 Å resolution, plausible proton transfer pathways across the membrane could be investigated. First, the pathway from the IMS to the c-ring involves the following two steps. (1) From the IMS aqueous environment, protons enter the a-subunit through the aqueous space around aE172, aH248, aH252, and aE288 (yellow oval in Figure 1C). (2) Then, protons pass through the space between aN243 (H5) and aE288 (H6), reaching the side chain of cE111 (chain-i in Figure 1B), which is protonated (the proton-carrier residue of the c-ring). The water-accessible region ends around aR239, which is a strictly conserved arginine residue of the a-subunit located between the two half-channels. Based on this pathway, aR239 plays a role in preventing direct proton jump between the two half-channels, which corresponds to proton leakage. Next, the pathway from the c-ring to the matrix-side aqueous space likely involves the following steps. (1) The proton-carrying cE111 (chain-a in Figure 1B) moves from the lipid-facing environment to the region that contacts the a-subunit (particularly H5) via c-ring rotation. (2) Then, cE111 releases the protons to exit into the matrix space. Because the side chain of aE225(H5) faces the c-ring and is expected to function as the end of the half-channel that reaches the matrix-side aqueous space, the proton passes from aE225 to the inside of the a-subunit and is released into the matrix space (yellow oval in Figure 1C).

Notably, the orientation of the funnel-like space with H4–loop–H5 of the a-subunit with respect to the c-ring should be unaltered. Especially, it may be so essential for ATP synthase activity that H5 is positioned along the c-ring as discussed in (Klusch et al., 2017). For because residues aW189(H4), aT193(H4), and aY229(H5) are thought to contribute to maintaining the angle of the funnel-like tip, and mutations of these residues (or those present at the same position in other species) resulted in severe mitochondrial diseases (De Meirleir et al., 1995; Castagna et al., 2007; Cortés-Hernández et al., 2007). The mutations might cause wrong orientation of the H5 curvature along the c-ring, it would separate aE225 (matrix channel) from cE111 (proton carrier of the c-ring), and the distance between the c-ring and the channel will make it difficult for protons to transport. By the way, the subtlety of this pathway warrants discussion. Roh et al. (2020) identified a tightly bound lipid molecule in the cavity between the c-ring and a-subunit in the cytosol of the VO motor (corresponding to the matrix side in the case of mitochondrial FO). This tightly bound lipid molecule prevents the direct access of water molecules from the cytoplasm to the c-ring. It should be noted that VO motor conformation has some notable difference from those of the FO motor. For example, one of the c-subunits in the VO is different from the others. Thus, it is not clear how well the mechanisms of Vo can be generalized to FO. Therefore, it is unlikely that protons are directly released by the c-ring proton-carrying residues into the matrix-side aqueous space, as anticipated in Fo. Instead, the proton in the c-ring residue would be first transferred to the a-subunit proton-carrying residue, followed by release on the matrix side.

If atomic-resolution electron maps are available, water molecules can be directly traced to the channels. Roh et al. (2020) marked water molecules on a 2.7 Å map using the “Find Waters” routine available in Coot (Emsley et al., 2010) (blue dots in Figure 2A). The authors also performed all-atom MD simulations from a randomly arranged water molecule configuration and mapped the frequency of the water molecule positions (red spheres in Figure 2A). Thus, Coot mapping, in conjugation with all-atom MD simulations, is an effective method to evaluate where water molecules are released inside the water-accessible channel. The authors also found that water molecules reside between the half-channels and c-subunit, possibly enabling proton relay between the a- and c-subunits. Interestingly, the placement of relaying water molecules strongly depended on the protonation state of the proton-relaying residues; thus, the authors examined two different setups for the protonation states of glutamate. In the first setup, cE137 (the proton-carrying residue of the yeast VO motor c-ring) was protonated and aE789 (the representative glutamate on the lumen side half-channel of the yeast VO motor) was deprotonated (left panel in Figure 2B). In the second setup, the protonation states of the two glutamates were swapped (right panel in Figure 2B). A “water wire”—a single-file arrangement of water molecules—was stably formed between the protonated cE137 and the deprotonated aE789 (left panel in Figure 2B), but not in the other setup. Unidirectional transport of protons may occur through this wire.

Pogoryelov et al. (2010) illustrated the structure of proton-coupled c-ring in S. platensis, which gives structural insights on the proton-transfer pathway. The side chains of the c-ring proton-carrier residues assume variable conformations depending on the protonation state and surrounding environment. Moreover, all-atom MD simulations confirmed that the orientation of the side chains of c-ring acidic proton-carrying residues changes depending on whether the residue faces the lipid membrane or a-subunit. When it faces the lipid membrane, the protonated side chain of the c-ring proton-carrier residue tends to face the inner side of the c-ring (proton-locked conformation) (left panel in Figure 2C). Meanwhile, when it faces the a-subunit, the deprotonated side chains face outward (deprotonated-extended conformation), facilitating proton transfer from the c-ring to the half-channels of the a-subunit (right panel in Figure 2C). These two conformations have also been reported in yeast VO motors (Roh et al., 2020). Overall, the proton relay can be summarized as follows (Figure 2D). (1) The c-subunit glutamate in the membrane-facing region is protonated and exists in a closed state. (2) When it faces the a-subunit, it releases protons, undergoing deprotonation-coupled conformational change of the side chain to the open state. (3) When the deprotonated glutamate comes close to the other half-channel via c-ring rotation, it receives a proton, followed by a conformational change to the closed state. (4) Upon protonation, glutamate in the closed state enters the lipid-facing region.

ATP synthesis by the action of FOF1 ATP synthase is driven by the proton motive force, which is the sum of contributions from the proton concentration gradient and membrane electrical potential as pointed out by Mitchell (1961). For the former, the pH values on the IMS and matrix sides are ~7.0 and 8.0, respectively, corresponding to a free energy difference of 2.3 kBT. Typically, the mitochondrial membrane electrical potential is 150 mV, corresponding to 5.4 kBT. Notably, Soga et al. (2012) found that the sum of the two factors, namely the contribution from the pH difference across the membrane and membrane electrical potential, rather than their individual contribution is relevant to ATP synthesis kinetics. This suggests that not only the thermodynamics, but also kinetics are largely controlled by the sum of the two contributions. Notably, Soga et al. (2012) used a bacterial ATP synthase that was engineered with a deletion of an endogenous inhibitory domain (epsilon subunit's C-terminal domain), so they eliminated another factor that might control kinetics in vivo.

To understand the free energy profile of proton transfer through the transport pathway, in addition to the overall driving force, another key factor is the intrinsic affinity of protons toward every proton carrier residue; this is quantified in terms of the pKa value, which is proportional to the protonation free energy of the corresponding chemical group and calculated as VpKa = −(ln 10) kBT pKa.

Based on high-resolution structures, Srivastava et al. (2018) discussed the pKa values of glutamate residues of two half-channels in yeast ATP synthase (aE223 for the IMS channel and aE162 for the matrix channel) and the c-ring glutamate responsible for proton transport (cE59). If the pKa of glutamate is 5.0, the standard value in an aqueous solution as they mentioned in the paper, the difference in pH on the matrix side would be 3.0. Note, glutamate pKa value in water is, typically, treated around 4 (Tollinger et al., 2002). In this case, proton transfer from aE162 to the matrix side during the ATP synthesis reaction would be easy, although reverse proton transfer during ATP hydrolysis would be difficult. Thus, the authors inferred that the pKa values of glutamates and c-ring proton-carrier residues of the two half-channels should be ~7.0. The pKa value of carboxylates shifted to ~7.0 through glutamate–glutamate and histidine–glutamate interactions (Koeppe and Stroud, 1976; Root and Mackinnon, 1994; Morrill and MacKinnon, 1999; Harris and Turner, 2002). In addition, although the distance between the side chains of cE59 and aE223/aE162 was not small in their structure, they confirmed that it could be brought to ~4 Å through specific c-ring rotation. Therefore, the authors speculated that c-ring rotation brings cE59 and aE223/aE162 closer, shifting the pKa value to near 7.0. In fact, as shown in Figure 2D, when the c-ring is close to the a-subunit, the orientation of its proton-carrier side chains changes in the direction in which the reaction is more likely to occur; therefore, the shift in pKa values is expected to occur at the same time. Most recently, Marciniak et al.

(2022) calculated the shift in pKa values of glutamates using all-atom MD simulations and reported that pKa values of the matrix and IMS sites faced c-subunits' glutamates were ~2 and 8, respectively. Of note, however, accurate de novo estimation of the pKa value is very challenging owing to its sensitivity to the local environments and approximations used.

In previous sections, we discussed how the protonation and deprotonation of glutamates in the FO motor are controlled by the environment, such as pKa, pH, and membrane potential, as well as physical interactions, such as whether they faces the a-subunit or lipid molecules. Now, we shall focus on the actual free energy of the FO motor along the rotary angle. Recently, for some representative protonation states obtained from our MC/MD simulations (details are described later), we drew the free energy surfaces of different protonation states (Figures 3A,B) (Kubo et al., 2020). For each fixed protonation state, we performed coarse-grained MD simulations to obtain the free energy surface. Using these trajectories, we employed the Bennett acceptance ratio method (Bennett, 1976) and calculated the free energy differences among different protonation states. Based on the results, we illustrated the preferred proton transfer pathways for both ATP hydrolysis and ATP synthesis. Starting from the state in which the proton-carrying residues of the two c-subunits are deprotonated, we propose two plausible pathways of ATP synthesis: the deprotonation-first pathway (Figure 3A) and the protonation-first pathway (Figure 3B). We speculate that the deprotonation-first pathway occurs more frequently than the protonation-first pathway. Warshel group also obtained free energy surfaces using fixed protonated states and coarse-grained MD; Using Metropolis MC proton transfer approach (Vorobyov et al., 2016) to determine the protonation state of each c-subunit of the chloroplast ATP synthase FO motor and determined the free energy surface for each angle of the c-ring (Bai and Warshel, 2019).

Using all-atom MD simulations, Roh et al. (2020) determined free energy surfaces for c-ring angles based on the yeast VO motor structures they obtained. The authors selected a few representative residues for (de)protonation and rotated the c-ring for each protonation setup by adding a force to the residues. The string method was used to refine the pathway. Finally, 50 intermediate structures along the pathway were determined in a rotation of ~14° and connected by successive all-atom MD simulations. Successive all-atom MD simulations revealed that the free energy surface of c-ring rotation showed five intermediate states (A to E) and a transition state (TS) between states C and D (Figure 3C). At the TS, c′′E108 completely lost hydrogen bonds with aR735 and aR799 (Figure 3D). These contacts were maintained until state C. Thus, the loss of these connections is the rate-limiting step in the reaction. In addition, Marciniak et al. (2022) determined free energy surfaces depending on the c-ring rotational angle for the yeast FO motor.

Technically, although coarse-grained MD simulations can easily sample various conformations, they are intrinsically less accurate. Meanwhile, while all-atom MD simulations are more accurate, they are limited in sampling for large molecular machines, such as ATP synthase. In a recent study (Unarta et al., 2021), the merits of the two approaches were combined: a wide range of structures sampled by coarse-grained MD simulations were back-mapped to all-atom models, followed by all-atom MD simulations.

Furthermore, we investigated how proton transfer is dynamically coupled to c-ring rotation. The rotation of the FO motor is realized through proton transfer between the two half-channels and the c-ring via protonation and deprotonation of their side chains. In particular, the electrostatic interaction between the conserved arginine residue of the a-subunit and the c-subunit acidic proton-carrying residue is thought to be important in driving the rotation of the FO motor during ATP synthesis. However, how they are dynamically coupled remains unclear.

Recently, we performed hybrid Metropolis MC/MD simulations, where proton transfer was mimicked by an MC move and the rotational motion of the FO motor was realized through MD simulations (Figure 4A) (Kubo et al., 2020). We conducted MD and MC steps alternatively; in the MD step, only the protein conformations were updated, while in the MC step, the protonation state for a fixed conformation was updated. In this section, we briefly describe the computational approach.

We used the yeast mitochondrial FO structure (PDB ID: 6CP6) (Srivastava et al., 2018). Our simulation system comprised an a-subunit and 10 c-subunits, forming a c10-ring. The proton carrier residue in the c-subunit was cE59. In the IMS and matrix channels, the residues that directly transferred protons to and from the c-ring were aE223 and aE162, respectively. We defined these 12 glutamates (10 cE59 residues and 2 residues in the half-channels) as proton-carrier sites and dynamically updated their protonation states. Their protonation states are represented by a 12-dimensional vector {hi+}, where each element hi+ is either zero when protonated or one when deprotonated. For simulation, we assigned -hi+ charge to each glutamate.

where R represents all protein structure coordinates. On the right-hand side, the first term is the protein interaction energy, except for electrostatic interactions, and it is, therefore, independent of the protonation state (H+); the second term is the electrostatic interaction, which depends on both the conformation and protonation state; and the last term represents the intrinsic preference for protonation, corresponding to the intrinsic pKa, which is assumed to be independent of the conformation.

During the MD step, protein conformation dynamics were determined as Vnon−es(R) and Ves(R, H+). For Vnon−es(R), we used a well-established structure-based model that maintains both c-ring and a-subunit structures (Li et al., 2014). This model uses a coarse-grained representation of proteins, where each amino acid is simplified as one bead located at the Cα position. Ves(R, H+) includes two terms: the Coulomb interaction between any charged residue term VC(R, H+), and the membrane-environment term Vmem(R, H+). The latter term approximates the electrostatic penalty to embed a charged residue, deprotonated glutamate in this case, into the lipid membrane environment. The simulations did not explicitly include lipid molecules. Instead, we assumed that the c-ring glutamate faces the lipid membrane not the a-subunit. When we reduced the lipid membrane area in the control simulation, rotation activity was markedly reduced, which suggests the importance of the funnel-like structure of the matrix-side half-channel, corroborating the view put forth by Klusch et al. (2017). Approximation of the lipid membrane area can be refined using all-atom models. For instance, using the CHARMM-GUI membrane builder (Jo et al., 2008; Wu et al., 2014; Lee et al., 2016), lipid molecules can be explicitly placed around the FO motor, the range within which lipids are present can be estimated, and the free energies of protonation/deprotonation can be obtained. In a recent study, Marciniak et al. (2022) reported this direction. Also, Novitskaia et al. (2019) used CHARMM-GUI membrane builder for evaluation of the number of lipid molecules inside c-ring.

The MC step begins with probabilistic selection of the protonation states of two glutamates in the a-subunit, namely aE223 and aE162, for yeast mitochondrial ATP synthase, followed by probabilistic proton transfer between the a-subunit and c-subunit glutamates. Here, an underlying assumption is that water molecules in the half-channels can move protons fast enough to equilibrate the protonation states of aE223 and aE162 for the IMS and matrix-side half-channels, respectively. The protonation energy for each residue can be calculated as follows:

The pH values of the IMS and matrix aqueous space were set to 7.0 and 8.0, respectively, and the membrane potential (Ψ) to 150 mV. As all glutamates are located near the center of the membrane bilayer, we assumed the electrical potential at that site to be Ψ/2. Accordingly, the local equilibrium probabilities of protonation at each site Pi can be calculated as follows: