Chloroplast ATP synthase: From structure to engineering.

F-type ATP synthases are extensively researched protein complexes because of their widespread and central role in energy metabolism. Progress in structural biology, proteomics, and molecular biology has also greatly advanced our understanding of the catalytic mechanism, post-translational modifications, and biogenesis of chloroplast ATP synthases. Given their critical role in light-driven ATP generation, tailoring the activity of chloroplast ATP synthases and modeling approaches can be applied to modulate photosynthesis. In the future, advances in genetic manipulation and protein design tools will significantly expand the scope for testing new strategies in engineering light-driven nanomotors.

Dynamic modulation of transthylakoid electric potential by chloroplast ATP synthases.

The light-induced transthylakoid membrane potential (ΔΨm) can function as a driving force to help catalyzing the formation of ATP molecules, proving a tight connection between ΔΨm and the ATP synthase. Naturally, a question can be raised on the effects of altered functioning of ATP synthases on regulating ΔΨm, which is attractive in the area of photosynthetic research. Lots of findings, when making efforts of solving this difficulty, can offer an in-depth understanding into the mechanism behind. However, the functional network on modulating ΔΨm is highly interdependent. It is difficult to comprehend the consequences of altered activity of ATP synthases on adjusting ΔΨm because parameters that have influences on ΔΨm would themselves be affected by ΔΨm. In this work, a computer model was applied to check the kinetic changes in polarization/depolarization across the thylakoid membrane (TM) regulated by the modified action of ATP synthases. The computing data revealed that under the extreme condition by numerically "switching off" the action of the ATP synthase, the complete inactivation of ATP synthase would markedly impede proton translocation at the cytb6f complex. Concurrently, the KEA3 (CLCe) porter, actively pumping protons into the stroma, further contributes to achieving a sustained low level of ΔΨm. Besides, the quantitative consequences on every particular component of ΔΨm adjusted by the modified functioning of ATP synthases were also explored. By employing the model, we bring evidence from the theoretical perspective that the ATP synthase is a key factor in forming a transmembrane proton loop thereby maintaining a propriate steady-state ΔΨm to meet variable environmental conditions.

Chloroplast ATP synthase biogenesis requires peripheral stalk subunits AtpF and ATPG and stabilization of atpE mRNA by OPR protein MDE1.

Chloroplast ATP synthase contains subunits of plastid and nuclear genetic origin. To investigate the coordinated biogenesis of this complex, we isolated novel ATP synthase mutants in the green alga Chlamydomonas reinhardtii by screening for high light sensitivity. We report here the characterization of mutants affecting the two peripheral stalk subunits b and b', encoded respectively by the atpF and ATPG genes, and of three independent mutants which identify the nuclear factor MDE1, required to stabilize the chloroplast-encoded atpE mRNA. Whole-genome sequencing revealed a transposon insertion in the 3'UTR of ATPG while mass spectrometry shows a small accumulation of functional ATP synthase in this knock-down ATPG mutant. In contrast, knock-out ATPG mutants, obtained by CRISPR-Cas9 gene editing, fully prevent ATP synthase function and accumulation, as also observed in an atpF frame-shift mutant. Crossing ATP synthase mutants with the ftsh1-1 mutant of the major thylakoid protease identifies AtpH as an FTSH substrate, and shows that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits. In mde1 mutants, the absence of atpE transcript fully prevents ATP synthase biogenesis and photosynthesis. Using chimeric atpE genes to rescue atpE transcript accumulation, we demonstrate that MDE1, a novel octotricopeptide repeat (OPR) protein, genetically targets the atpE 5'UTR. In the perspective of the primary endosymbiosis (~1.5 Gy), the recruitment of MDE1 to its atpE target exemplifies a nucleus/chloroplast interplay that evolved rather recently, in the ancestor of the CS clade of Chlorophyceae, ~300 My ago.

The ATP Synthase γ Subunit ATPC1 Regulates RNA Editing in Chloroplasts.

RNA editing is the process of modifying RNA molecules by inserting, deleting, or substituting nucleotides. In flowering plants, RNA editing occurs predominantly in RNAs encoded by the organellar genomes of mitochondria and chloroplasts, and the main type of editing involves the substitution of cytidine with uridine at specific sites. Abnormal RNA editing in plants can affect gene expression, organelle function, plant growth, and reproduction. In this study, we report that ATPC1, the gamma subunit of ATP synthase in Arabidopsis chloroplasts, has an unexpected role in the regulation of editing at multiple sites of plastid RNAs. The loss of function of ATPC1 severely arrests chloroplast development, causing a pale-green phenotype and early seedling lethality. Disruption of ATPC1 increases the editing of matK-640, rps12-i-58, atpH-3'UTR-13210, and ycf2-as-91535 sites while decreasing the editing of rpl23-89, rpoA-200, rpoC1-488, and ndhD-2 sites. We further show that ATPC1 participates in RNA editing by interacting with known multiple-site chloroplast RNA editing factors, including MORFs, ORRM1, and OZ1. The transcriptome in the atpc1 mutant is profoundly affected, with a pattern of defective expression of chloroplast development-related genes. These results reveal that the ATP synthase γ subunit ATPC1 is involved in multiple-site RNA editing in Arabidopsis chloroplasts.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit ß.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit ß in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPß may be involved in proper ATP synthase accumulation during complex assembly.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly, photosynthesis, and chloroplast development in Arabidopsis.

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CFO) and a soluble coupling factor (CF1). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF1-CFO assembly. In plants that lacked AtCGL160N, CF1-CFO content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF1 subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF1-ß subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF1 subcomplex, which is critical for the modulation of CF1-CFO activity and photosynthesis.

Chloroplast inner envelope protein FtsH11 is involved in the adjustment of assembly of chloroplast ATP synthase under heat stress.

The maintenance of a proton gradient across the thylakoid membrane is an integral aspect of photosynthesis that is mainly established by the splitting of water molecules in photosystem II and plastoquinol oxidation at the cytochrome complex, and it is necessary for the generation of ATP in the last step of photophosphorylation. Although environmental stresses, such as high temperatures, are known to disrupt this fundamental process, only a few studies have explored the molecular mechanisms underlying proton gradient regulation during stress. The present study identified a heat-sensitive mutant that displays aberrant photosynthesis at high temperatures. This mutation was mapped to AtFtsH11, which encodes an ATP-dependent AAA-family metalloprotease. We showed that AtFtsH11 localizes to the chloroplast inner envelope membrane and is capable of degrading the ATP synthase assembly factor BFA3 under heat stress. We posit that this function limits the amount of ATP synthase integrated into the thylakoid membrane to regulate proton efflux from the lumen to the stroma. Our data also suggest that AtFtsH11 is critical in stabilizing photosystem II and cytochrome complexes at high temperatures, and additional studies can further elucidate the specific molecular functions of this critical regulator of photosynthetic thermotolerance.

Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins.

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔµH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔµH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔµH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔµH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

Enhanced abundance and activity of the chloroplast ATP synthase in rice through the overexpression of the AtpD subunit.

ATP, produced by the light reactions of photosynthesis, acts as the universal cellular energy cofactor fuelling all life processes. Chloroplast ATP synthase produces ATP using the proton motive force created by solar energy-driven thylakoid electron transport reactions. Here we investigate how increasing abundance of ATP synthase affects leaf photosynthesis and growth of rice, Oryza sativa variety Kitaake. We show that overexpression of AtpD, the nuclear-encoded subunit of the chloroplast ATP synthase, stimulates both abundance of the complex, confirmed by immunodetection of thylakoid complexes separated by Blue Native-PAGE, and ATP synthase activity, detected as higher proton conductivity of the thylakoid membrane. Plants with increased AtpD content had higher CO2 assimilation rates when a stepwise increase in CO2 partial pressure was imposed on leaves at high irradiance. Fitting of the CO2 response curves of assimilation revealed that plants overexpressing AtpD had a higher electron transport rate (J) at high CO2, despite having wild-type-like abundance of the cytochrome b6f complex. A higher maximum carboxylation rate (Vcmax) and lower cyclic electron flow detected in transgenic plants both pointed to an increased ATP production compared with wild-type plants. Our results present evidence that the activity of ATP synthase modulates the rate of electron transport at high CO2 and high irradiance.

Disentangling chloroplast ATP synthase regulation by proton motive force and thiol modulation in Arabidopsis leaves.

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain that contains a cysteine couple which interacts with the torque-transmitting ßDELSEED-loop. This thiol modulation equips CF1Fo with an important environmental fine-tuning mechanism. In vitro, disulfide formation in the γ-redox domain slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient ( [Formula: see text] ), which agrees with its proposed role as chock based on recently solved structure. The γ-dithiol formation at the onset of light is crucial to maximize photosynthetic efficiency since it lowers the [Formula: see text] activation level for ATP synthesis in vitro. Here, we validate these findings in vivo by utilizing absorption spectroscopy in Arabidopsis thaliana. To do so, we monitored the [Formula: see text] present in darkness and identified its mitochondrial sources. By following the fate and components of light-induced extra [Formula: see text] , we estimated the ATP lifetime that lasted up to tens of minutes after long illuminations. Based on the relationship between [Formula: see text] and CF1Fo activity, we conclude that the dithiol configuration in vivo facilitates photosynthesis by driving the same ATP synthesis rate at a significative lower [Formula: see text] than in the γ-disulfide state. The presented in vivo findings are an additional proof of the importance of CF1Fo thiol modulation, reconciling biochemical in vitro studies and structural insights.

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana.

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

Structural basis of redox modulation on chloroplast ATP synthase.

In higher plants, chloroplast ATP synthase has a unique redox switch on its γ subunit that modulates enzyme activity to limit ATP hydrolysis at night. To understand the molecular details of the redox modulation, we used single-particle cryo-EM to determine the structures of spinach chloroplast ATP synthase in both reduced and oxidized states. The disulfide linkage of the oxidized γ subunit introduces a torsional constraint to stabilize the two ß hairpin structures. Once reduced, free cysteines alleviate this constraint, resulting in a concerted motion of the enzyme complex and a smooth transition between rotary states to facilitate the ATP synthesis. We added an uncompetitive inhibitor, tentoxin, in the reduced sample to limit the flexibility of the enzyme and obtained high-resolution details. Our cryo-EM structures provide mechanistic insight into the redox modulation of the energy regulation activity of chloroplast ATP synthase.

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins.

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF1-γ. Previous studies showed that the light-dependent reduction of CF1-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF1-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.

The decline in photosynthetic rate upon transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase in Bletilla striata.

Upon a sudden transition from high to low light, the rate of CO2 assimilation (AN) in some plants first decreases to a low level before gradually becoming stable. However, the underlying mechanisms remain controversial. The activity of chloroplast ATP synthase (gH+) is usually depressed under high light when compared with low light. Therefore, we hypothesize that upon a sudden transfer from high to low light, the relatively low gH+ restricts ATP synthesis and thus causes a reduction in AN. To test this hypothesis, we measured gas exchange, chlorophyll fluorescence, P700 redox state, and electrochromic shift signals in Bletilla striata (Orchidaceae). After the transition from saturating to lower irradiance, AN and ETRII decreased first to a low level and then gradually increased to a stable value. Within the first seconds after transfer from high to low light, gH+ was maintained at low levels. During further exposure to low light, gH+ gradually increased to a stable value. Interestingly, a tight positive relationship was found between gH+ and ETRII. These results suggested that upon a sudden transition from high to low light, AN was restricted by gH+ at the step of ATP synthesis. Taken together, we propose that the decline in AN upon sudden transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas (Chlamydomonas r einhardtii), chloroplast gene expression is tightly regulated posttranscriptionally by gene-specific trans-acting protein factors. Here, we report the identification of the octotricopeptide repeat protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase oligomycin-sensitive chloroplast coupling factor. Unlike most trans-acting factors characterized so far in Chlamydomonas, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5' untranslated regions of both the atpH and atpI genes. Coimmunoprecipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5' end of triphosphorylated atpH mRNA. A very similar sequence, located ∼60 nucleotides upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in Chlamydomonas. Such a dual-targeted trans-acting factor provides a means to coregulate the expression of the two proton hemi-channels.

Does the stromal concentration of Pi control chloroplast ATP synthase protein amount in contrasting growth environments?

Changes in the growth environment can generate imbalances in chloroplast photosynthetic metabolism. Under water deficit, stomatal closure limits CO2 availability such that the production of ATP and NADPH by the thylakoid membrane-localized electron transport chain may not match the consumption of these energy intermediates by the stroma-localized Calvin-Benson cycle, thus challenging energy balance. Alternatively, in an elevated CO2 atmosphere, carbon fixation by the Calvin-Benson cycle may outpace the activity of downstream carbohydrate-utilizing processes, thus challenging carbon balance. Our previous studies have shown that, in both of the above scenarios, a mitochondrial alternative oxidase contributes to maintaining energy or carbon balance, highlighting the importance of photosynthesis-respiration interactions in optimizing photosynthesis in different growth environments. In these previous studies, we observed aberrant amounts of chloroplast ATP synthase protein across the different transgenic plant lines and growth conditions, compared to wild-type. Based on these observations, we develop here the hypothesis that an important determinant of chloroplast ATP synthase protein amount is the stromal concentration of inorganic phosphate. ATP synthase is a master regulator of photosynthesis. Coarse control of ATP synthase protein amount by the stromal inorganic phosphate status could provide a means to coordinate the electron transport and carbon fixation reactions of photosynthesis.

Revisiting the protomotive vectorial motion of F0-ATPase.

The elucidation of the detailed mechanism used by F0 to convert proton gradient to torque and rotational motion presents a major puzzle despite significant biophysical and structural progress. Although the conceptual model has advanced our understanding of the working principles of such systems, it is crucial to explore the actual mechanism using structure-based models that actually reproduce a unidirectional proton-driven rotation. Our previous work used a coarse-grained (CG) model to simulate the action of F0 However, the simulations were based on a very tentative structural model of the interaction between subunit a and subunit c. Here, we again use a CG model but with a recent cryo-EM structure of cF1F0 and also explore the proton path using our water flooding and protein dipole Langevin dipole semimacroscopic formalism with its linear response approximation version (PDLD/S-LRA) approaches. The simulations are done in the combined space defined by the rotational coordinate and the proton transport coordinate. The study reproduced the effect of the protomotive force on the rotation of the F0 while establishing the electrostatic origin of this effect. Our landscape reproduces the correct unidirectionality of the synthetic direction of the F0 rotation and shows that it reflects the combined electrostatic coupling between the proton transport path and the c-ring conformational change. This work provides guidance for further studies in other proton-driven mechanochemical systems and should lead (when combined with studies of F1) to a complete energy transduction picture of the F0F1-ATPase system.