Variants in Human ATP Synthase Mitochondrial Genes: Biochemical Dysfunctions, Associated Diseases, and Therapies.

Mitochondrial ATP synthase (Complex V) catalyzes the last step of oxidative phosphorylation and provides most of the energy (ATP) required by human cells. The mitochondrial genes MT-ATP6 and MT-ATP8 encode two subunits of the multi-subunit Complex V. Since the discovery of the first MT-ATP6 variant in the year 1990 as the cause of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome, a large and continuously increasing number of inborn variants in the MT-ATP6 and MT-ATP8 genes have been identified as pathogenic. Variants in these genes correlate with various clinical phenotypes, which include several neurodegenerative and multisystemic disorders. In the present review, we report the pathogenic variants in mitochondrial ATP synthase genes and highlight the molecular mechanisms underlying ATP synthase deficiency that promote biochemical dysfunctions. We discuss the possible structural changes induced by the most common variants found in patients by considering the recent cryo-electron microscopy structure of human ATP synthase. Finally, we provide the state-of-the-art of all therapeutic proposals reported in the literature, including drug interventions targeting mitochondrial dysfunctions, allotopic gene expression- and nuclease-based strategies, and discuss their potential translation into clinical trials.

Novel Regioselective Synthesis of 1,3,4,5-Tetrasubstituted Pyrazoles and Biochemical Valuation on F1FO-ATPase and Mitochondrial Permeability Transition Pore Formation.

An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F1FO-ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a, 6h, and 6o on the F1FO-ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor (ESI). The dissociation constant of the ESI complex (Ki') in the presence of the 6a had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F1FO-ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o. Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a. On the contrary, 1 mM 6o had stronger desensitization of mPTP formation than 0.1 mM 6o. The F1FO-ATPase is a target of Pzs blocking mPTP formation.

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction.

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

Protein folding and unfolding: proline cis-trans isomerization at the c subunits of F1 FO -ATPase might open a high conductance ion channel.

The c subunits, which constitute the c-ring apparatus of the F1 FO -ATPase, could be the main components of the mitochondrial permeability transition pore (mPTP). The well-known modulator of the mPTP formation and opening is the cyclophilin D (CyPD), a peptidyl-prolyl cis-trans isomerase. On the loop, which connects the two hairpin α-helix of c subunit, is present the unique proline residue (Pro40 ) that could be a biological target of CyPD. Indeed, the proline cis-trans isomerization might provide the switch that interconverts the open/closed states of the pore by pulling out the c-ring lipid plug.

Ca2+ as cofactor of the mitochondrial H+ -translocating F1 FO -ATP(hydrol)ase.

The mitochondrial F1 FO -ATPase in the presence of the natural cofactor Mg2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H+ . Interestingly, Mg2+ can be replaced by Ca2+ , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca2+ inserts in F1 , the torque generation built by the chemomechanical coupling between F1 and the rotating central stalk was reported as unable to drive the transmembrane H+ flux within FO . However, the failed H+ translocation is not consistent with the oligomycin-sensitivity of the Ca2+ -dependent F1 FO -ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F1 FO -ATPase distortion driven by ATP hydrolysis sustained by Ca2+ is consistent with the permeability transition pore signal propagation pathway. The Ca2+ -activated F1 FO -ATPase, by forming the pore, may contribute to dissipate the transmembrane H+ gradient created by the same enzyme complex.

1,5-Disubstituted-1,2,3-triazoles as inhibitors of the mitochondrial Ca2+ -activated F1 FO -ATP(hydrol)ase and the permeability transition pore.

The mitochondrial permeability transition pore (mPTP), a high-conductance channel triggered by a sudden Ca2+ concentration increase, is composed of the F1 FO -ATPase. Since mPTP opening leads to mitochondrial dysfunction, which is a feature of many diseases, a great pharmacological challenge is to find mPTP modulators. In our study, the effects of two 1,5-disubstituted 1,2,3-triazole derivatives, five-membered heterocycles with three nitrogen atoms in the ring and capable of forming secondary interactions with proteins, were investigated. Compounds 3a and 3b were selected among a wide range of structurally related compounds because of their chemical properties and effectiveness in preliminary studies. In swine heart mitochondria, both compounds inhibit Ca2+ -activated F1 FO -ATPase without affecting F-ATPase activity sustained by the natural cofactor Mg2+ . The inhibition is mutually exclusive, probably because of their shared enzyme site, and uncompetitive with respect to the ATP substrate, since they only bind to the enzyme-ATP complex. Both compounds show the same inhibition constant (K'i ), but compound 3a has a doubled inactivation rate constant compared with compound 3b. Moreover, both compounds desensitize mPTP opening without altering mitochondrial respiration. The results strengthen the link between Ca2+ -activated F1 FO -ATPase and mPTP and suggest that these inhibitors can be pharmacologically exploited to counteract mPTP-related diseases.

Incoming news on the F-type ATPase structure and functions in mammalian mitochondria.

•Recent findings of cryo-EM structures of mammalian F1FO-ATPase.•The membrane-embedded domain of the F1FO-ATPase and the permeability transition pore.•The Ca2+-activated 1FO-ATPase role in the mPTP is consistent with recent cryo-EM findings.•The membrane-embedded FO participates in mPTP formation in mammalian mitochondria.•Conformational changes within FO modify the inner mitochondrial membrane shape.

Mitochondrial F-type ATP synthase: multiple enzyme functions revealed by the membrane-embedded FO structure.

Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic FO domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The FO complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes. The FO membrane domain, essential in the enzyme machinery, also participates in the bioenergetic cost of synthesizing ATP and in the formation of the cristae, thus contributing to mitochondrial morphology. The recent enzyme involvement in a high-conductance channel, which forms in the inner mitochondrial membrane and promotes the mitochondrial permeability transition, highlights a new F-type ATP synthase role. Point mutations which cause amino acid substitutions in FO subunits produce mitochondrial dysfunctions and lead to severe pathologies. The FO variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.

The mitochondrial permeability transition pore in cell death: A promising drug binding bioarchitecture.

Bioenergetic failure often features programmed cell death involved in some severe pathologies. When the cell is fated to die, the inner mitochondrial membrane becomes permeable to ions and solutes, due to the formation and opening of a channel known as mitochondrial permeability transition pore (mPTP). Up to now, the still-elusive mPTP structure and mechanism prevented any attempt to identify/design drugs to rule its formation and limit cell death. Latest advances, which strongly suggest that the F1 FO -ATPase can coincide with the mPTP, open new perspectives in therapy. Compounds targeting and inhibiting cyclophilin D, a known mPTP promoter, could be exploited to block mPTP formation. Moreover, if the mPTP-F1 FO -ATPase connection will be consolidated, selected F1 FO -ATPase inhibitors could represent novel therapeutic options to attenuate mPTP-related diseases by directly acting on mPTP molecular mechanism. This intriguing perspective, which raises new hopes to counteract mPTP-related diseases, stimulates further studies to clarify the mPTP architecture and mechanism.

Mitochondrial Ca2+ -activated F1 FO -ATPase hydrolyzes ATP and promotes the permeability transition pore.

The properties of the mitochondrial F1 FO -ATPase catalytic site, which can bind Mg2+ , Mn2+ , or Ca2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca2+ -activated F1 FO -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg2+ activates the F1 FO -ATPase in competition with Mn2+ , Ca2+ is a noncompetitive inhibitor in the presence of Mg2+ . Selective F1 inhibitors (Is-F1 ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca2+ - or Mg2+ -activated F1 FO -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg2+ is replaced by Ca2+ . In a partially purified F1 domain preparation, Ca2+ -activated F1 -ATPase maintained Is-F1 sensitivity, and enzyme inhibition was accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the structural relationship between Ca2+ -activated F1 FO -ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes.

The N-terminal region of the ϵ subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity.

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a ß-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.

Mitochondrial matrix pH acidifies during anoxia and is maintained by the F1Fo-ATPase in anoxia-tolerant painted turtle cortical neurons.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine-123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF-1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo-ATPase was inhibited with oligomycin-A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

Inhibition of F1 -ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1.

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression.

Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of Trypanosoma brucei on its mitochondrial genome (kinetoplast DNA [kDNA]), a target of the antitrypanosomatid drug isometamidium. Here, we report that V-ATPase inhibition with bafilomycin A1 (BafA) provides partial resistance to genetic knockdown of mitochondrial gene expression. BafA does not promote long-term survival after kDNA loss, but in its presence, isometamidium causes less damage to kDNA.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation.

The F1F0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα(521-540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mtα(521-540) comes in close proximity with subunit γ residues 104-109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα(514-549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.

Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli.

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors.

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3ß3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 µs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.

Mitochondrial responses to prolonged anoxia in brain of red-eared slider turtles.

Mitochondria are central to aerobic energy production and play a key role in neuronal signalling. During anoxia, however, the mitochondria of most vertebrates initiate deleterious cell death cascades. Nonetheless, a handful of vertebrate species, including some freshwater turtles, are remarkably tolerant of low oxygen environments and survive months of anoxia without apparent damage to brain tissue. This tolerance suggests that mitochondria in the brains of such species are adapted to withstand prolonged anoxia, but little is known about potential neuroprotective responses. In this study, we address such mechanisms by comparing mitochondrial function between brain tissues isolated from cold-acclimated red-eared slider turtles (Trachemys scripta elegans) exposed to two weeks of either normoxia or anoxia. We found that brain mitochondria from anoxia-acclimated turtles exhibited a unique phenotype of remodelling relative to normoxic controls, including: (i) decreased citrate synthase and F1FO-ATPase activity but maintained protein content, (ii) markedly reduced aerobic capacity, and (iii) mild uncoupling of the mitochondrial proton gradient. These data suggest that turtle brain mitochondria respond to low oxygen stress with a unique suite of changes tailored towards neuroprotection.

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria.

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599-608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F1-ATPase and F1-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1-ATPase at the open catalytic αE/ßE interface, and two partial γ rotations lock the N terminus of ζ in an "inhibition-general core region," blocking further γ rotation, while the ζ globular domain anchors it to the closed αDP/ßDP interface. Heterologous inhibition of the F1-ATPase of P. denitrificans by the mitochondrial IF1 supported both the modeled ζ binding site at the αDP/ßDP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF1 or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.