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Abstract: Background: The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases. Methods: The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction. Results: When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I 1 and I 2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI 1 I 2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT≥1μM. The TBT-driven enzyme desensitization was reversed by DTE. Conclusions: The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT. General significance: The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy. [Copyright &y& Elsevier]

Abstract: The antibiotic oligomycin is known to inhibit mitochondrial F-type ATP synthases. The antibiotic inhibits both ATP synthesis and hydrolysis by blocking the H+ translocation through FO which is coupled to the catalytic activity of F1. The amphiphilic organotin tri-n-butyltin (TBT), a known mitochondrial poison, can penetrate into biological membranes and covalently bind to electron-donor atoms of biomolecules such as sulfur. This study aims at exploring the mechanism(s) involved in the enzyme desensitization to oligomycin which occurs at concentrations >1 μM TBT. This poorly known effect of TBT, which only appeared at temperatures above the break in the Arrhenius plot of the enzyme activity, was found to be accompanied by the oxidation of isolated thiol groups of the mitochondrial complex. The oligomycin sensitivity was restored by the reducing agents glutathione and dithioerythritol and not influenced by antioxidants. The whole of data is consistent with the hypothesis that thiol oxidation is caused by TBT covalent binding to cysteine residues in a low-affinity site on FO and not by other possible oxidative events. According to this putative model, the onset of tin–sulfur bonds would trigger conformational changes and weaken the oligomycin interaction with FO. [Copyright &y& Elsevier]

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F 1 F O -ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg 2+ and at 150 μM when it is activated by Ca 2+ . The PAO-driven F 1 F O -ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE). Conversely, 300 μM DBrB decreases the F 1 F O -ATPase activity by 25% when activated by Mg 2+ and by 50% when activated by Ca 2+ . In both cases, the F 1 F O -ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca 2+ -dependent F 1 F O -ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F 1 F O -ATPase, with (ir)reversible redox states, in the mPTP modulation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mitochondrial permeability transition pore (PTP), which drives regulated cell death when Ca 2+ concentration suddenly increases in mitochondria, was related to changes in the Ca 2+ -activated F 1 F O -ATPase. The effects of the gadolinium cation (Gd 3+ ), widely used for diagnosis and therapy, and reported as PTP blocker, were evaluated on the F 1 F O -ATPase activated by Mg 2+ or Ca 2+ and on the PTP. Gd 3+ more effectively inhibits the Ca 2+ -activated F 1 F O -ATPase than the Mg 2+ -activated F 1 F O -ATPase by a mixed-type inhibition on the former and by uncompetitive mechanism on the latter. Most likely Gd 3+ binding to F 1 , is favoured by Ca 2+ insertion. The maximal inactivation rates (k inact ) of pseudo-first order inactivation are similar either when the F 1 F O -ATPase is activated by Ca 2+ or by Mg 2+ . The half-maximal inactivator concentrations (K I ) are 2.35 ± 0.35 mM and 0.72 ± 0.11 mM, respectively. The potency of a mechanism-based inhibitor (k inact /K I ) also highlights a higher inhibition efficiency of Gd 3+ on the Ca 2+ -activated F 1 F O -ATPase (0.59 ± 0.09 mM -1 ∙s -1 ) than on the Mg 2+ -activated F 1 F O -ATPase (0.13 ± 0.02 mM -1 ∙s -1 ). Consistently, the PTP is desensitized in presence of Gd 3+ . The Gd 3+ inhibition on both the mitochondrial Ca 2+ -activated F 1 F O -ATPase and the PTP strengthens the link between the PTP and the F 1 F O -ATPase when activated by Ca 2+ and provides insights on the biological effects of Gd 3+ ., (Copyright © 2021. Published by Elsevier B.V.)

The mitochondrial F 1 F O -ATPase in the presence of the natural cofactor Mg 2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H + . Interestingly, Mg 2+ can be replaced by Ca 2+ , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca 2+ inserts in F 1 , the torque generation built by the chemomechanical coupling between F 1 and the rotating central stalk was reported as unable to drive the transmembrane H + flux within F O . However, the failed H + translocation is not consistent with the oligomycin-sensitivity of the Ca 2+ -dependent F 1 F O -ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F 1 F O -ATPase distortion driven by ATP hydrolysis sustained by Ca 2+ is consistent with the permeability transition pore signal propagation pathway. The Ca 2+ -activated F 1 F O -ATPase, by forming the pore, may contribute to dissipate the transmembrane H + gradient created by the same enzyme complex., (© 2020 Wiley Periodicals LLC.)

In mammalian cells enzymatic and non-enzymatic pathways produce H 2 S, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the H 2 S donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the F 1 F O -ATPase activated by the natural cofactor Mg 2 , but, when Mg 2+ was replaced by Ca 2+ , a slight 15% enzyme inhibition at 100 µM NaHS was shown. Conversely, both the NADH-O 2 and succinate-O 2 oxidoreductase activities were totally inhibited by 200 μM NaHS with IC 50 values of 61.6 ± 4.1 and 16.5 ± 4.6 μM NaHS, respectively. Since the mitochondrial respiration was equally inhibited by NaHS at both first or second respiratory substrates sites, the H 2 S generation may prevent the electron transfer from complexes I and II to downhill respiratory chain complexes, probably because H 2 S competes with O 2 in complex IV, thus reducing membrane potential as a consequence of the cytochrome c oxidase activity inhibition. The Complex IV blockage by H 2 S was consistent with the linear concentration-dependent NADH-O 2 oxidoreductase inhibition and exponential succinate-O 2 oxidoreductase inhibition by NaHS, whereas the coupling between substrate oxidation and phosphorylation was unaffected by NaHS. Even if H 2 S is known to cause sulfhydration of cysteine residues, thiol oxidizing (GSSG) or reducing (DTE) agents, did not affect the F 1 F O -ATPase activities and mitochondrial respiration, thus ruling out any involvement of post-translational modifications of thiols. The permeability transition pore, the lethal channel which forms when the F 1 F O -ATPase is stimulated by Ca 2+ , did not open in the presence of NaHS, which showed a similar effect to ruthenium red, thus suggesting a putative Ca 2+ transport cycle inhibition., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The mitochondrial permeability transition pore (mPTP), a high-conductance channel triggered by a sudden Ca 2+ concentration increase, is composed of the F 1 F O -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 Ca 2+ -activated F 1 F O -ATPase without affecting F-ATPase activity sustained by the natural cofactor Mg 2+ . 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 Ca 2+ -activated F 1 F O -ATPase and mPTP and suggest that these inhibitors can be pharmacologically exploited to counteract mPTP-related diseases., (© 2020 New York Academy of Sciences.)

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca 2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F 1 F O -ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg 2+ by Ca 2+ in the F 1 F O -ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg 2+ - or Ca 2+ -dependent F 1 F O -ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg 2+ -dependent F 1 F O -ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F 1 F O -ATPase activities, but desensitizes the mPTP to Ca 2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F 1 F O -ATPase activities and a failed relationship between the Ca 2+ -dependent F 1 F O -ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F 1 F O -ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide., Competing Interests: Declaration of interest None., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic F O domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The F O 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 F O 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 F O subunits produce mitochondrial dysfunctions and lead to severe pathologies. The F O variability in different species, pointed out by cryo-EM analysis, mirrors the multiple enzyme functions and opens a new scenario in mitochondrial biology.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F 1 F O -ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F 1 F O -ATPase either sustained by the natural cofactor Mg 2+ or by Ca 2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg 2+ and 0.64 ± 0.05 mM in the presence of Ca 2+ ). Multiple inhibitor analysis indicates that features of the F 1 catalytic sites and/or the F O proton binding sites are apparently unaffected by PGO. However, PGO and F 1 or F O inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg 2+ -activated F 1 F O -ATPase, whereas they do not mutually exclude to bind the Ca 2+ -activated F 1 F O -ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F 1 F O -ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation., Competing Interests: Declaration of competing interest No competing interests declared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The properties of the mitochondrial F 1 F O -ATPase catalytic site, which can bind Mg 2+ , Mn 2+ , or Ca 2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca 2+ -activated F 1 F O -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg 2+ activates the F 1 F O -ATPase in competition with Mn 2+ , Ca 2+ is a noncompetitive inhibitor in the presence of Mg 2+ . Selective F 1 inhibitors (Is-F 1 ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca 2+ - or Mg 2+ -activated F 1 F O -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg 2+ is replaced by Ca 2+ . In a partially purified F 1 domain preparation, Ca 2+ -activated F 1 -ATPase maintained Is-F 1 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 Ca 2+ -activated F 1 F O -ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes., (© 2019 New York Academy of Sciences.)

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg 2+ effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F 1 F O -ATPase (F-ATPase) activity promoted by micromolar Hg 2+ concentrations were investigated. Hg 2+ was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg 2+ doses which inhibited the NADH-O 2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg 2+ dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg 2+ interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Increasing evidence highlights the role of the ATP synthase/hydrolase, also known as F1FO-complex, as key molecular and enzymatic switch between cell life and death, thus increasing the enzyme attractiveness as drug target in pharmacology. Being inhibition of ATP production usually linked to antiproliferative properties, drugs targeting the enzyme complex have been mainly considered to fight pathogen parasites and cancer. In recent years, a number of natural macrolides, produced by bacterial fermentation and structurally related to the classical enzyme inhibitor oligomycin, have been shown to bind to the membrane-embedded FO sector and to inhibit the enzyme complex by an oligomycin-like mechanism, namely by interacting with the c-ring. Other than natural macrolide antibiotics, which display variegated inhibition power on different F1FO-complexes, synthetic compounds from the diarylquinoline and organotin families also target the c-ring and strongly inhibit the enzyme. Bioinformatic insights address drug design to target FO subunits. Additionally, the possible modulation of the drug inhibition power, by amino acid substitutions or post-translational modifications of c-subunits, adds further interest to the target. The present survey on compounds targeting the c-ring and bi-directionally blocking the transmembrane proton flux which drives ATP synthesis/hydrolysis, discloses new therapeutic options to fight cancer and infections sustained by therapeutically recalcitrant microorganisms. Additionally, c-ring targeting compounds may constitute new tools to eradicate undesired biofilms and to address at the molecular level the therapy of mammalian diseases linked to mitochondrial dysfunctions. In summary, studies on the only partially known molecular interactions within the c-ring of the F1FO-complex may renew hope to counteract mammalian diseases.

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman's reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In vivo and in vitro experiments elicited different responses to ammonia nitrogen (ammonia-N) of gill and mantle Na,K-ATPase and ouabain-insensitive Na-ATPase activities in the Philippine clam Tapes philippinarum. Short-term (120 h) exposed clams to sublethal ammonia-N (NH(3)+NH (4) (+) ) concentrations (1.5 and 3.0 mg/L ammonia-N) showed enhanced gill and mantle ouabain-insensitive ATPase activity and decreased mantle Na,K-ATPase activity with respect to unexposed clams, while gill Na,K-ATPase was unaffected. In vitro experiments showed that NH (4) (+) could efficiently replace Na(+) in ouabain-insensitive ATPase activation and K(+), but not Na(+), in Na, K-ATPase activation. Simple saturation kinetics was constantly followed with similar K (0.5) values to that of the substituted cation. The same maximal ouabain-insensitive ATPase activation was obtained at 80 mM Na(+) or NH (4) (+) in the gills and at 50 mM Na(+) or NH (4) (+ ) in the mantle and that of Na,K-ATPase at 10 mM K(+) or NH (4) (+) in the presence of 100 mM Na(+) in both tissues. The two coexistent ATPase activities maintained their typical response to ouabain also when stimulated by NH (4) (+) : when activated by Na(+)+K(+) or by Na(+)+NH (4) (+) the ATPase activity was completely suppressed by 10(-3 )M ouabain, whereas the Na(+)- or NH (4) (+) -stimulated ATPase activity was unaffected by up to 10(-2 )M ouabain. The whole of the data suggests a possible involvement of the two ATPase activities in NH (4) (+) transmembrane transport.

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.