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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.)

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.)