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Based on recent advances on the Ca2+-activated F1FO-ATPase features, a novel multistep mechanism involving the mitochondrial F1FO complex in the formation and opening of the still enigmatic mitochondrial permeability transition pore (MPTP), is proposed. MPTP opening makes the inner mitochondrial membrane (IMM) permeable to ions and solutes and, through cascade events, addresses cell fate to death. Since MPTP forms when matrix Ca2+ concentration rises and ATP is hydrolyzed by the F1FO-ATPase, conformational changes, triggered by Ca2+ insertion in F1, may be transmitted to FO and locally modify the IMM curvature. These events would cause F1FO-ATPase dimer dissociation and MPTP opening.

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg2+ or, with lower efficiency, by Ca2+. The latter may play a role when calcium concentration rises in mitochondria, a condition associated with cascade events leading to cell death. Common and distinctive features of these differently activated mitochondrial ATPases were pointed out in swine heart mitochondria. When Ca2+ replaces the natural cofactor Mg2+, the enzyme responsiveness to the transmembrane electrochemical gradient and to the classical F-ATPase inhibitors DCCD and oligomycin as well as the oligomycin sensitivity loss by thiol oxidation, are maintained. Consistently, the two mitochondrial ATPases apparently share the F1FO complex basic structure and mechanism. Peculiar cation-dependent properties, which may affect the F1 catalytic mechanism and/or the FO proton binding site features, may be linked to a different physiological role of the mitochondrial Ca-activated F-ATPase with respect to the Mg-activated F-ATPase.

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

The antibiotic oligomycin is known to inhibit mitochondrial F-type ATP synthases. The antibiotic inhibits both ATP synthesis and hydrolysis by blocking the proton 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 mM 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.

Based on recent advances on the Ca

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss.

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg

The lipophilic pollutant tributyltin (TBT), other than inhibiting the DCCD (N,N'-dicyclohexylcarbodiimide) and oligomycin-sensitive Mg-ATPase activities in digestive gland mitochondria from the Mediterranean mussel Mytilus galloprovincialis, at higher than 1.0 μM concentrations in vitro promotes an increase in the ATPase activity fraction refractory to inhibitors of F(O) moiety, namely oligomycin and DCCD. By exploring the mechanisms involved in the TBT promoted enzyme desensitization to DCCD, we pointed out intriguing differences in the enzyme desensitization to the two inhibitors. Differently from oligomycin, the TBT promoted enzyme desensitization to DCCD is independent of the redox state of thiol groups of the enzyme complex and strongly temperature dependent, being significantly elicited only at temperatures above the break of the Arrhenius plots (around 18 °C). Such differences may cast light on multiple TBT interaction modes with the enzyme complex. The TBT-driven increase in the activation energy of the Mg-ATPase activities insensitive to inhibitors of F(O) sector suggests that the temperature-dependent incorporation of the lipophilic toxicant within the lipid bilayer may deeply affect the membrane-bound complex functionality. Accordingly, incorporated TBT may cause structural changes in the intramembrane F(O) subunits, thus weakening or even preventing DCCD binding to the enzyme complex.