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The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives
No full textThe F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H+ or Na+) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the “molecular currency unit” of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme’s potential role as a drug target
The inhibition kinetics of the Ca2+-activated F-ATPase by F1 inhibitors strengthens its role in the mitochondrial permeability transition pore formation
No full textPhenylglyoxal inhibition of the mitochondrial F1FO-ATPase activated by Mg2+ or by Ca2+ provides clues on the mitochondrial permeability transition pore
Full text linkPhenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ 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 Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation
Mini-review. Nitrite as novel pore-shutter: hints from the preferential inhibition of the mitochondrial ATP-ase when activated by Ca2+
No full textSmall inorganic compounds able to prevent the mitochondrial permeability transition, the master player in apoptosis and necrosis, are increasingly considered as beneficial tools in cytoprotection. Nitrite, a known cellular nitric oxide reservoir, has a recognized role in cardioprotection, but the molecular mechanisms of its action are not thoroughly understood. Mitochondrial permeability changes are known to constitute the molecular bases of human cardiac diseases and pathologies related to mitochondrial dysfunctions. In turn oxidative stress and mitochondrial damage are related issues in degenerative and cardiovascular diseases. Assumed that the mitochondrial F1FO complex is structurally or functionally involved in the mitochondrial permeability transition pore (MPTP), which triggers the mitochondrial permeability transition, nitrite effects on the enzyme complex may be exploited to shut the MPTP. Many clues suggest that nitrite may prevent or limit cell death by modulating the F1FO complex. Accordingly, nitrite decreases the ATPase activity stimulated by Ca2+, it is ineffective on the Mg-ATPase up to 2 mM and the enzyme inhibition is apparently enhanced under oxidative stress conditions. Through the inhibition of the calcium-activated F1FO complex, nitrite would shut the MPTP, which is likely to be related to the calcium-dependent functioning mode of the F1FO complex, and limit mitochondrial impairment and cell death under physio-pathological conditions
Preferential nitrite inhibition of the mitochondrial F1FO-ATPase activities when activated by Ca(2+) in replacement of the natural cofactor Mg(2.)
Full text linkThe ATP synthase can be imagined as a reversible H+-translocating channel embedded in the membrane, FO portion, coupled to a protruding catalytic portion, F1. Under physiological conditions the F1FO complex synthesizes ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. Alternatively, under other patho-physiological conditions it exploits ATP hydrolysis to energize the membrane by uphill pumping protons. The reversibility of the mechanism is guaranteed by the structural coupling between the hydrophilic F1 and the hydrophobic FO. Which of the two opposite processes wins in the energy-transducing membrane complex depends on the thermodynamic balance between the protonmotive force (Δp) and the phosphorylation potential of ATP (ΔGP). Accordingly, while Δp prevalence drives ATP synthesis by translocating protons from the membrane P-side to the N-side and generating anticlockwise torque rotation (viewed from the matrix), ΔGP drives ATP hydrolysis by chemomechanical coupling of FO to F1 with clockwise torque. The direction of rotation is the same in all the ATP synthases, due to the conserved steric arrangement of the chiral a subunit of FO. The ability of this coupled bi-functional complex to produce opposite rotations in ATP synthesis and hydrolysis is explained on the basis of the a subunit asymmetry
Putative role of the calcium-dependent F1FO-ATPase activity in mitochondrial permeability transition
No full textThe mitochondrial F1FO-ATPsynthase/ase is a bi-powered enzymatic engine, skilled for massive ATP production by oxidative phosphorylation, but also able to operate in reverse by hydrolyzing ATP and acting as a proton pump to re-energize the membrane [1]. Universally known as the enzyme of life, recently this complex has also been defined as enzymatic switch between life and death, due to its implication with the mitochondrial permeability transition pore (MPTP), in turn involved in cell death [2]. Since Ca2+ is essential for MPTP opening, when its concentration rises in the matrix Ca2+ may replace the natural Mg2+ cofactor in the F1FO-ATPase activation. The Mg2+ and Ca2+-dependent F1FO-ATPase activities, already shown to be both oligomycin-sensitive [3], have a similar pH dependence. However pH differently modulates the enzyme inhibition by DCCD depending on the activating cation and NAD+ only inhibits the Ca2+ dependent F1FO-ATPase. The enzyme desensitization to oligomycin by thiol oxidation, both when either Ca2+ or Mg2+ activate the enzyme, shoulders the enzyme involvement in MPTP, reported as oligomycin-insensitive when specific Cys are oxidized [2]. Taken together the findings suggest that Ca2+-activated F1FO-ATPase may be functionally involved in the MPTP
From the Ca2+-activated F1FO-ATPase to the mitochondrial permeability transition pore: an overview
Full text linkBased 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 F1FO-ATPase exploits the dithiol redox state to modulate the permeability transition pore
Full text linkThe dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 μM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE). Conversely, 300 μM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-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 F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation
Crucial aminoacids in the F O sector of the F 1 F O -ATP synthase address H + across the inner mitochondrial membrane: molecular implications in mitochondrial dysfunctions
Full text linkThe eukaryotic F 1 F O -ATP synthase/hydrolase activity is coupled to H + translocation through the inner mitochondrial membrane. According to a recent model, two asymmetric H + half-channels in the a subunit translate a transmembrane vertical H + flux into the rotor rotation required for ATP synthesis/hydrolysis. Along the H + pathway, conserved aminoacid residues, mainly glutamate, address H + both in the downhill and uphill transmembrane movements to synthesize or hydrolyze ATP, respectively. Point mutations responsible for these aminoacid changes affect H + transfer through the membrane and, as a cascade, result in mitochondrial dysfunctions and related pathologies. The involvement of specific aminoacid residues in driving H + along their transmembrane pathway within a subunit, sustained by the literature and calculated data, leads to depict a model consistent with some mitochondrial disorders
How the mitochondrial F1FO-ATPase works when Ca2+ replaces Mg2+
No full textThe mitochondrial F1FO-ATPase is a reversible and biologically unique energy-transducing mechanism with coupled capabilities of H+ translocation across the hydrophobic FO domain and ATP synthesis/hydrolysis by the hydrophilic F1 domain. When the Ca2+ replaces the natural cofactor Mg2+ in the catalytic site [1], most of the enzyme features are preserved, namely the inhibition by the FO blockers DCCD and oligomycin, the oligomycin desensitization by thiol oxidation and the dependence on the electrochemical gradient. The two differently activated F-ATPases share the basic structure and function mechanism of H+ translocation. However, the mixed type inhibition of the Ca-F1FO-ATPase by ADP and azide suggests that, while the overall F1 catalysis is apparently divalent ion-independent, the intimate molecular mechanism of catalysis can be altered by the replacement of Mg2+ with Ca2+, probably due to the higher steric hindrance of Ca2+ with respect to Mg2+ in the cofactor binding sites [2]. Apparently, the two F-ATPases are two distinct functioning modes of the same F1FO complex. The putative involvement of enzyme in membrane permeability changes [3] hints that the Ca-dependent F1FO-ATPase modulation may open/close the permeability transition pore
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