Phenylglyoxal inhibition of the mitochondrial F1FO-ATPase activated by Mg2+ or by Ca2+ provides clues on the mitochondrial permeability transition pore

Abstract

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 for 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 for 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.

Molecular and Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System: Implications for Pathology

“…Moreover, the adenine nucleotide translocase isoforms could form a secondary lowconductance mPTP inhibited by both CsA and BKA [205]. Notably, the mPTP activity can be enhanced by Ca 2+ and pharmacologically modulated by selective inhibitors of F 1 [196] and F O domain [206][207][208]. Newly, purified and functionally active F 1 F O -ATPase monomers [209] and dimers [210] act as a voltage-gated ion channel endowed with mPTPlike properties.…”

Ca2+ as cofactor of the mitochondrial H+‐translocating F1FO‐ATP(hydrol)ase

“…Therefore oligomycin blocks H + translocation coupled to ATP hydrolysis irrespective of the divalent cation (Figure 2C). Consistently, these data suggest that mechanochemical coupling of Ca 2+ ‐dependent F 1 ‐ATP(hydrol)ase works as a rotary chemical motor to drive H + translocation in the F O domain 11,16 . The fact that the H + ‐pumping activity driven by Ca 2+ may not energize IMM is not surprising, being supported by the new “bent‐pull” model of the c ‐ring gated channel 32 and by the cryo‐EM maps of the enzyme exposed to Ca 2+ 4 .…”

“…To evaluate the mitochondrial F 1 F O ‐ATPase activities, the mitochondrial preparations obtained as described by Nesci et al, 11 were added to the reaction system that contained 3 mM ATP and 2 mM Ca 2+ or Mg 2+ in 75 mM ethanolammine‐HCl buffer, pH 8.8. The enzyme activity was spectrophotometrically detected and evaluated after subtraction of the nonspecific ATP hydrolysis in the blank 16 . The sensitivity of the F 1 F O ‐ATPase activity, either sustained by Mg 2+ or by Ca 2+ , to the specific F 1 F O ‐ATPase inhibitor oligomycin witnessed the functional and structural coupling between the two sectors F 1 and F O 14 …”

1,5‐Disubstituted‐1,2,3‐triazoles as inhibitors of the mitochondrial Ca2+‐activated F1FO‐ATP(hydrol)ase and the permeability transition pore

“…mPTP opening was evaluated by the increase in the fluorescence intensity ratio (Fura‐FF high Ca 2+ )/(Fura‐FF low Ca 2+ ), which indicates a decrease in CRC. All measurements were processed by LabSolutions RF software 40 …”