Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone.

BACKGROUND AND PURPOSE: 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F(0) F(1) -ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH: Kinetic analyses were performed on F(0) F(1) -ATP synthase in sub-mitochondrial particles and soluble F(1) -ATPase. Activity assays and immunodetection of the inhibitor protein IF(1) were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS: T1AM was a non-competitive inhibitor of F(0) F(1) -ATP synthase whose binding was mutually exclusive with that of the inhibitors IF(1) and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF(1) binding site, causing IF(1) release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F(0) F(1) -ATP synthase consistent with displacement of endogenous IF(1,) , reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS: Effects of T1AM on F(0) F(1) -ATP synthase were twofold: IF(1) displacement and enzyme inhibition. By targeting F(0) F(1) -ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF(1) and aurovertin binding sites are described.

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase.

Upon separation of a crude preparation of beta subunit ("beta fraction") from mitochondrial F(1)-ATPase containing one equivalent of Fe(III) in the nucleotide-independent site (1Fe(III)-loaded MF(1)), Fe(III) is almost completely recovered. CD spectra show that "beta fraction" maintains the structural changes induced by Fe(III) in the whole enzyme. In accordance, EPR reveals that the Fe(III) site geometry is conserved in "beta fraction." Moreover, the EPR spectra of 1Fe(III)-loaded MF(1) and its "beta fraction" undergo similar changes of the line-shape upon Pi binding at the catalytic site, indicating that the Pi and Fe(III) are proximal on beta. Highly purified beta in nucleotide-free form binds 1mol of Fe(III)/mol of protein. MF(1) "freezed" by inhibitors with two beta in closed conformation and one beta in open or half-closed conformation binds 1mol of Fe(III)/mol of enzyme. Therefore, the Fe(III) site location in the unique beta subunit not adopting the closed conformation is proposed.

Effects of Fe(III) binding to the nucleotide-independent site of F1-ATPase: enzyme thermostability and response to activating anions.

Mitochondrial F1-ATPase was induced in different conformations by binding of specific ligands, such as nucleotides. Then, Fourier transform infrared spectroscopy (FT-IR) and kinetic analyses were run to evaluate the structural and functional effects of Fe(III) binding to the nucleotide-independent site. Binding of one equivalent of Fe(III) induced a localised stabilising effect on the F1-ATPase structure destabilised by a high concentration of NaCl, through rearrangements of the ionic network essential for the maintenance of enzyme tertiary and/or quaternary structure. Concomitantly, a lower response of ATPase activity to activating anions was observed. Both FT-IR and kinetic data were in accordance with the hypothesis of the Fe(III) site location near one of the catalytic sites, i.e. at the alpha/beta subunit interface.

Fe(III) binding to Bacillus PS3 F(1)ATPase, alphabeta subcomplexes and isolated alpha- and beta-subunits.

Isolated alpha- and beta-subunits of Thermophilic Bacillus PS3 F(1)ATPase (TF(1)) bind about 1 Fe(III) equivalent. Upon reassembling in the symmetric alpha(3)beta(3) hexamer, Fe(III) binding capacity decreases, as this complex binds about three Fe(III) equivalents. In accordance, when the hexamer is dissociated in the alpha(1)beta(1) heterodimer, each heterodimer binds about one Fe(III) equivalent. On the contrary, native TF(1) exhibits a single Fe(III) site. CD spectra in far UV indicate that upon Fe(III) binding both the whole complex and the isolated beta-subunit undergo structural modifications accompanied by decrease of alpha-helix content, while alpha-subunit doesn't. As in alpha(3)beta(3) and in the whole enzyme the number of bound Fe(III) equivalents is consistent with the number of beta-subunits in the "empty" conformation, it is inferred that the single Fe(III) site in TF(1) is probably located in beta(E).

Effect of inhibitor binding to beta subunits of F1ATPase on enzyme thermostability: a kinetic and FT-IR spectroscopic analysis.

FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.