Novel approaches towards characterization of the high-affinity nucleotide binding sites on mitochondrial F1-ATPase by the fluorescence probes 3'-O-(1-naphthoyl)adenosine di- and triphosphate.
The fluorescence properties of 3'-O-(1-naphthoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial F1-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and 'ground-state' quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg2+ 120 nM; N-ATP/Mg2+ 160 nM; N-ADP/EDTA 560 nM; N-ATP/EDTA 3500 nM. For bound ligand the environment was found to be rather unipolar; the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg2+, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg2+). After the first fast step (kon greater than 1 X 10(6) M-1 X s-1), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D000244:Adenosine Diphosphate; C032458:3'-O-(naphthoyl-1)adenosine diphosphate; C037102:naphthoyl-ATP; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases