Subunit-Subunit-Interactions in F₀F₁ as Revealed by Ligand Binding and Intrinsic Fluorescence

Abstract

For a better understanding of the molecular mechanism of the H⁺-ATP-synthase, a prerequisite is to know more about the correlation between structural and functional aspects. One possibility to study this correlation is to compare the properties of the subunits of this enzyme complex with the changes that occur upon reconstitution of the bigger complexes. Because of its extreme stability, the ATP-synthase of the thermophilic bacterium PS3 is especially well suited for these kinds of experiments (1, 2). Using this enzyme we have compared structural properties, ligand binding and ATP hydrolysis of the holoenzyme with those of the isolated subunits. For experiments concerning ligand binding and functional properties, the adenine-nucleotide analogue naphthoyl-ATP (N-ATP) has been used (3, 4); the advantages of this ligand are: (1) being fluorescent, (2) having a high affinity for the ATP-binding sites, (3) affecting the intrinsic fluorescence of TF₁, (4) being a potent inhibitor of the ATPase reaction. Preparation procedures and the methods have been described in detail elsewhere (5–7).