Modification of domains of α and β subunits of F₁-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms, by 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP)

Photoaffinity labeling by 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed α and β subunits of F₁-ATPase from the thermophilic bacterium PS3 (TF₁) is described. BzATP binds to both isolated α and β subunits, to complexed β subunit but not to complexed α subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [γ-³²P]BzATP-labeled α subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422-Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably αQ422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed β subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments βG10-M64, βE75-M233, and βD390-M469 were labeled with the isolated β subunit. With complexed β subunit the label was found only in CNBr fragments: βBE75-M233 and βG339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the α and β subunits the amino-and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When α combines with β to form the α₃β₃ core complex the new conformation of the subunits is such that covalent labeling by BzATP of α and of the amino terminal of β subunit is excluded.