MODIFICATION OF DOMAINS OF ALPHA-SUBUNIT AND BETA-SUBUNIT 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'-triphos phate (BzATP) of the adenine nucleotide binding site(s) on isolated an d complexed alpha and beta subunits of F-1-ATPase from the thermophili c bacterium PS3 (TF1) is described. BzATP binds to both isolated alpha and beta subunits, to complexed beta subunit but not to complexed alp ha subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [gamma-P-32]BzATP-labeled alpha subunit indicates that residues on both the amino-terminal (residues A 41-E67) and carboxy-terminal (residues Q422-4476) were modified by BzA TP. One of the residues in the carboxy-terminal modified by BzATP is m ost probably alpha Q422. Although the binding stoichiometry of 1 mol o f BzATP incorporated by either isolated or complexed beta subunit was maintained, the spatial conformation of the polypeptide determines whi ch amino acid residue(s) is more accessible to the reactive radical. C NBr derived fragments beta G10-M64, beta E75-M233, and beta D390-M469 were labeled with the isolated beta subunit. With complexed beta subun it the label was found only in CNBr fragments: beta E75-M233 and beta G339-M389. The locations where the covalently bound BzATP was found, i n the soluble and assembled subunits, indicate that different conforma tional states exist. In the isolated form of the alpha and beta subuni ts the amino- and carboxy-termini can fold and reach the central domai n of the polypeptide, the domain containing the adenine nucleotide bin ding site. When alpha combines with beta to form the alpha(3) beta(3) core complex the new conformation of the subunits is such that covalen t labeling by BzATP of alpha and of the amino terminal of beta subunit is excluded.