Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-}{}^3\text{H]aminobutyryl-Ado}\text{PP}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPPNH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPPNH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of ${}^3\text{H]Ado}\text{PP}\text{NH}\text{]P}$. The photoactivable derivative of AdoPPNH]P was found to bind to F₁ with high affinity, like AdoPPNH]P. Once ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPPNH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPPNH]P, ADP, or ATP prevented the covalent labeling of the enzyme by ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPPNH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound ${}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.