Azidonaphthoyl‐ADP: a specific photolabel for the high‐affinity nucleotide‐binding sites of F₁‐ATPase

3'-O-[5-azidonaphthoyl]-ADP has been synthesized as a photoreactive analog to 3'-O-naphthoyl(1)-ADP which is known to bind to the high-affinity nucleotide sites of mitochondrial F1-ATPase, considered to be the catalytic sites. The photolabel in the dark acts as a ligand to F1-ATPase and as a competitive inhibitor with Ki = 11 microM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy of 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both alpha and beta polypeptides of F1; it is accompanied by corresponding removal of reversible high-affinity sites for ADP or naphthoyl-ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.