Localization of the tight ADP-binding site on the membrane-bound chloroplast coupling factor one.

The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.