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

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Abstract
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 μM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 μM) was similar to that observed for ADP (2 μM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[β-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-[β-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the β subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the β subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.