A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF₁) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkötter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [¹⁴C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [¹⁴C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [¹⁴]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [¹⁴C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [¹⁴C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [¹⁴C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [¹⁴C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.