[Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast...

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.