Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF 1): The effect on the CF 1-ATPase activity

Abstract

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF 1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19–24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[β- 32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF 1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the β subunit of the CF 1 and since there are probably three β subunits per CF 1, these results indicate strong cooperativity among β subunits and between the site of tightly bound nucleotides and the catalytic sites.