Stability of bound ADP functioning as a phosphoryl donor in ATP synthesis by chloroplasts.

Upon illumination of spinach chloroplasts in the presence of [32P]orthophosphate and AMP and absence of substrate ADP, coupling factor-bound [32P]ADP is formed. This bound r3’P]ADP is stable enough to withstand purification of the coupling factor to homogeneity. However, the ability of this specially bound [““PIADP to serve as the ultimate phosphoryl donor to substrate ADP for the synthesis of [32P]ATP is labile. That is, addition of substrate ADP to the reaction immediately after the light is turned off causes quantitative transfer of the /3-phosphoryl group from this special [32P]ADP to the substrate ADP to yield [Y-~~P]ATP. As progressively longer periods of darkness are imposed between the formation of the coupling factorbound [32P]ADP and the addition of unlabeled substrate ADP, correspondingly less [32P]ATP is produced. This decay has a half-life of approximately 10 s. After the bound [32P]ADP has lost its competence to serve as a phosphoryl donor, it still can exchange with added unlabeled ADP. This exchange also decays in the dark, over an interval of several minutes. After a period of steady state photophosphorylation and several washes of the membranes in darkness to remove remaining [32P]phosphate, the donor function of the bound [32P]ADP can be reactivated in a second illumination period. It appears that, at the end of a period of steady state photophosphorylation, the chloroplasts are left with ADP bound in the donor site of the coupling factor. This bound ADP serves as the source of the y-phosphoryl group of the first ATP molecules synthesized at the start of a subsequent period of photophosphorylation. In addition, evidence was found that phosphate is sequestered by the chloroplasts in some way during illumination. An expanded model for ATP synthesis and a preliminary flip-flop (alternating site) mechanism for phosphorylation that involves four catalytic nucleotide binding sites are presented.