Probes of initial phosphorylation events in ATP synthesis by chloroplasts

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety. The results point to ADP as the initial acceptor of phosphate for ATP synthesis, with a slow side reaction in which bound ATP phosphorylates bound AMP to give a bound ADP. The phosphorylation of bound ADP by medium [32P]phosphate in the absence of added ADP is confirmed, but the reaction is too slow to serve as an intermediate in photophosphorylation. The appearance of label from [32P]phosphate in ATP in the acid-base transition at 25 degrees shows a lag of only about 3 to 7 ms, consistent with the absence of any phosphorylated intermediate. The lag is followed by a linear rate of [gamma-32]ATP formation that is about as fast as that observed in steady photophosphorylation, consistent with a proton gradient serving for transmission of energy from electron transfer reactions to the ATP-synthesizing complex.