Demonstration of a transitory tight binding of ATP and of committed P(i) and ADP during ATP synthesis by chloroplasts.

Rapid mixing, quenching, and filtration experiments with chloroplast thylakoid membranes, with energization by acid-base transition, demonstrate that an ATP tightly bound to the isolated membranes is a transient intermediate in the catalytic sequence for ATP synthesis. The experiments also show that most of the P(i) and ADP bound at a catalytic site is committed to ATP formation without interchange with medium P(i) or ADP. Other results give evidence that upon energization, the tightly bound ADP that is detectable in isolated thylakoid membranes or coupling factor ATPase is rapidly released to the medium from a catalytic site. These findings support an alternating site model in which an energy-requiring conformational transition loosens ATP binding at one site and simultaneously promotes P(i) and ADP binding at the other site in a manner favoring ATP formation.