Cooperation among electron-transfer complexes in ATP synthesis in chloroplasts.

We have investigated the extent to which redox reactions of thylakoid membranes cooperate in ATP synthesis. This was done by measuring the onset of ATP synthesis following a series of single-turnover light-flashes at various levels of electron transport inhibition. In the presence of antibiotics that prevent the formation of a membrane potential, the onset of ATP synthesis seems to depend entirely on the formation of an adequately large delta pH. Under our conditions, the accumulation of about 60 mmol H+ X mol chlorophyll-1 is then necessary to form the requisite delta pH, which in turn requires about 15 saturating flashes in uninhibited thylakoid samples. Inhibition of some of the electron transfer centers by limiting the light intensity of the flashes, by dichlorophenyldimethylurea, by heat treatment, or by NH2OH-treatment caused an increase in the number of flashes required for the onset of ATP synthesis. The increase in the requisite number of flashes reflected the decreased number of electrons transferred in each flash, almost exactly the same number of electrons being transferred before ATP synthesis could begin. This effect of inhibitors was true when the two photosystems were operating in unison and when either of the two photosystems was acting alone. However, when either photosystem acted alone, there was an increase in the number of flashes required for the onset of ATP synthesis, an increase which was consistent with the observed lower flash-induced proton accumulation. A mathematical analysis of the onset of flash-induced ATP synthesis shows that at least several hundred proton-translocating electron transport complexes must be cooperating to form the threshold delta pH. In spite of this evidence for extensive cooperation among different electron transport complexes in ATP formation, the implied pooling of H+ ions does not seem to involve inner vesicle regions accessible to exogenous buffers. Thus, even when the number of H+ ions accumulated per flash was reduced by 70% through attenuation of the intensity of the flashes, exogenous hydrogen ion buffers present within the lumen of the thylakoid vesicle had no effect on the number of flashes required for the onset of ATP synthesis.