The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light

1. Photophosphorylation was studied in spinach chloroplasts on illumination, from the dark state, with saturating short ("single turnover") flashes of light. 2. At rapid flash rates (100 Hz), phosphorylation began within the first five flashes. The ATPase inhibitor protein appeared to be displaced from its inhibitory site on the ATPase also within five flashes, as deduced from the flash-induced ATPase activity. 3. At slower flash rates, or if the rate of electron transfer were reduced with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), phosphorylation began only after a larger number (50--60) of flashes. The displacement of the ATPase inhibitior protein was similarly delayed. 4. Partial displacement of the inhibitor protein from its inhibitory site on the ATPase (by pretreatment with dithioerythritol) allowed phosphorylation to proceed without a perceptible lag, even in the presence of DCMU. It was concluded that the ATPase inhibitor protein must be displaced on the ATPase before phosphorylation can begin, and that this process is energy dependent. 5. During the flash regime used, release of inhibitor from its inhibitory site seemed to be governed largely by the membrane potential. The light-induced pH gradient seemed to have little effect under these conditions. Our results are not compatible with a direct conformational interaction between the electron transfer chain and the ATPase causing displacement of the inhibitor. 6. The maximal rate of photophosphorylation induced by less than 200 flashes was 0.12--0.15 mol ATP made/mol ATPase per flash. This rate seemed to be limited not be the supply of energy to the ATPase molecules, nor by the maximal turnover capacity of the ATP synthesising system, but by the number of ATPase molecules which were active in synthesis, i.e., which lacked the inhibitor protein. 7. The bound nucleotides of the coupling ATPase exchanged with added nucleotides during single turnover flashes. At high flash rates, exchange began within 5 flashes. The average amount of nucleotide exchanged per flash over 100 flashes was about one tenth the amount of ATP synthesised in each flash. 8. It is concluded that, during phosphorylation, a steady state level of active coupling ATPases is set up. The energy-dependent displacement of the inhibitor protein, and its (energy-independent) relaxation back on to the inhibitory site are the two opposing factors involved in this steady state.