Photophosphorylation in Chloroplasts With Varied Proton Motive Force (PMF): II. Phosphorylation and the PMF

Measurements of ATP (luciferase assay) formed by class C pea chloroplasts in illumination times varying from 10 ms to 30 s are reported for control, + valinomycin and + nigericin conditions. ATP was made at a constant rate in controls following a time lag of a few milliseconds which varied with the illumination. Valinomycin increased the time lag to ~100 ms after which therate approached controls. Nigericin caused a gradual decrease in rate of ATP synthesis over a period of ~1s . In the steady state the rate was a different function of the transthylakoid pH difference (ΔpH) with nigericin and with valinomycin, with thresholds at ΔpH = 2.9 and 3.5 respectively. The time lags and thresholds are shown to be consistent with a threshold proton motive force (PMF) of 140-190 mV in various experiments. It is argued that this PMF corresponds to that required to poise the phosphorylation reaction to the point of ATP net synthesis at the prevailing dark phosphorylation potential. The experiments could not decide between a stoichiometry of 2 or 3 protons per ATP. Data suitable for use in constructing a kinetic model are briefly discussed. The findings generally are interpreted as showing a close correlation between phosphorylation and the PMF estimated as the mean potential energy of protons in the intrathylakoid spaces relative to the outside. It is concluded that Mitchellian coupling between bulk protons and the ATP synthetase is not yet to be discarded.