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Notizen: Abstract Two modes of chloroplast membrane post-illumination phosphorylation were detected, using the luciferin-luciferase ATP assay, one of which was not influenced by added permeable buffer (pyridine). That finding provides a powerful new tool for studying proton-membrane interactions during energy coupling. When ADP and Pi were added to the thylakoid suspension after a train of flashes [similar to the traditional post-illumination phosphorylation protocol (termed PIP− here)], the post-illumination ATP yield was influenced by pyridine as expected, in a manner consistent with the ATP formation, in part, being driven by protons present in the bulk inner aqueous phase, i.e., through a delocalized protonmotive force. However, when ADP and Pi were present during the flash train (referred to as PIP+), and ATP formation occurred during the flash train, the post-illumination ATP yield was unaffected by the presence of pyridine, consistent with the hypothesis that localized proton gradients were driving ATP formation. To test this hypothesis further, the pH and flash number dependence of the PIP− and PIP+ ATP yields were measured, the results being consistent with the above hypothesis of dual compartment origins of protons driving post-illumination ATP formation. Measuring proton accumulation during the attainment of the threshold energization level when no Δψ component was allowed to form (+ valinomycin, K+), and testing for pyridine effects on the proton uptake, reveals that the onset of ATP formation requires the accumulation of about 60 nmol H+ (mg Chl)−1. Between that level and about 110–150 nmol H+ (mg Chl)−1, the accumulation appears to be absorbed by localized-domain membrane buffering groups, the protons of which do not equilibrate readily with the inner aqueous (lumen) phase. Post-illumination phosphorylation driven by the dissipation of the domain protons was not affected by pyridine (present in the lumen), even though the effective pH in the domains must have been well into the buffering range of the pyridine. That finding provides additional insight into the localized domains, namely that protons can be absorbed by endogenous low pK buffering groups, and released at a low enough pH (≤5.7 when the external pH was 8, ≤4.7 at pH 7 external) to drive significant ATP formation when no further proton production occurs due to the redox turnovers. We propose that proton accumulation beyond the 110–150 nmol (mg Chl)−1 level spills over into the lumen, interacting with additional, lumenal endogenous buffering groups and with pyridine, and subsequent efflux of those lumenal protons can also drive ATP formation. Such a dual-compartment thylakoid model for the accumulation of protons competent to drive ATP formation would require a gating mechanism to switch the proton flux from the localized pathway into the lumen, as discussed by R. A. Dilley, S. M. Theg, and W. A. Beard (1987)Annu. Rev. Plant Physiol. 38, 348–389, and recently suggested by R. D. Horner and E. N. Moudrianakis (1986)J. Biol. Chem. 261, 13408–13414. The model can explain conflicting data from past work showing either localized or delocalized gradient coupling patterns.