Protons in the thylakoid membrane-sequestered domains can directly pass through the coupling factor during ATP synthesis in flashing light.

Thylakoid membranes contain sequestered domains in which protons are held in a metastable state out of equilibrium with those in the inner (lumen) or the outer aqueous bulk phases unless the membranes are made leaky by, for instance, the addition of uncouplers. Previously, it has not been clear whether such sequestered domain protons are: 1) directly on a localized pathway into the CF0-CF1; 2) on an ultimately delocalized pathway comprising domains----lumen----CF0-CF1; or 3) perhaps not importantly involved in any way with proton gradient-linked ATP formation. Recent developments now permit a test of the above possibilities. The test for the possible mode of involvement of domain protons in energy coupling utilized single turnover flashes to energize electric field-driven ATP formation, as influenced by both the proton depletion level of the sequestered domains and whether the thylakoids were prepared in a way to show either a localized or delocalized proton gradient response in ATP formation (this response is reversibly controlled by incubation in low (localized coupling mode) or high KCl (delocalized mode) incubations (Beard, W. A., and Dilley, R. A. (1986) FEBS Lett. 201, 57-62; Chiang, G. and Dilley, R. A. (1987) Biochemistry 26, 4911-1916). Using thylakoids showing delocalized coupling responses, there was no influence of the reversible depletion of the sequestered domain buffering pool on the delta psi-driven ATP formation onset lag. Lumenal protons rather than domain protons in that case appear to be the first to be driven through the CF0-CF1 complex by the delta psi field. However, using thylakoids prepared to be in the localized gradient coupling mode, protons in the domains, rather than those in the lumen, are the first to pass through the CF0-CF1 complex in the onset of energization. We conclude that the sequestered domain protons, in the latter case only, are obligatorily in the main proton diffusion pathway for energization of ATP formation and are the first protons (rather than lumen protons) driven through the CF1-CF0 complex by a delta psi field.