Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

Abstract

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P e 2 = 0.3–0.4 ). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H 2O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II. 2. The initial phase of H + uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H + uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination. 3. Dibromothymoquinone reduction, observed as O 2 evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O 2 evolution agreeing well with the steady-state rate observed under continuous illumination. 4. Such measurements of the initial rates of O 2 evolution and H + uptake yield an H + e − ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H + e − ratio of 1.7 at pH 7.6.) 5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H + uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P i and hexokinase, when added individually, have no effect on proton uptake in this system. 6. The involvement of a proton uptake reaction with an H + e − ratio of 0.5 in the Photosystem II partial reaction H 2O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e 2) and proton uptake ( H + e − ) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.