Further investigation on the lateral and transversal proton currents at the thylakoid membrane level by hydrogen-deuterium exchange

Abstract

Energetically-coupled processes (electron flow, proton uptake and correlated pH gradient) were investigated on envelope-free chloroplasts of lettuce suspended in 1H 2O or 2H 2O media. Study of the light-intensity and temperature dependencies of these phenomena led to the following observations: 1. At neutral pH, 2H 2O diminishes the transmembrane H + gradient in strong light (chain Photosystem II + Photosystem I) but not in low light; the total H + uptake is increased at all light intensities: the buffering capacity of the inner compartment is increased in heavy water, possibly through enhancement of interactions between membranous titrable groups and the aqueous phase. 2. 2H 2O does not affect the photochemical events of the redox chain, whatever the electron pathway (PSII, PSI or PSII + PSI): only thermal steps are inhibited. The diminution of the apparent quantum yield, sometimes observed, may be ascribed to the dual site of action of the artificial redox carrier (ferricyanide) then used. 3. 2H 2O does not modify the activation energy of the limiting step of the electron flow (PSII + PSI) in uncoupled (44 vs. 47 kJ · mol −1) or — but less clearly — in coupled, i.e., ‘basal’, state (55 vs. 59 kJ · mol −1). 2H 2O does not either change the temperature of the phase transition of the membrane (17°C) for the uncoupled flow. However, a low-temperature transition, observed only for the coupled chain, is slightly increased by 2H 2O; this thermal transition is attributed to the freezing of some bound water near the plastoquinone pool. 4. Δp 2H is smaller than Δp 1H at all temperatures (PSII + PSI chain). ΔpH is quasi-constant from 0°C to 10°C, then decreases when temperature rises. 2H 2O does not change the activation energy of the dark passive H + efflux, which is almost twice that of the coupled electron flow. The phase transition at low temperature suggests that the proton efflux occurs via two parallel pathways, one temperature-dependent and the other temperature-independent. Except for the increase of the internal buffering capacity, the effects of 2H 2O on the membrane conformation seem limited, as shown by the unchanged activation energies of the electron flow and of the H + leakage. The null activation energy observed at low temperature emphasizes the role of the bound water in these processes; however, the different effects of 2H 2O on the transition temperatures indicate that this bound water has different properties when associated with the translocation sites or with the H + leakage ones. This ‘microcompartmentation’ of the membranes is consistent with the concept of lateral pH heterogeneity we have previously suggested (de Kouchkovsky, Y., and Haraux, F. (1981) Biochem. Biophys. Res. Commun. 99, 205–212). The theoretical computations and the experimental results suggest that in the steady state, the internal pH would be several tenths of a ‘unit’ lower near the plastoquinones than near the H + efflux sites (coupling factors); this difference would be increased when 2H + replaces 1H +, owing to the lower mobility of the deuteron. It is concluded that local, and not average, pH (and ΔpH) should be considered for the understanding of the energy transduction processes.