Yaroslav de Kouchkovsky

Adjustable microchemiosmotic character of the proton gradient generated by systems I and II for photosynthetic phosphorylation in thylakoids

To clarify the debate on the localized character of proton gradients in energy-transducing membrane systems, the way in which the osmolarity and ionicity of the medium, which should affect the thylakoid lumen properties, may modulate the relative efficiency of Δ\gmH+ generated by PS I or PS II (restricted here to ΔpH: valinomycin present) was examined. Although the results depended on the preparations and the conditions, a trend towards proton delocalization, especially in 50 mM KCl vs. the usual 5–10 mM, was observed when thylakoids were suspended in a sorbitol-free buffer for only a short time before the experiment. It was also verified that the better efficiency of PS I vs. PS II protons was not due to the 9-aminoacridine method used to quantify ΔpH. One main argument is that similar results were found when the proton gradient was estimated by total H+ translocation, measured with a glass electrode, and by probe partitioning, followed in parallel. Lastly, it was observed that, even when protons are emitted by water-splitting enzymes, i.e., far from coupling factors, the rate of ATP synthesis is less sensitive to nigericin than expected from the ΔpH decrease. This suggests that protons are flowing, from redox to phosphorylating pumps, in an anisotropic medium. The role of vesicular configuration and topological organization of energy-transducing membranes in the microchemiosmotic behaviour of organelles is stressed. It is suggested that besides water, polypeptide chains, rather than lipid heads (owing to the limited effectiveness of lipophilic nigericin), may ensure the lateral H+ transport between their points of influx and efflux.

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

Energetically-coupled processes (electron flow, proton uptake and correlated pH gradient) were investigated on envelope-free chloroplasts of lettuce suspended in 1H2O or 2H2O media. Study of the light-intensity and temperature dependencies of these phenomena led to the following observations: 1. At neutral pH, 2H2O 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. 2H2O 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. 2H2O 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). 2H2O 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 2H2O; this thermal transition is attributed to the freezing of some bound water near the plastoquinone pool. 4. Δp2H is smaller than Δp1H at all temperatures (PSII + PSI chain). ΔpH is quasi-constant from 0°C to 10°C, then decreases when temperature rises. 2H2O 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 2H2O 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 2H2O 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.

A microchemiosmotic interpretation of energy-dependent processes in biomembranes based on the photosynthetic behaviour of thylakoids

Abstract The way in which protons couple the redox chain activity to ATP synthesis in biomembranes is controversial. (1) An important problem is the determination of the proton electrochemical potential difference, Δ μ (H+), between the two sides of the membrane. A critical analysis of the methods to estimate its osmotic component ΔpH (with amine probes, viz. fluorescent 9-aminoacridine), and its electric component Δφ (by electrochromic spectral-shift of endogeneous pigments) is presented. The influence of probe partitioning in the membrane and the effect of Δφ indicate that the experimentally computed ΔpH is probably related more to the interfacial than to the bulk ΔpH, but overestimates it. After continuous illumination, the electrochromic absorbance change (520 nm) in thylakoids decays in two phases; it is suggested that the slow phase reflects internal surface potential changes resulting from deprotonation of the inner membrane buffering groups. Neither phase may be reliably calibrated. Even though the combination of the measured ΔpH and Δφ to give true Δ μ (H+) is therefore rather questionable, it does not prevent the separate use of these two terms for comparative studies, such as presented below. (2) The kinetic and thermodynamic correlations between fluxes (electron flow and phosphorylation) and forces (essentially here Δ pH, since it largely predominates in Δ μ (H+) at steady-state) were studied under different conditions: isotope substitution (replacement of 1H+ by the slower 2H+); membrane topography manipulation (use of System I and System II chains to change the distance between H+ ports of entry and exit); and proton-permeability-increase of the membrane (nigericin addition) or of the coupling factor (nucleotide addition). (3) The simplest interpretation of these results is the existence of multiple H+ resistances, especially lateral, which cause different local Δ μ (H+) at various membrane points (H+ input and output): the measured Δ pH and Δφ reflect average values. This microchemiosmotic hypothesis is illustrated by an electrical analogue circuit; in this view, Mitchells delocalized chemiosmosis and Williamss direct coupling theories may be formally considered as particular cases of a more flexible mechanism. Without excluding it, the proposed hypothesis does not require the one-to-one relationship between primary and secondary proton pumps proposed by others (mosaic chemiosmosis).

Quantitative estimation of the photosynthetic proton binding inside the thylakoids by correlating internal acidification to external alkalinisation and to oxygen evolution in chloroplasts

The external alkalinisation delta pHe, or the rate of oxygen evolution vO2, of a suspension of envelope-free chlorplasts was correlated with their internal acidification, estimated from the transmembrane delta pHei. Knowing the external buffer value, the concentration of the total protons moved Hi was calculated from the delta pHe, measured with a glass electrode ([Hi] was also obtained from vO2), and the free proton concentration [Hi+] was determined from delta pHei, measured with 9-aminoacridine. This gives a ratio gamma i = theta [Hi]/theta [Hi+], which is independent of the thylakoids internal volume. Within a large pHi range, scanned by varying the light intensity, gamma i was kept reasonably constant; it was hardly sensitive to pHi. This apparent invariability implies a continuous change of the internal buffer value beta i with pHi, since beta i/gamma i = -2.3.....10pHi, a relationship which inlcudes neither the total concentration of protonizable groups [Ai] nor pKi. As gamma i approximately Ki[Ai]/(Ki + [Hi+i]2, to keep gamma i constant when pHi drops, pKi and [Ai] must increase. This may be achieved by a progressive unmasking of anionic functions, initially inaccessible in the membrane. The relative slowness of this process may explain why gamma i calculated from the initial kinetics was sometimes smaller in high than in low light, where it always equalled that measured from the steady-state amplitude at all intensities. A small deficit of [Hi+] deduced from what could have been expected from delta pHe may reflect a limited binding of protons in the membrane itself, about 1 H+ for 30--130 chlorophylls (gamma i could be between 70 and 240, more frequently around 100); these numbers varied depending on the samples, but were constant for a given preparation.

Study of the photosynthetic electron transfer reactions in chloroplasts and algae with the plastoquinone antagonist dibromothymoquinone.

Abstract 1. As is known, low concentrations of the plastoquinone antagonist dibromothymoquinone partly inhibit the Hill reaction (with dichlorophenolindophenol or ferricyanide) and completely the electron transfer from water or NH 2 OH to methylviologen. At high concentrations (where dibromothymoquinone serves as an electron acceptor), it was found that dibromothymoquinone also inhibits the System I-dependent electron transfer from reduced 2,6-dichlorophenolindophenol to methylviologen (less readily in uncoupled chloroplasts, suggesting two sites of reduced 2,6-dichlorophenolindophenol entry in the redox chain). This practically means that, by varying dibromothymoquinone concentration, one may have chloroplasts where each system is active independently of the other or where only System II remains efficient. 2. The concentrations of dibromothymoquinone for which the different inhibitions are seen depend on the chlorophyll concentration and the inhibition is not reversed by simple washing. 3. Dibromothymoquinone cancels completely the O 2 burst, a purely System II-dependent phenomenon, thought to measure the plastoquinone pool “A”. The slope of the concentration curve of O 2 -burst inhibition is less than that of the NH 2 OH to methylviologen electron transfer, a System II+I reaction. This suggests that at low concentrations dibromothymoquinone acts on the plastoquinone molecules closest to photoreaction I oxidants and that the progressive increase of dibromothymoquinone concentration disconnects the plastoquinone pool nearer and nearer to Q, the primary electron acceptor of photoreaction II. Finally all plastoquinone is “out-circuit”, and then dibromothymoquinone may react rather close to Q, an hypothesis supported by the fact that 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits the Hill reaction less efficiently with dibromothymoquinone (at high concentration) than with 2,6-dichlorophenolindophenol or ferricyanide. 4. The flash-yield O 2 oscillation pattern is not changed by dibromothymoquinone; therefore, it does not act at the oxidizing side of the reaction center II. 5. The effect of dibromothymoquinone on the O 2 evolution by Chlorella cells (burst and steady-state photosynthesis) is similar to that reported above for chloroplasts.

THE 518-mμ ABSORBANCE CHANGE AND ITS RELATION TO THE PHOTOSYNTHETIC PROCESS

Abstract— A study of the 518‐mμ light‐induced absorbance change in green cells and a comparison with photosynthetic O2 evolution were made. The effect of various chemical agents was also investigated. On the one hand, no antagonistic two‐light effect was observed on the absorbance change, and DCMU had only a partial inhibitory effect on it. On the other hand, it was possible to observe in some cases an indirect kinetic relationship between O2 evolution and 518‐mμ change. It is suggested that probably at least two substances absorb around 518‐mμ, one (XI) belonging to system I and another (XII) to system II. The existence and the function of XI are hypothetical and the identity of XII with the first photoproduct of system II is yet not well established.

Study of the chlorophyll fluorescence in chloroplasts and algae with the plastoquinone antagonist dibromothymoquinone

Abstract The effect of the plastoquinone antagonist dibromothymoquinone on chlorophyll fluorescence in vitro and in vivo was investigated. 1. 1. With chlorophyll a in solution quenching is observed, more efficient than that of p -benzoquinone (the Stern-Volmer constant K = 200 M −1 ); ascorbate removes this effect. 2. 2. With isolated chloroplasts, a dramatic enhancement of quenching occurs (also abolished by ascorbate) in the following order of importance: thermal step, photochemical step (of variable fluorescence), and constant fluorescence; K for the total variable fluorescence ≈ 480 000 M −1 , for constant ≈ 190 000 M −1 . Parallel to the quenching effect, an enlargement of the complementary area of the variable fluorescence is observed. Addition of ascorbate suppresses the quenching and this enlargement. If this area in the presence of reduced dibromothymoquinone is similar to that of the control, this means that A, the pool of oxidant next to Q, remains connected to it, a result contradicted by the O 2 -burst measurement. However, when the excess of unbound dibromothymoquinone is washed out, which removes the quenching effect, a significant decrease of the area is seen, in agreement then with the burst results. Therefore, the meaning of the complementary area and the nature of the controlling factors may be questioned. 3. 3. With whole cells ( Chlorella ), no quenching is observed; instead, an enhancement of the thermal step is noticed, together with an even larger increase of the complementary area. Dibromothymoquinone being in an oxidized form at the thylakoid level, it is proposed that in intact membranes, the chlorophyll molecules are not readily accessible to it: thus, no quenching is observable, and the predominant effect is on the redox chain. The specific effect of dibromothymoquinone on the thermal step supports the hypothesis that it is normally controlled by a secondary quencher, related to plastoquinone.

Ionic composition of the medium, surface potential and affinity of the membrane-bound chloroplast ATPase for its charged substrate ADP

Abstract Because the membrane-bound ATPase and its substrate ADP are both electronegative, the effect of the ionic concentration on the kinetic parameters of photophosphorylation Vmax and Km (ADP) was examined in lettuce thylakoids in various conditions. The initial rate of ATP synthesis was measured by the “scalar” H+ consumption in the external medium, in conditions where the “vectorial” electrochemical proton gradient (restricted here to ΔpH) was maintained constant; ΔpH was estimated from the 9-aminoacridine fluorescence quenching. This signal was calibrated by experiments where the “phosphate potential” ΔGp was in equilibrium with ΔpH. With a fixed H+/P stoichiometry of 3, a strong dependence of the probe response on the ionic conditions was found. On the other hand, at a given ΔpH, the Km for ADP was unexpectedly found insensitive to the ionic conditions. Especially, lowering the Mg2+ concentration from 5 mM to 0.5 mM diminished Vmax considerably but did not affect Km. Attempts to measure the membrane surface electrical potential ψs by salt-induced release of 9-aminoacridine and by particle electrophoresis showed important discrepancies between the two methods. This seems not to be due to the difference between the potential measured at the membrane surface, more precisely at the Stern or Gouy levels, and that at the plane of shear only. It is more likely a consequence of side effects with 9-aminoacridine. Taking the electrokinetic potential ζ as representative of ψs, it is then shown that the membrane surface potential is already small in the minimum salt medium (ca. −15 mV) and, consequently, that the ADP concentration near the membrane, and hence Km, can barely change with the ionic concentration increase. This makes that the Km determinations, based on ADP concentrations in the bulk phase, are close to the true Km, which must take into account the actual ADP concentration at the membrane level.

Proton Currents and Local Energy Coupling in Thylakoids: A Survey

A survey of the experimental and conceptual basis of a microchemiosmotic interpretation of energy-dependent processes in thylakoids is presented. It assumes that protons circulate from their active transport points to their backflow ports in a heterogeneous medium, having a resistivity not only transversal, from membrane to isopotential bulk phase, but also lateral, in or on the membrane, an idea central to our hypothesis. Consequently, the proton electrochemical potential ΔμH at the H+-generators (redox carriers) is higher than ΔμH at H+-leaks (coupling factors and membrane pores), and the measured ΔμH is an average of local values. However, some flexibility in this microscopic coupling is possible, and a delocalized behaviour may sometimes be obtained by increasing the lumen volume (osmolarity decrease) and its conductivity (ionicity increase). A one-to-one link between primary and secondary pumps, as advocated by mosaic chemiosmosis, seems therefore improbable here.

Comparative Photochemical and Electrochemical Properties of Thylakoids, Stromal Lamellae, Inside-in and Inside-out Vesicles (Granal and Randomized)

The local events in energy transduction (see Haraux, de Kouchkovsky, 1983) must have a material support. Thus, the recent isolation of inside-out and normal inside-in vesicles from grana stacks (Andersson, Akerlund, 1978) or “randomized” thylakoids (Andersson et al., 1980) is of great interest. But before using these fractions for such problems, we have characterized them by their electrostatic properties and their chlorophyll fluorescence, both good witnesses of membrane state and functional integrity.