Francis Haraux

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.

The efficiency of energized protons for ATP synthesis depends on the membrane topography in thylakoids

In thylakoids system II water‐splitting proton generation is mainly localized in grana stacks, whereas system I plastoquinol reoxidation, is essentially restricted to non‐appressed regions, such as stromal lamellae; the same is true for the coupling factor. For a given mean proton gradient, a system II chain was found to be less able to drive phosphorylation than a system I or a system I + II chain. These results support our microchemiosmotic hypothesis, based on the existence of lateral resistances to H+ movements. They confirm that the proton gradients at the redox chain and at the coupling factor are unequal and that both are different from their mean experimentally measured value.

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).

Energy coupling and ATP synthase

This review is focused on the chloroplast (i.e. thylakoid) ATP synthase-hydrolase (ATPase) but also refers to the bacterial and mitochondrial cases when relevant. (1) A proton-translocating F-ATPase (CF-ATPase in chloroplasts) comprises an intramembrane proton channel, F 0, and an extramembrane catalytic head, F 1, the latter forming a ‘ball’ of 3 alternative couples of α andβ subunits plus one copy of each γ , δ, subunit;α andβ subunits bear regulatory and catalytic nucleotide binding sites, respectively. F 0 acts as a motor, with a stator made of subunits IV (a in prokaryotes), I and II ( b, b′), and a rotor arranged in a crown of 9–12 subunits III ( c). Subunitγ , anchored to the rotor, thus turns with the latter while its other extremity, entering into F 1, induces periodical conformational changes that allow binding of substrates on one β and release of products from another. The energy is supplied by the proton gradient built by the redox chain. Thus, one H + from the high potential compartment (thylakoid lumen) protonates, via part of subunit a, a neighboring subunit c while anotherc nearby, facing the other part of a, is deprotonated and the resulting H + escapes into the low potential compartment (chloroplast stroma). (2) The mechanistic stoichiometry H +/ATP of protons transported per ATP formed was initially found to equal 2–3 but is now considered to equal 4. Such a shift may be due to the diversity of material (chloroplasts, mitochondria, bacteria, liposomes), techniques (steady-state vs. pH or salt jumps) and approaches (kinetic: ‘flow–flow’, or thermodynamic: ‘force–force’ methods). In mitochondria, after subtraction of the H + due to Pi translocation, H+/ATP is close to 3, perhaps due to a smaller number of c subunits (H+/ATP = c/β). (3) The osmotic ( 1pH) and electric ( 19) components of total 1μ̃H+ are thermodynamically interconvertible and, despite some conflicting results, also seem kinetically equivalent. This is thanks to a ‘proton well’, a scheme of which is presented. A related problem is whether phosphorylation depends on pH in and pHout or solely on their difference (=1pH). No consensus exists on how the enzyme affinity for ADP, expressed by its K m, may depend on1μ̃H+. Two models are discussed. One is the simultaneous binding of an ADP and release of an ATP, driven by the simultaneous translocation of 4 protons while the other introduces an intermediary step. (4) Besides being an energetic source for ATP synthesis, 1μ̃H+ switches ATPase from inactive to active state and, specifically for CF 1, exposes a disulfide bridge of γ to thiolreducing agents, thioredoxin i vivo (reduced by Photosystem I), DTT in vitro. This reduction lowers the activation for threshold1μ̃H+ and at the same time prolongs the life-time of the active enzyme from milliseconds to minutes after dissipation of 1μ̃H+. By using ATPase inhibitors before or after activation, we have suggested that activating and catalytic protons are different (actually, activation does not necessarily require a proton flow). Activation is also accompanied – or governed – by conformational changes. In mitochondria, there is an unbinding of a special subunit IF1 from F1; its equivalent for CF1 could be a conformational change of . A final remark concerns the physiological role of (de)activation, if any. It was proposed that inactive state of CF could prevent futile hydrolysis of ATP in dark, but in fact, thanks to the low 1μ̃H+ threshold required for activation of reduced CF 1 and to low membrane permeability to protons in vivo, a minute consumption of ATP is sufficient to maintain a high enough 1μ̃H+ to oppose a counter-protonmotive force limiting sustained ATP hydrolysis.

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.

Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxin†

Difficulties in synthesis make natural cyclopeptides challenging targets for chemists. Our interest focused on two natural toxic cyclopeptide series produced by pathogenic fungi: tentoxin, [cyclo-(N-MeAla 1 -Leu 2 -N-MeΔ z Phe 3 -Gly 4 )] and the destruxins [cyclo-(Pro 1 -Ile 2 -N-MeVal 3 -N-MeAla 4 -β-Ala 5 -HA 6 )]. The total syntheses of these two bioactive series were optimised, and several analogues were designed and synthesised to establish structure-activity relationships. The importance of synthetic analogues in the identification of molecular targets and the explanation of mechanisms of action was demonstrated. Such systematic investigations can determine the crucial features responsible for the activity of the natural compound and help the design of more powerful or more selective products.

Shift from localized to delocalized protonic energy coupling in thylakoids by permeant amines

Abstract We have suggested that in energy-transducing organelles structural constraints may hinder H+ transport from their sites of active, redox translocation to their sites of passive or phosphorylating escape (microchemiosmosis). We could modulate these constraints first by affecting the physico-chemical properties of the medium, and now by adding permeant amine buffers to a suspension of thylakoids. The following results are obtained. (1) Whether driven by Photosystem I or by Photosystem II, phosphorylation is stimulated by amines (imidazole, hexylamine, NH4CI) at concentrations low enough hardly to modify the proton gradient: ΔpH is stable if not reduced, Δψ is slightly increased but still negligible; this extends the observations made by Giersch. (2) The concentration curves of phosphorylation stimulation by amines exhibit a minor peak before the main one previously reported. (3) ATP synthesis and ΔpH are decreased by amines at higher concentrations, but the dependence of the phosphorylation rate (flow) versus ΔpH (force) is then shifted towards lower ΔpH values. (4) The normally less efficient Photosystem II-driven phosphorylation is comparable to Photosystem I with amines. (5) The flow-force curves, which are distinct when traced by limiting ΔpH by an H+ influx decrease (light) or efflux increase (nigericin), are much closer with either photosystem when amines are added. The Photosystem I curve produced by increasing nigericin beyond approx. 200 nM becomes insensitive to amines. (6) In general, Photosystem II requires significantly less nigericin or amines than Photosystem I to obtain similar effects. It is proposed that amines have a double effect. By efficiently carrying protons along the membrane, amines lower the protonic resistances and thereby delocalize the proton gradient. At higher amine concentrations, uncoupling occurs, probably by an indirect backflow of protons more than by a buffering effect, as generally admitted. In conclusion, the multiple-resistances microchemiosmotic scheme which we have proposed earlier is strengthened; it predicts that intermediate states may link delocalized (canonical chemiosmosis) and localized coupling modes, which is established here.

2H2O effect on the electron and proton flow in isolated chloroplasts: An indication for lateral heterogeneity of membrane pH

Abstract To determine how the actual pH on the membrane surfaces regulate the redox reactions, a study of the electron and proton flow in thylakoids was made in heavy water, in which 2H+ has a smaller mobility than 1H+. The decrease of the redox rates by 2H2O is stronger in coupled than in uncoupled conditions, although the bulk ΔpH is slightly diminished; therefore changing 1H+ by 2H+ enhances the electron transfer control. This is particularly so when the two systems are linearly connected: i.e. the plastoquinone pool, not the water-splitting complex, is the main point of the redox chain regulation by ΔpH and of the deuterium action. The role of the proton diffusion barriers is emphasized and the concept of a heterogeneity—accentuated in 2H2O - of the pH along the membrane is proposed. The corresponding local pH , different at the points of H+ active translocation and passive leakage, would be the real factors controlling the membrane-bound processes.

ΔpH-activation of the thiol-modified chloroplast ATP hydrolase: Nucleotide binding effects

Abstract The activation of the membrane-bound ATP hydrolase by the electrochemical proton gradient Δ \ gm H + was studied with dithiothreitol-treated, dark-adapted lettuce thylakoids. After reactivation, carried out at different light intensities, hydrolysis of ATP or GTP was measured upon fast membrane deenergization and substrate addition. The following results were obtained. (1) The initial rate of ATP hydrolysis depends on both the ΔpH attained during the light-reactivation stage and on the external pH (ΔpH was measured by the 9-aminoacridine technique, Δψ being collapsed by valinomycin). (2) When hexylamine is present the initial rate of ATP hydrolysis, as that of GTP hydrolysis measured in the presence of 50 μM GDP, only depends on the ΔpH. (3) ADP addition is able to inhibit a fraction of the ATPases within less than a few seconds. The affinity for the inhibitor is increased if ADP is added 10 s after complete membrane deenergization. From the results obtained, with either ATP or GTP in delocalized conditions (hexylamine present), it is proposed that the number of active thiol-reduced ATPases is a simple function of the ΔpH, independent of the external pH. The results without hexylamine are interpreted as being due to an incomplete delocalization of the proton gradient. The mechanistic implications of this ΔpH-activation are discussed. The rapid deactivation of only a fraction of active ATPases by ADP binding, and more especially the decrease in affinity for this inhibitor by ΔpH, suggests the existence of different active states which are discriminated by their deactivation and not their catalytic properties.