Claude Sigalat

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.

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).

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.

Effect of hydrogendeuterium exchange on energy-coupled processes in thylakoids: A new illustration of the hypothesis of local proton gradients with the energy-transducing biomembranes

The redox chain control of envelope-free chloroplasts suspended in a deuterated medium is enhanced despite a diminished proton gradient [ 1,2]. This observation, and others [2,3], led us to hypothesize that, superimposed to the classical ‘chemiosmotic’ mean transmembrane ApHT, a lateral pH difference, ApH,, of some tenths of ‘units’ (negative inside, positive outside), may exist between the sites of H’ ‘pumping’ (plastoquinone, PQ) and leakage (coupling factor, CF), due to their spatial separation and to the existence of a significant lateral resistance to protons [3]. Such a resistance would be increased in ‘H20, owing to the lower mobility of deuterons along the membrane. Thus, the back-pressure at the plastoquinone level would be exerted by an internal pH locally lower than the average value measured in the inner compartment, this difference, pH, pHF, being enhanced in 2H20. A corollary of our hypothesis is that for a similar ApH, with both isotopes, the local ApHT at the CF level should be smaller in 2H20 than in ’ H20, which should reduce the rate of ATP synthesis. We have therefore investigated the effect of heavy water on the photophosphorylation, measured correlatively with the A& and the electron flow. Earlier works on mitochondria gave inconsistent results: the ‘P/O’ ratio was found unchanged [4,5], decreased [6] or increased [7,8] in deuterated media, but no attention was then paid to ApH, the existence and role of which were not known at that time. Our results indicate that the isotopic substitution slows down the ATP synthesis even more than the electron flow, resulting in a decrease of the ‘P/e’ ratio, (number of ATP molecules formed per electron transferred). That is, the P/e vs Ap*plot is translated in 2H20 towards the positive ApH, values, which is, as expected, a shift opposite to that seen for the redox chain control [ 1,2]. These results are therefore consistent with our concept of a steady-state lateral pH heterogeneity along the membrane, and the increase of the lateral resistance to protons (i.e., deuterons) in 2H20 should enhance the difference between local and mean transmembrane ApH,.

The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide ROLE OF CATALYTIC INTERMEDIATES AND ENZYME TURNOVER

The mechanism of inhibition of yeast mitochondrial F1-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (β-Y345W). Fluorescence quenching of a β-Trp345 indicates that the binding of MgADP to F1 can be described as 3 binding sites with dissociation constants of Kd1 = 10 ± 2 nm, Kd2 = 0.22 ± 0.03 μm, and Kd3 = 16.3 ± 0.2 μm. In addition, the ATPase activity of the β-Trp345 enzyme followed simple Michaelis-Menten kinetics with a corresponding Km of 55 μm. Values for the Kd for MgATP were estimated and indicate that the Km (55 μm) for ATP hydrolysis corresponds to filling the third catalytic site on F1. IF1 binds very slowly to F1-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 μm, but decreased thereafter. The rate of inhibition was half-maximal at 5 μm MgATP, which is 10-fold lower than the Km for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.

Proton coupling is preserved in membrane-bound chloroplast ATPase activated by high concentrations of tentoxin.

The effect of tentoxin at high concentrations was investigated in thylakoids and proteoliposomes containing bacteriorhodopsin and CF0CF1. Venturicidin‐sensitive ATP hydrolysis, ATP‐generated ΔpH and ATP synthesis were practically 100% inhibited at 2 μM tentoxin, and restored to various extents beyond 50 μM. With respect to the native enzyme, tentoxin‐reactivated ATPase had the following properties: (i) a higher ΔpH requirement to synthetise ATP; (ii) a decreased futile proton flow through CF0CF1 (without ADP), which remains 100% blocked by ADP. It is concluded that despite its altered kinetic performances, tentoxin‐modified CF0CF1 preserves its mechanism and remains a tightly coupled proton pump.

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.

ENZYME TURNOVER IS ESSENTIAL FOR DEACTIVATION OF F0F1-ATPASE IN PLANT MITOCHONDRIA

Abstract In potato tuber mitochondria, ATPase deactivates immediately after treatment with an uncoupler or with polyoxyethylene 9-lauryl ether (Lubrol), a non-ionic detergent. Deactivation was completely prevented by another non-ionic detergent, lauryldimethylamine oxide (LDAO). LDAO also induced slow reactivation of inactive ATPase formed in deenergized mitochondria. Freezing of the active state by LDAO was used to study the process of ATPase deactivation following deenergization in intact mitochondria. Deactivation was slowed down by carboxyatractyloside (CATR), which prevents ATP import into the matrix, and by ATPase inhibitors. ATP hydrolysis was also triggered by Lubrol with CATR-treated mitochondria. The initial rate was close to the capacity for ATP synthesis but rapidly decayed. The rate of decay increased with the concentration of MgATP and no decay was observed in the presence of EDTA. The following conclusions were drawn. (1) Deenergization in itself is not sufficient for ATPase deactivation in plant mitochondria: enzyme turnover is also required. The probability of one enzyme to be deactivated at each turnover is much higher in potato tuber than in pea leaf organelles. (2) Enzyme turnover probably shifts the IF1-F1 complex from an active to an inactive form; the rate of deactivation indeed does not seem to be controlled by the binding of the inhibitory peptide. (3) The short-term effect (protection) and the long-term effect (reactivation) of LDAO on MF0MF1 may tentatively be used to titrate the activated versus total amounts of these enzymes in cells.