Light-induced proton slip and proton leak at the thylakoid membrane

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H⁺/e⁻-ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF₁-γ of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF₁-γ was only 36%, which indicates that in vivo the reduction of CF₁-γ could be limited by fast reoxidation and/or restricted accessibility of CF₁-γ to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.     

The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex

Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site.     

The synthesis of NPQ-effective zeaxanthin depends on the presence of a transmembrane proton gradient and a slightly basic stromal side of the thylakoid membrane

In the present study we address the question which factors during the synthesis of zeaxanthin determine its capacity to act as a non-photochemical quencher of chlorophyll fluorescence. Our results show that zeaxanthin has to be synthesized in the presence of a transmembrane proton gradient. However, it is not essential that the proton gradient is generated by the light-driven electron transport. NPQ-effective zeaxanthin can also be formed by an artificial proton gradient in the dark due to ATP hydrolysis. Zeaxanthin that is synthesized in the dark in the absence of a proton gradient by the low pH-dependent activation of violaxanthin de-epoxidase is not able to induce NPQ. The second important factor during the synthesis of zeaxanthin is the pH-value of the stromal side of the thylakoid membrane. Here we show that the stromal side has to be neutral or slightly basic in order to generate zeaxanthin which is able to induce NPQ. Thylakoid membranes in reaction medium pH 5.2, which experience low pH-values on both sides of the membrane, are unable to generate NPQ-effective zeaxanthin, even in the presence of an additional light-driven proton gradient. Analysing the pigment contents of purified photosystem II light-harvesting complexes we are further able to show that the NPQ ineffectiveness of zeaxanthin formed in the absence of a proton gradient is not caused by changes in its rebinding to the light-harvesting proteins. Purified monomeric and trimeric light-harvesting complexes contain comparable amounts of zeaxanthin when they are isolated from thylakoid membranes enriched in either NPQ-effective or ineffective zeaxanthin.     

Fluorescence induction in chloroplasts isolated from the green alga Bryopsis maxima III. A fluorescence transient indicating proton gradient across the thylakoid membrane

The yield of chlorophyll a fluorescence in dark-adapted intactchloroplasts isolated from the green alga, Bryopsis maxima,showed, after the first wave of the fluorescence induction wasover, a peak labelled M₁ at about 10th sec of illumination.The time to reach M₁ during continuous illumination inverselydepended upon exciting light intensity. The appearance of thepeak M₁ was accelerated by the addition of methyl viologen aselectron acceptor and delayed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.KCN had no effect on the peak M₁ at a concentration where photosyntheticoxygen evolution was completely suppressed. Thus, the peak M₁appears to be related to electron transport but not to the carbonreducing cycle. Carbonylcyanide m-chlorophenylhydrazone, NH₄Cl and methylaminediminished or eliminated M₁. On the other hand, an enhancementof the fluorescence yield at M₁ was observed in the presenceof energy transfer inhibitors. Valinomycin plus KCl also increasedheight of the peak M₁. However, the combined addition of valinomycinand dinitrophenol resulted in the complete elimination of thepeak M₁. These results indicate that the fluorescence peak M₁occurring at about 10 sec of illumination is linked to a protongradient across the thylakoid membrane. (Received July 7, 1977; )     

The proton gradient across mycoplasma membranes

The proton gradient across mycoplasma membranes was determined by using different probes which distribute between the intracellular space and the suspension medium in response to a transmembrane proton gradient. The intracellular pH of intact glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: pH_ext=7 and pH_int=7.4; hence, pH=0.4. The size of the proton gradient depended upon the extracellular pH. Without nutrient substrate, the mycoplasmas were unable to maintain a transmembrane proton gradient, i.e., pH approximated O.N, N-dicyclohexylcarbodiimide, an inhibitor of membrane-bound ATPase, carbonyl cyanide-m-chlorophenyl hydrazone, a proton conductor, and gramicidin, an antibiotic forming cation conduction channels across membranes, strongly affected and even abolished the proton gradient across mycoplasma membranes. These substances also impaired the metabolic activity and viability of mycoplasmas.     

Correlation between photosynthesis and the transthylakoid proton gradient

In isolated intact chloroplasts, maximal rates of photosynthetic O₂ evolution (in saturating HCO^?₃) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO₂ fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical ΔpH is 3.9.CO₂-dependent O₂ evolution is inhibited by increases of 0.1–0.2 in ΔpH that occur when catalase is omitted from the medium, NO^?₂ is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O₂. Low concentrations of antimycin (0.33 μM) or NH₄Cl (0.33 mM) decrease ΔpH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower ΔpH or relieve the inhibition.A decrease of ΔpH below 3.9 by weaker illumination, millimolar levels of NH₄Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate.These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO₂ assimilation.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

In vivo regulation of thylakoid proton motive force in immature leaves

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.     

Sigmoidal stimulation by solutes of light-induced proton translocation in thylakoid membranes

A sigmoidal curve was obtained for the relationship betweenthe stimulation of light-induced proton uptake and the concentrationof salts in a suspending medium for thylakoid membranes. Substitutionof sucrose for the salts also resulted in a sigmoidal curve.It changed into a hyperbolic curve with salts when the mediumalready contained sucrose. The results are discussed in relationto the structural arrangement of the thylakoid membranes bythe osmotic effect of the solutes. (Received February 10, 1975; )     

Light-induced proton translocation through thylakoid and cytoplasmic membranes of Plectonema boryanum

Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine. The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from the cytoplasm into the incubation medium.     

Measurement of the electrochemical proton gradient in submitochondrial particles

The pH gradient and membrane potential of submitochondrial particles from bovine heart were estimated by the uptake of [14C]ethylamine and [36Cl]perchlorate, using filtration through a glass fiber prefilter and Millipore filter without washing to separate the vesicles from the medium. An external volume probe of [3H] sucrose was also used. Internal volume of the vesicles was measured by the extent of uptake of glucose, which equilibrates slowly across the membrane. The electrochemical potential gradient of H+ (delta micro H+) calculated from uptake of ethylamine and perchlorate, assuming the ions taken up were free in solution inside the vesicles, was 23 to 24 kJ/mol of H+ (240-250 mV) during respiration in the absence of ATP. The ratio of the free energy of ATP synthesis (delta GATP) to delta micro H+ was 2.2 to 2.3 during oxidative phosphorylation and only slightly higher during ATP hydrolysis indicating that the H+-translocating ATPase is close to equilibrium under both conditions. The nonintegral ratio suggests there is a systematic error in the measurement of delta micro H+. The value of delta micro H+ calculated from ion uptake could be too high if some of the ions taken up are bound to the membrane or concentrated into the electric double layer at the inner membrane-water interface. The effects of vesicle volume (varied osmotically) and permeant ions (which affect internal ionic strength and pH) on the ratio of delta GATP to delta micro H+ suggested that ion association with the membrane in fact caused significant overestimation of delta micro H+. Association of ethylammonium and perchlorate ions with unenergized submitochondrial particles was measured by centrifugation, in the presence of a high concentration of impermeant salt to minimize association with the external surface. The results were used to estimate the extent of binding during the ion uptake assays, and delta micro H+ was recalculated taking this binding into account. The resulting values were between 19 and 20 kJ/mol of H+ (197-207 mV) during respiration in the absence of ADP, and the ratio of delta GATP to delta micro H+ was about 3 during oxidative phosphorylation.     

A new electrochemical gradient generator in thylakoid membranes of green algae

Using a new method of delayed luminescence digital imaging, mutants of Chlorella sorokiniana lacking the chloroplast CF0CF1 ATP synthase were isolated for the first time. Biochemical characterization of these strains indicates a lack of detectable synthesis and accumulation of the ATP synthase subunits alpha-CF1 and beta-CF1. Functional characterization indicates the presence of a permanent electrochemical gradient (DeltaMu) across the thylakoid membrane in the dark-adapted state, which is not suppressed under anaerobic conditions. Contrary to what is observed in the presence of the CF0CF1 ATP synthase, this gradient is essentially due to an electric field component DeltaPsi with no detectable DeltapH component, under both aerobic and anaerobic conditions. Neither the CF0CF1 ATP synthase nor a respiratory process can thus be responsible for a permanent gradient detected under these conditions. The previous proposal of a new ATP-dependent electrogenic pump in thylakoid membranes is supported by these results that, in addition, indicate a specificity of this new pump for ions other than protons.     

Distinguishing between luminal and localized proton buffering pools in thylakoid membranes

The dual gradient energy coupling hypothesis posits that chloroplast thylakoid membranes are energized for ATP formation by either a delocalized or a localized proton gradient geometry. Localized energy coupling is characterized by sequestered domains with a buffering capacity of approximately 150 nmol H(+) mg(-1) chlorophyll (Chl). A total of 30 to 40 nmol mg(-1) Chl of the total sequestered domain buffering capacity is contributed by lysines with anomolously low pK(a)s, which can be covalently derivatized with acetic anhydride. We report that in thylakoid membranes treated with acetic anhydride, luminal acidification by a photosystem I (duraquinol [DQH(2)] to methyl viologen [MV]) proton pumping partial reaction was nearly completely inhibited, as measured by three separate assays, yet surprisingly, H(+) accumulation still occurred to the significant level of more than 100 nmol H(+) mg Chl(-1), presumably into the sequestered domains. The treatment did not increase the observed rate constant of dark H(+) efflux, nor was electron transport significantly inhibited. These data provide support for the existence of a sequestered proton translocating pathway linking the redox reaction H(+) ion sources with the CF(0) H(+) channel. The sequestered, low-pK(a) Lys groups appear to have a role in the H(+) diffusion process and chemically modifying them blocks the putative H(+) relay system.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.     

Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H⁺ electrochemical gradient (Δ_μ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δ_μ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.     

Light dependence of the decay of the proton gradient in broken chloroplasts

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetic analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant k_L is equal to k_D + mR₀, where R₀ is the initial rate of proton uptake which is a function of light intensity, k_D is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K⁺ increases k_D without affecting m. Treatment with N,N′-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affecting k_D. Treatment with glutaraldehyde makes k_L independent of light intensity and hence m = 0. These results suggest that the light-dependent part (mR₀) of k_L is due to leakage of protons through the coupling factor (CF₁-CF₀) complex which can open or close depending on light intensity and that the light-independent part (k_D) of the decay constant k_L is due to proton leakage elsewhere.     

Gradation of the magnitude of the electrochemical proton gradient in Mycoplasma cells

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non-metabolizable analogue methyl alpha-D-glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+-dependent ATPase (phi (cHxN)2C H+) and the respective amplitudes of the components of delta mu H+. When Mycoplasma cells are incubated with 10 mM methyl alpha-D-glucoside, the amplitude of phi (cHxN)2C H+, of the electrical potential delta psi and of the chemical gradient delta pH become continuous functions of external glucose concentration within the limits of the non-energized and fully energized states. Analysis of the relationships between graduated amplitudes of delta psi, delta pH and phi (cHxN) 2C H+ show that the primary form of energy stored by a delta mu H+ generator is the electrical potential.     

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (～7.6) and ΔpH(m) (～0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (～5 mm, pH 7.8) compared with the cytosol (～20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.