The distance between thiol groups in the γ subunit of coupling factor 1 influences the proton permeability of thylakoid membranes

Spinach chloroplast thylakoids treated in the light with bifunctional maleimides were previously shown to be uncoupled. The increase in proton permeability by these reagents is caused by the cross-linking of an accessible group on the subunit of coupling factor 1 (CF₁) to a group that becomes exposed to reaction with maleimides only when the thylakoids are energized. In this study, several bifunctional maleimides, includingo-,m-, andp-phenylenebismaleimides, 2,3- and 1,5-naphthalenebismaleimides, and azophenylbismaleimide, were tested for their ability to form cross-links and to uncouple photophosphorylation. These reagents form cross-links from about 6 to 19 Å. Each reagent was found to form cross-links in the light and to inhibit photophosphorylation. However, the effectiveness of these compounds as uncouplers decreased as the distance between the cross-linked groups increased, indicating that the distance between two groups on the subunit of CF₁ can regulate proton flux through the membrane. Monofunctional maleimides cause a light-dependent energy transfer type of inhibition of photophosphorylation. Although this inhibition was correlated to the reaction of the maleimide with a group on the subunit that is exposed only in energized thylakoids, the accessible group on this subunit was also modified by the reagent. However, we show here that the accessible group plays no role in the inhibition of photophosphorylation. This group may be blocked by incubating thylakoids in the dark with methyl methanethiolsulfonate. The light-dependent inhibition of photophosphorylation byN-ethylmaleimide was unaffected by this treatment or by the subsequent removal of the methanethiol moiety from the accessible group.     

Sulfhydryl reagents and energy-linked reactions in monocot thylakoids

Monofunctional maleimides have been used to covalently modify the coupling factor protein of monocot thylakoid membranes. As with dicot thylakoids, incubation of the monocot thylakoids with maleimides in the light but not in the dark results in inhibition of both ATP synthesis and hydrolysis. In the dark, sites on the γ and ε subunits of maize Zea mays coupling factor 1 are modified after incubation of maize mesophyll thylakoids with the fluorescent maleimide N-(anilinonaphthyl-4) maleimide. A light accessible site localized solely to the γ subunit has also been demonstrated. In contrast to the case with dicot thylakoids (spinach [Spinacia oleracea] and pea [Pisum sativum]) treatment of monocot thylakoids (maize, barley [Hordeum vulgare], crabgrass [Digitaria sanguinalis]) with bifunctional maleimides or thiol oxidants in the light does not result in functional uncoupling, i.e the bifunctional reagents act more like energy transfer inhibitors. The lack of functional uncoupling could be due either to a failure of the reagents to cross-link key sulfhydryl residues in the γ subunit or to the continued ability of the γ subunit to gate proton movements through the chloroplast coupling factor complex even though its conformation has been altered by sulfhydryl reagents.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the gamma subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the gamma subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the epsilon subunit. Our results suggest that overcoming inhibition by the epsilon subunit costs energy.     

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.     

Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling-resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro

The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. “Marketer”) and spinach (Spinacia oleracea L. “Bloomsdale”) were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.     

Die Wirkung von Dinactin auf die cyclische Photophosphorylierung,die lichtinduzierte ATP-Pa-Austauschreaktion und den lichtinduzierten Protonentransport in Chloroplasten

Summary Dinactin, an antibiotic forming complexes with K⁺ ions, uncouples phosphorylation in chloroplasts without requiring the presence of a substance increasing the permeability of the membrane for protons. To inhibit photophosphorylation, less Dinactin is necessary in the absence than in the presence of K⁺.When added before the light phase, Dinactin affects the light-triggered ATP-P_i exchange reaction in the same way as it does the complete photophosphorylation. Addition of the antibiotic after the activation by light inhibits the exchange reaction independently of the presence of K⁺, possibly by blocking the energy transfer to ATP.The inhibition of the light-induced proton transport by Dinactin is more pronounced in the presence of K⁺ than of Na⁺ ions. The manner in which changes in the permeability of the chloroplast membrane for K⁺ ions caused by Dinactin may influence photophosphorylation and reactions coupled with it is discussed.

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Verwendete Abkürzungen ATP Adenosintriphosphat - ADP Adenosindiphosphat - P_a anorganisches Phosphat - PMS Phenazinmethosulfat - DCPIP Dichlorphenolindophenol - FeCy Ferricyanid - DNP Dinitrophenol - FCCP Carbonylcyanid-p-trifluormethoxyphenylhydrazon - SQ 15859 Squibb Compound 15859     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores.

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation. (2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl2 + phosphate, or ADP + MgCl2 + arsenate during preillumination provides full protection against this effect. (3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished. (4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate. (5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction

Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.     

Effects of Diffusion and Topological Factors on the Efficiency of Energy Coupling in Chloroplasts with Heterogeneous Partitioning of Protein Complexes in Thylakoids of Grana and Stroma. A Mathematical Model

In this work, we studied theoretically the effects of diffusion restrictions and topological factors that could influence the efficiency of energy coupling in the heterogeneous lamellar system of higher plant chloroplasts. Our computations are based on a mathematical model for electron and proton transport in chloroplasts coupled to ATP synthesis in chloroplasts that takes into account the nonuniform distribution of electron transport and ATP synthase complexes in the thylakoids of grana and stroma. Numerical experiments allowed the lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids to be simulated under different metabolic conditions (in the state of photosynthetic control and under conditions of photophosphorylation). This model also provided an opportunity to simulate the effects of steric constraints (the extent of appression of thylakoids in grana) on the rates of non-cyclic electron transport and ATP synthesis. This model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: 1) slowing down of non-cyclic electron transport due to a decrease in the intra-thylakoid pH, and 2) retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana. Numerical experiments for model systems that differ with respect to the arrangement of thylakoids in grana allowed the effects of osmolarity on the photophosphorylation rate in chloroplasts to be explained.     

A simple, quantitative approach to the coupling of photophosphorylation to electron flow in terms of proton fluxes.

A simple relationship between observed phosphorylation efficiencies (P/e ratios) and internal proton concentration in spinach chloroplast thylakoids has been derived. P/e ratios, varked by either changing the light intensity or by adding the energy transfer inhibitor, 4'-deoxyphlorizin, were found to change with internal proton concentration in accordance with this relationship. A quantitative prediction of the effect of uncouplers on the P/e ratio can probably also be made. By extrapolation of plots of observed P/e ratios against internal proton concentration divided by the overall rate of electron flow, a maximum intrinsic P/e of about 0.66 is obtained. Assuming that two protons appear inside thylakoids per electron transferred, a P/e ratio of 0.66 suggests that three internal protons are consumed for each ATP formed. Internal protons may be considered to be substrates for the phosphorylation reaction. Hill plots of phosphorylation rate vs. internal proton concentration also indicate that three protons are consumed for each ATP synthesized. Thus, the H+ concentration gradient behaves quantitatively, as well as qualitatively, as if it is the connecting link between electron flow and phosphorylation in illuminated thylakoids.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation.(2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl₂ + phosphate, or ADP + MgCl₂ + arsenate during preillumination provides full protection against this effect.(3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished.(4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate.(5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.     

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.     

Inhibition of photophosphorylation by kaempferol

Kaempferol, a naturally occurring flavonol, inhibited coupled electron transport and both cyclic and noncyclic photophosphorylation in isolated pea (Pisum sativum) chloroplasts. Over a concentration range which gave marked inhibition of ATP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids. It is suggested that kaempferol acts as an energy transfer inhibitor.     

Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics

The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-P_i exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-P_i exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-P_i exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD⁺ reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling.     

Quantitative aspects of adenosine triphosphate-driven proton translocation in spinach chloroplast thylakoids

After illumination in the presence of dithiothreitol, chloroplast thylakoids catalyze ATP hydrolysis and an exchange between ATP and Pi in the dark. ATP hydrolysis is linked to inward proton translocation. The relationships between ATP hydrolysis, ATP-Pi exchange, and proton translocation during the steady state were examined. The internal proton concentration was found to be proportional to the rate of ATP hydrolysis when these parameters were varied by procedures that do not alter the proton permeability of the thylakoid membranes. A linear relationship between the internal proton concentration and the rate of nonphosphorylating electron flow was previously verified. By determining the constant relating internal proton concentration to both ATP hydrolysis and electron flow, the proton/ATP ratio for the chloroplast ATPase complex was calculated to be 3.4 +/- 0.3. The presence of Pi, which allows ATP-Pi exchange to occur, lowers the internal proton concentration, but does not alter the relationship between the net rate of ATP hydrolysis and internal proton concentration. ATP-Pi exchange shows a dependence on the proton activity gradient very similar to that of ATP synthesis in the light. These results suggest that ATP-Pi exchange resembles photophosphorylation. In agreement with this idea, it is nucleoside diphosphate from the medium that is phosphorylated during exchange. Moreover, the energy-linked incorporation of Pi and ADP into ATP during exchange occurs at a similar rate. Thus, ATP synthesis from medium ADP and Pi takes place at the expense of the pH gradient generated by ATP hydrolysis.     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Quantitative relationships between phosphorylation, electron flow, and internal hydrogen ion concentrations in spinach chloroplasts.

1. Further evidence that the uptake of [14C]hexylamine, determined by centrifugal filtration of spinach chloroplast thylakoids through silicone fluid layers, gives precise estimations of light-induced H+ concentration gradients (deltapH) is presented. DeltapH was independent of the amount of thylakoids used or of the concentration of hexylamine. Moreover, hexylamine uptake was sensitive to the osmolarity of the suspending medium. 2. Internal H+ concentration ([H+]in) is proportional to the rate of electron flow when light intensity was used to vary these parameters. Proportionality was still observed in the presence of 0.1 and 1.0 muM gramicidin D. When, however, [H+]in and electron flow were varied by increasing the concentration of gramicidin D, at constant light intensity the rate of electron flow was approximately proportional to 1/[H]in. 3. The phosphorylation efficiency (P/e2 ratio) falls with decreasing light intensity or increasing concentrations of the phosphorylation inhibitor, 4'-deoxyphlorizin. The proportionality between the rate of electron flow and [H+]in allows the calculation of the rate of nonphosphorylating (basal) electron flow if [H+]in under phosphorylating conditions is known. The contribution of basal electron flow, a consequence of passive efflux of H+ from the thylakoids, to the overall rate of electron flow increases as the rate of phosphorylation decreases. P/e2 ratios calculated using rates of electron flow from which the basal component has been subtracted are constant. A calculated P/e2 ratio of about 1.3 is obtained. 4. It is shown that the reciprocal of the phosphorylation efficiency should be proportional to 1/[H+]in2 when these parameters are varied using light intensity. This relationship was verified and provided an estimate of the P/e2 at infinite [H+]in. This value was 1.3. These results provide further evidence that a H+ electrochemical gradient serves to couple photophosphorylation to electron flow and that the rate of phosphorylation is proportional to [H+]in3. That is, three H+ are translocated out of thylakoids for each adenosine triphosphate formed.     

叶绿体atpE基因表达产物的生化特性

在细菌中表达的叶绿体ａｔｐＥ基因产物ε亚基蛋白对不同方式激活的叶绿体ＡＴ－Ｐａｓｅ均有抑制作用,而其抗血清则促进ＡＴ－Ｐａｓｅ活力。Ｅ．ｃｏｌｉ中表达的ε亚基蛋白在光合磷酸化反应中对循环和非循环光合磷酸化都有促进作用,其抗血清对循环光合磷酸化有抑制作用,而对非循环光合磷酸化则起促进作用。