Localized or delocalized protons in photophosphorylation? On the accessibility of the thylakoid lumen for ions and buffers

The deposition of protons inside thylakoids after flash excitation was measured photometrically with neutral red as pH indicator. In continuation of previous work (Junge, W., Ausländer, W., McGeer, A. and Runge, T. (1979) Biochim. Biophys. Acta 546, 121–141), we studied the influence of salts on neutral red binding and on the pK of the heterogeneous protonation-deprotonation of inside-bound neutral red as a function of salts. With freeze-thawed (cryoprotective dimethyl sulphoxide) or aged chloroplasts, we observed that the heterogeneous pK of inside-bound neutral red was salt dependent in a way which suggested that neutral red was bound close to the plane of negative fixed charges and that the adjacent inner aqueous phase could accommodate an extended ionic double layer. This, together with the known extremely rapid proton exchange between surface layer and adjacent bulk phase, led us to conclude that inside-deposited protons rapidly reached an aqueous inner bulk phase. This conclusion was corroborated by the observation that extremely hydrophilic buffers like phosphate quenched the transient internal acidification independent of whether proton deposition was due to water oxidation or to plastohydroquinone oxidation. Very different behaviour was observed for freshly prepared chloroplasts with broken outer envelope. Here, inside-bound neutral red was seemingly unaffected by salts and hydrophilic buffers failed to quench the internal acidification. The electrical conductivity and proton permeability of the thylakoid membrane, on the other hand, were as usual. We attributed the seeming inaccessibility of the internal phase to the failure to accommodate a sufficiently extended ionic cloud between the tightly appressed membranes. In such material we observed hindered lateral mobility of protons at the outer side of the thylakoid membrane. This was tentatively attributed to multiple binding-debinding at buffering groups. The consequences for the chemiosmotic theory are: There is one type of damaged chloroplast material, which is competent in photophosphorylation and where protons are deposited into an internal aqueous bulk phase in the orthodox sense. In more intact material, however, the internal space lacks the characteristic properties of an aqueous bulk phase and there is evidence for lateral diffusion limitation for protons. Here, the thermodynamics of photophosphorylation may be inadequately described by the proton-motive force between two aqueous phases which are each isopotential.     

Determination of H+e− ratios in chloroplasts with flashing light

Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H₂O₂ formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratios of 3 and 4 for ferricyanide and methyl viologen, respectively.In continuous light of low intensity, we obtained the same $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio approached 2 as a limit for both acceptors.A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a ≈ 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6–10 ms.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

Nigericin-induced stimulation of photophosphorylation in chloroplasts

Amines have been shown recently to stimulate at low concentrations the steady-state rate of photophosphorylation by unbroken chloroplasts (Giersch, C. (1982) Z. Naturforsch. 37c, 242–250). In the present contribution it is demonstrated that not only amines but also the carboxylic ionophores nigericin and monensin at concentrations of 10 and 150 nM, respectively, stimulate the phosphorylation rate. The $\text{ATP}\text{2}\text{e}$ ratio is not decreased upon the addition of nigericin at concentrations that stimulate phosphorylation. Nigericin-induced stimulation is observed only in the presence of sufficient external potassium, indicating that the observed stimulation is unlikely to be a side-effect of the uncoupler but is related to H⁺-K⁺ exchange. The proton permeability of the thylakoid membrane is increased and the proton gradient decreased by amounts of nigericin that stimulate phosphorylation. The membrane potential is not affected in the steady state, indicating that the proton-motive force is slightly reduced upon addition of the ionophore. Data on the proton-motive force were related to maximum values of the phosphorylation potential, which was 45 000–50 000 M^?1 in the absence and 30 000–35 000 M^?1 in the presence of 10 nM nigericin. The observation that the $\text{ATP}\text{2}\text{e}$ ratio is not decreased in the presence of uncoupler-induced proton leakage is suggested to indicate that the thylakoid lumen does not represent a homogeneous phase of constant proton electrochemical potential. The results presented here are in agreement with the chemiosmotic concept as far as energetic aspects are concerned but seem to be at variance with the postulated free mobility of protons inside the thylakoids. A tentative model of uncoupler-induced stimulation of phosphorylation is presented.     

Light-, pH- and uncoupler-dependent association of chloride with chloroplast thylakoids

Studies of the association of Cl^? with Photosystem (PS) II in CF₁-containing thylakoid membranes revealed that photosynthetically active Cl^? is retained in a Cl^?-free medium unless it is sufficiently alkaline, uncoupling conditions are established and light is excluded. After treatment under such conditions, electron transport from water became dependent on added Cl^? under all conditions. Quantitative measurements of ³⁶Cl^? retention in the light revealed that there were about five Cl^? anions present in Cl^?-sufficient chloroplasts per PS II reaction center, and one-fourth of that in Cl^?-deficient samples. Uncouplers representing three different types of uncoupling mechanism were found to be effective mediators of Cl^? release from thylakoids. Since the ability to collapse a proton gradient probably is the only property shared by all the tested uncouplers, a proton gradient may be involved in the retention of Cl^?. As uncoupler-mediated Cl^? release did not depend on preillumination of our samples, a long-lived proton gradient must exist in dark-adapted chloroplasts which may not span the whole thickness of the thylakoid membrane. It is postulated that the Cl^? active in PS II reactions resides in a special membrane domain from which protons slowly equilibrate with those in the bulk solutions. Cl^? is thought to be released to the bulk phases only when the pH of the membrane domain is raised above a certain threshold by the action of uncouplers. This domain may be identical to the intramembranous compartment which has been postulated to be associated with PS II (Prochaska, L.J. and Dilley, R.A., (1978) Front. Biol. Res. Energ. 1, 265–274).     

Electron transport control in chloroplasts. Effects of photosynthetic control monitored by the intrathylakoid pH

(1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pH_in). A decrease in pH_in due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg²⁺-ADP with P_i); Mg²⁺-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700⁺ reduction. In chloroplasts, without adding Mg²⁺-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 ($\text{Δ}\text{pH}\text{? 0.5–1.0}$). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700⁺ reduction in the presence of Mg²⁺-ADP (with P_i) and without adding Mg²⁺-ADP is dependent on external pH variations, showing a maximum value of $\text{K ? 3.5}$ at pH_out 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg²⁺-ADP and P_i.     

Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium.

A procedure for the isolation of membrane vesicles after sonication of $\text{Halobacterium halobium}$ is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In the light, these vesicles extruded protons. However, on addition of low concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.     

The accumulation of neutral red in illuminated thylakoids

Thylakoids isolated from spinach (Spinacia oleracea L.) bind only a small fraction of neutral red in the dark whereas they accumulate large amounts of the protonated dye in their inner space under light. Light-induced neutral red uptake depends on the size of the proton gradient across the thylakoid membrane but does not follow the mechanism established for amines. Instead, the correlation between pH gradient and neutral red uptake can be predicted quantitatively assuming that protonated neutral red is accumulated mainly as dimer species.Under appropriate conditions, accumulation of protonated neutral red in the inner thylakoid space is proportional to an absorbance increase at 520 nm. This 520-nm change can be used for the continuous measurement of pH changes in thylakoids during steady-state illumination.     

Nonequilibration of membrane-associated protons with the internal aqueous space in dark-maintained chloroplast thylakoids

Isolated spinach thylakoids retain a slowly equilibrating pool of protons in the dark which are predominantly bound to buffering groups, probably amines, with low pKa values. We have measured the effects of permeant buffers, salts, sucrose, and uncouplers on the retention of the proton pool. Acetic anhydride, which reacts with neutral primary amine groups, was used to determine the protonation state of the amine buffering groups. It was previously shown by Bakeret al. that the extent of inhibition of photosystem II water-oxidizing capacity by acetic anhydride and the increase in derivatization by the anhydride are proportional to, and dependent on, the deprotonated state of the amine buffering pool. Therefore, acetic anhydride inhibition of water oxidation activity may be used as a measure of the protonation state of the amine buffering pool. By this method it is inferred that protons, in a metastable state, were retained by membranes suspended in high pH buffer for several hours in the dark. When both the internal and external aqueous phases were equilibrated with pH 8.8 buffer, the proton pool was released only upon addition of a protonophore. The osmotic strength of the suspension buffer affected uncoupler-induced proton release while ionic strength had little influence. The acetic anhydride-sensitive buffering group(s) of the water-oxidizing apparatus had an apparent pKa of 7.8. We conclude that an array of protein buffering groups reside either within the membrane matrix, or in proteins at the membrane surface, not in equilibrium with the bulk aqueous phases, and is responsible for the retention of the proton pool in dark maintained chloroplasts.     

Gated proton conduction via the coupling factor of photophosphorylation modified by N,N-orthophenyldimaleimide

The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient > 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. II. Two modes of post-illumination phosphorylation driven by either delocalized or localized proton gradient coupling

Two modes of chloroplast membrane post-illumination phosphorylation were detected, using the luciferin-luciferase ATP assay, one of which was not influenced by added permeable buffer (pyridine). That finding provides a powerful new tool for studying proton-membrane interactions during energy coupling. When ADP and P_i were added to the thylakoid suspension after a train of flashes [similar to the traditional post-illumination phosphorylation protocol (termed PIP^– here)], the post-illumination ATP yield was influenced by pyridine as expected, in a manner consistent with the ATP formation, in part, being driven by protons present in the bulk inner aqueous phase, i.e., through a delocalized protonmotive force. However, when ADP and P_i were present during the flash train (referred to as PIP⁺), and ATP formation occurred during the flash train, the post-illumination ATP yield was unaffected by the presence of pyridine, consistent with the hypothesis that localized proton gradients were driving ATP formation. To test this hypothesis further, the pH and flash number dependence of the PIP^– and PIP⁺ ATP yields were measured, the results being consistent with the above hypothesis of dual compartment origins of protons driving post-illumination ATP formation.Measuring proton accumulation during the attainment of the threshold energization level when no component was allowed to form (+ valinomycin, K⁺), and testing for pyridine effects on the proton uptake, reveals that the onset of ATP formation requires the accumulation of about 60 nmol H⁺ (mg Chl)^–1. Between that level and about 110–150 nmol H⁺ (mg Chl)^–1, the accumulation appears to be absorbed by localized-domain membrane buffering groups, the protons of which do not equilibrate readily with the inner aqueous (lumen) phase. Post-illumination phosphorylation driven by the dissipation of the domain protons was not affected by pyridine (present in the lumen), even though the effective pH in the domains must have been well into the buffering range of the pyridine. That finding provides additional insight into the localized domains, namely that protons can be absorbed by endogenous low pK buffering groups, and released at a low enough pH (5.7 when the external pH was 8, 4.7 at pH 7 external) to drive significant ATP formation when no further proton production occurs due to the redox turnovers. We propose that proton accumulation beyond the 110–150 nmol (mg Chl)^–1 level spills over into the lumen, interacting with additional, lumenal endogenous buffering groups and with pyridine, and subsequent efflux of those lumenal protons can also drive ATP formation. Such a dual-compartment thylakoid model for the accumulation of protons competent to drive ATP formation would require a gating mechanism to switch the proton flux from the localized pathway into the lumen, as discussed by R. A. Dilley, S. M. Theg, and W. A. Beard (1987)Annu. Rev. Plant Physiol. 38, 348–389, and recently suggested by R. D. Horner and E. N. Moudrianakis (1986)J. Biol. Chem. 261, 13408–13414. The model can explain conflicting data from past work showing either localized or delocalized gradient coupling patterns.     

Interaction of photosystem I-derived protons with the water-splitting enzyme complex. Evidence for localized domains

The induction of millisecond delayed fluorescence mediated by PS I-dependent proton pumping has been used as an indicator of the time course with which those protons equilibrate with sites on the oxygen-evolving enzyme complex (Bowes, J. M., and Crofts, A. R. (1978).Z. Naturforsch. 33C, 271–275). We found that the induction curves were retarded by a reversible exposure of non-energized thylakoids to low concentrations of the uncoupler, desaspidin, at alkaline, but not at neutral, pH. The induction curves were not retarded by increasing the buffering capacity of the thylakoid lumen with Tricine, and were inhibited by the energy transfer inhibitors, dicyclohexylcarbodiimide (DCCD) and triphenyltin chloride (TPT). These data suggest that (i) the catalytic site of the water-splitting complex is located in proton-sequestering membrane domains, rather than at the lumen-exposed inner membrane surface, (ii) protons released during PS I-mediated electron transport might equilibrate with protonatable sites on the oxygen-evolving complex without passing through the lumen, and (iii) those protons may travel over specific conducting pathways which can be blocked by DCCD and TPT.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Probing the micelle/water interface by rapid laser-induced proton pulse

The laser-induced pH jump (Gutman, M. and Huppert, D.J. (1979) Biochem. Biophys. Methods 1, 9–19) has a time resolution capable of measuring the diffusion-controlled rate constant of proton binding. In the present study we employed this technique for measuring the kinetics of protonation-deprotonation of surface groups of macromolecules.The heterogeneous surface of proteins excludes them from serving as a simple model, therefore we used micelles of a neutral detergent (Brij 58) as a high molecular weight structure. The charge was varied by the addition of a low concentration of sodium dodecyl sulfate and the surface group with which the protons react was an adsorbed pH indicator (bromocresol green or neutral red).The dissociation of a proton from adsorbed bromocresol green is slower than that from free indicator. This effect is attributed to the enhanced stabilization of the acid form of the indicator in the pallisade region of the micelle. The $\text{p}\text{K}$ shift of bromocresol green adsorbed on neutral micelles is thus quantitatively accounted for by the decreased rate of proton dissociation. Indicators such as neutral red, which are more lipid soluble in their alkaline form, do not exhibit such decelerated proton dissociation in their adsorbed state nor a $\text{p}\text{K}$ shift on adsorption to neutral micelles.The protonation of an indicator is a diffusion-controlled reaction, whether it is free in solution or adsorbed on micelles. By varying the electric charge of the micelle this rate can be accelerated or decelerated depending on the total charge of the micelle. The micellar charge calculated from this method was corroborated by other measurements which rely only on equilibrium parameters.The high time resulation of the pH jump is exemplified by the ability to estimate the diffusion coefficient of protons through the hydrated shell of the micelle.     

Receptor binding of glucagon and adenosine 3',5'-monophosphate accumulation in isolated rat fat cells.

The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient greater than 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.     

Factors defining the functional coupling of bacteriorhodopsin and ATP synthase in liposomes

Optimal conditions for the co-reconstitution of bacteriorhodopsin and yeast mitochondria ATP synthase were determined. Reconstitution was achieved with a quick two-step procedure. Preparations obtained by this method displayed in optimal cases 2–3-times higher activities (up to 500 nmol ATP/min per mg protein) compared with maximal values reported in the literature, when light-driven ATP synthesis was measured under similar conditions. The final activities depended on the purification method used for the ATP synthase, and it is shown that the oligomycin-sensitive ATP hydrolysis activity was not a good measure for the ability of the ATP synthase preparations to perform ATP synthesis after co-reconstitution. Light-driven ATP synthesis activities depended also on the type of phospholipid used, soybean phospholipid giving the best results. A close relation to the bacteriorhodopsin proton pump activity was found. Using different phospholipids, different $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratios were found, calculated from ATP synthesis activities and initial and steady-state light-driven proton pump activities. From this, together with the findings that the ATP synthase displayed the same ATP hydrolysis and ATP-³²P_i exchange activities with these different phospholipids used, it is concluded that the protein distribution for the two proteins among the liposomes is different relative to each other for the different phospholipids. The light-driven ATP synthesis activity did not correlate with the variation in leakiness of the membrane for protons when different phospholipids were used. An explanation is given by the finding that at high light intensities, the ATP synthesis became independent of the presence of protonophore.     

On the stimulation of the light-induced proton uptake by uncoupling aminoacridine derivatives in spinach chloroplasts.

1. Light-induced proton uptake by spinach chloroplasts is enhanced several-fold by 9-(4-diethylamino-1-methylbutylamino)-6-chloro-2-methoxyacridine (atebrin). This stimulation does not depend on the chlorophyll concentration. The amount of extra protons taken up in the presence of atebrin is determined by the pKa values of atebrin and the pH of the incubation medium. 2. Both the stimulation of the proton uptake and the maximal binding capacity for atebrin is sensitive to uncouplers. However, the ratio of bound to free atebrin does not depend on the presence of uncoupler up to the saturating atebrin concentration. 3. From simultanious kinetic measurements of atebrin fluorescence and proton movement it seems that after binding of the completely protonated atebrin the dye and the protons can move separately. This can also be inferred from the spectral behaviour of atebrin in illuminated chloroplasts. 4. The stimulation of the proton uptake by atebrin does not depend on the presence of salts in the incubation medium. However, the 'saturating' atebrin concentration increases strongly with increasing salt concentration in the medium. 5. It is concluded that the interaction of atebrin and other acridines with energized chloroplasts most likely occurs at the level of the membrane proper. 6. It is proposed that uncoupling by atebrin is a consequence of the creation of a high proton activity at the periphery of the thylakoid membrane, which opposes a proton gradient across the membrane. The uncoupling by atebrin is not of the protonophoric type according to this mechanism.     

Changes in the redox state of the secondary acceptor of Photosystem II associated with light-induced thylakoid protein phosphorylation

Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the $\text{B}{}^{\mathrm{?}}\text{B}$ ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B^?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.     

Temperature dependence of energy-transducing functions and inhibitor sensitivity in chloroplasts

A comparative analysis of the temperature dependence of energy-transducing reactions in spinach (Spinacia oleracea) chloroplasts and their sensitivity for uncouplers and energy-transfer inhibitors at different temperatures is presented. Arrhenius plots reveal two groups of transitions, around 19°C and around 12°C. Activities that show transitions around 19°C include linear electron flow from water to ferricyanide, its coupled photophosphorylation, the dark-release of the fluorescent probe atebrin, and the slow component of the 515 nm (carotenoid) absorbance decay after a flash. The transitions around 12°C are observed with pyocyanine-mediated cyclic photophosphorylation, light- and dithioerythritol-activated ATP hydrolysis, the dark-release of protons, and the fast 515 nm decay component. It is suggested that both groups of temperature transitions are determined by proton displacements in different domains of the exposed thylakoid membranes. The effects of various uncouplers and an energy-transfer inhibitor are temperature dependent. Some uncouplers also show a different relative inhibition of proton uptake and ATP synthesis at lower temperatures. The efficiency of energy transduction (ATP/e₂) varied with temperature and was optimal around 10°C.