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.     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. I. An assay using luciferin-luciferase luminescence

The great sensitivity of the luciferin-luciferase ATP detection system allows direct observation of ATP formation derived from single-turnover flashes in a thylakoid reaction mixture. The method can measure the energization threshold—the number of flashes required for the initiation of ATP formation—as well as detect post-illumination ATP formation after the last flash of a flash sequence. We describe the characteristics of this post-illumination phosphorylation which can be observed after a series of phosphorylating flashes (PIP⁺) or when the assay for ATP formation was performed in a traditional manner where the ADP and P_i were added after the flash-energization period (PIP^–).Comparing PIP⁺ yields and kinetics of the PIP⁺ decay under various treatments can give information about membrane energization events only if it is clearly established that different PIP⁺ yields and decay rates are not due to limitations of the luciferase-catalyzed reaction. Experiments showing that the PIP⁺ ATP yield and kinetics were due to membrane-limited deenergization events (proton efflux) rather than luciferase limitations include: (1) An uncoupler, nigericin, added after the last flash reduced the PIP⁺ yield, but had no effect on the luciferase reaction. (2) The kinetics of the luminescence after adding standard ATP were much faster than the PIP⁺ kinetics. (3) Valinomycin and K⁺ stimulated the PIP⁺ yield but had no influence on the luciferase reaction. (4) Lowering the pH from 8 to 7 increased both the PIP^– (an assay independent of luciferase kinetics) and the PIP⁺ ATP yields, an expected result owing to the greater endogenous buffering power encountered by the proton gradient when the external pH is 7.In spite of the last-mentioned point, the threshold flash number for ATP formation onset was the same for pH 7 and 8 (valinomycin, K⁺ present) at slow flash frequencies. This is consistent with a membrane-localized rather than a delocalized gradient. The accompanying reports (W. A. Beard, G. Chiang and R. A. Dilley, and W. A. Beard and R. A. Dilley,J. Bioenerg. Biomembr.) show that different conditions can lead to observing either localized or delocalized proton gradient coupling in the PIP⁺ event and the ATP onset threshold flash number.     

A shift in chloroplast energy coupling by KCl from localized to bulk phase delocalized proton gradients

Spinach thylakoids were resuspended in a buffer including either 200 mM sucrose or 100 mM KCl. singleturnover flash excitation and the luciferin-luciferase system were used to energize and follow ATP formation. The effect of the permeable buffer pyridine was measured on the ATP formation onset lag and on the post-illumination ATP yield. Consistent with a bulk phase delocalized proton gradient coupling model, thylakoids stored in 100 mM KCl exhibited an increase in these two parameters in the presence of pyridine. Thylakoids isolated in the absence of KCl showed no effect of pyridine on the two parameters indicating a localized energy coupling mode.     

Intact Chloroplasts Show Ca-Gated Switching between Localized and Delocalized Proton Gradient Energy Coupling (ATP Formation)

Intact chloroplasts were compared to isolated thylakoids as to whether storage of the organelle in high KCl medium caused the energy coupling reactions to show a delocalized or a localized proton gradient energy coupling response. With isolated thylakoids, the occurrence of one or the other energy coupling mode can be reversibly controlled by the concentration of mono- and divalent cations used for the thylakoid storage media. Calcium was shown to be the key ion and previous evidence suggested a Ca²⁺-controlled gating of H⁺ fluxes in the thylakoid membrane system (G Chiang, RA Dilley [1987] Biochemistry 26: 4911-4916). Isolated, intact chloroplasts, which retained the outer envelope membranes during the 30 min or longer storage treatments in various concentrations of KCl and CaCl₂ (with sorbitol to maintain iso-osmotic conditions), were osmotically burst in a reaction cuvette and within 3 minutes were assayed for either a localized or a delocalized proton gradient energy coupling (ATP formation) mode. The intact chloroplast system was analogous to isolated thylakoids, with regard to the effects of KCl and CaCl₂ on the energy coupling mode. For example, adding 100 millimolar KCl to the intact organelle storage medium resulted in the subsequent ATP formation assay showing delocalized proton gradient coupling just as with isolated thylakoids. Adding 5 millimolar CaCl₂ to the 100 millimolar KCl storage medium resulted in a localized proton gradient coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations and testing the energy coupling response showed that the stromal milieu has enough Ca²⁺ to cause the localized coupling response even though there was about 80 millimolar K⁺ in the intact chloroplasts used in this study (determined by atomic absorption spectrophotometry). Extrapolating the intact chloroplast data to the whole leaf level, we suggest that proton gradient energy coupling is normally of the localized mode, but under certain conditions it could be either localized or delocalized, depending on factors that affect the putative Ca²⁺-regulated proton flux gating function.     

Evidence that the intrinsic membrance protein LHCII in thylakoids is necessary for maintaining localized\Delta \tilde \mu _{H^ + } energy coupling

This work tested the hypothesis that thylakoid localized proton-binding domains, suggested to be involved in localized -driven ATP formation, are maintained with the involvement of several membrane proteins, including the LHCII (Laszlo, J. A., Baker, G. M., and Dilley, R. A. (1984) Biochim. Biophys. Acta 764, 160–169), which comprises about 50% of the total thylakoid protein. The concept we have in mind is that several membrane proteins cooperate to shield a localized proton diffusion pathway from direct contact with the lumen, thus providing a physical barrier to H⁺ equilibration between the sequestered domains and the lumen. A barely mutant,chlorina f ₂, that lacks Chl b and does not accumulate some of the LHCII proteins, was tested for its capacity to carry out localized-proton gradient-dependent ATP formation. Two previously developed assays permit clear discrimination between localized and delocalized gradient-driven ATP formation. Those assays include the effect of a permeable buffer, pyridine, on the number of single-turnover flashes needed to reach the energetic threshold for ATP formation and the more recently developed assay for lumen pH using 8-hydroxy-1,3,6-pyrene trisulfonic acid as a lumenally loaded pH-sensitive fluorescent probe. By those two criteria, the wild-type barley thylakoids revealed either a localized or a delocalized energy coupling mode under low- or high-salt storage conditions, respectively. Addition of Ca⁺⁺ to the high-salt storage medium caused those thylakoids to maintain a localized energy-coupling response, as previously observed for pea thylakoids. In contrast, thechlorina f ₂ mutant thylakoids had an active delocalized energy coupling activity but did not show localized energy coupling under any conditions, and added Ca⁺⁺ to the thylakoid storage medium did not alter the delocalized energy coupling mode. One interpretation of the results is that the absence of the LHCII polypeptides produces a leaky pathway for protons which allows the gradient to equilibrate with the lumen under all conditions. Another interpretation is possible but seems less likely, that being that the absence of the LHCII polypeptides in some way causes the proposed Ca⁺⁺ -gated H⁺ flux site on the membrane sector (CF₀) of the energy coupling complex to lose its gating function.     

Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids

Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20–30 single-turnover flashes and 8 min of dark before addition of ADP, P_i, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and P_i, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H⁺ (mg chl)^–1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequested amine group array may provide localized association-dissociation sites for proton movement to the coupling complex.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Factors affecting the development of the capacity for ATP formation in isolated chloroplasts

Full development of the capacity for ATP formation in isolated thylakoid membranes coincides with the beginning of illumination. Indeed, the yield of ATP per ms of illumination is about twice as great during the first 15 ms of high-intensity illumination as it is thereafter. The presence of valinomycin and K⁺ prevents the formation of a membrane potential (as indicated by the obliteration of most of the change in absorbance at 518 nm) and at the same time delays the formation of the capacity for ATP synthesis for many milliseconds. Presumably, phosphorylation is initially dependent on a rapidly formed membrane potential, whereas after a delay a ΔpH sufficient to drive ATP formation forms. The actual duration of this delay depends on the phosphoryl group transfer potential (i.e., ΔG_ATP) of the ATP-synthesizing reaction. If the delay in the presence of valinomycin and K⁺ represents the time required to develop a ΔpH capable of driving phosphorylation by itself, then the effect of ΔG_ATP on the duration of the delay suggests that the onset of phosphorylation is determined by the magnitude of the electrochemical potential of protons and not by factors affecting the activation of the coupling factor enzyme. The initial ATP formation, which is almost entirely dependent on the electrical potential, should not be affected by the electrically neutral exchange of cations catalyzed by nigericin. When the external pH is 7.0 this seems to be true, since the ATP synthesis which is initially sensitive to valinomycin and K⁺ is largely insensitive to nigericin and K⁺. However, when the external pH is 8.0 the response to nigericin is exactly the opposite and the ATP formation which is sensitive to valinomycin is also abolished by nigericin. These data suggest that there may be either an energetic requirement for both a ΔpH and membrane potential at alkaline pH or a non-energetic requirement for a minimum proton activity in the initiation of phosphorylation.     

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.     

Influence of Ca2+ on the thylakoid lumen violaxanthin de-epoxidase activity through Ca2+ gating of H+ flux at the CFo H+ channel

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H⁺ accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Δ~μ_H+) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca²⁺ binding to the lumen side of the thylakoid membrane; in particular, Ca²⁺ binding to the 8 kDA subunit III of the CF_o H⁺ channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca²⁺ from the CF_o H⁺ channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photophosphorylation and the chemiosmotic perspective

Photophosphorylation was discovered in chloroplasts by D. Arnon and coworkers, and in bacterial ‘chromatophores’ (intercytoplasmic membranes) by A. Frenkel. Initial low rates were amplified by adding electron-carrying compounds such as FMN, later shown to support the ‘pseudocyclic’ electron flow. ATP synthesis, and coupling to electron flow, was detected accompanying linear electron flow from H₂O to either NADP⁺ or ferricyanide. Another pattern of electron flow supporting photophosphorylation was that of a cycle around Photosystem I (PS I). Isolation and analysis of the ATP synthase showed, as with mitochondrial and bacterial analogues, an intrinsic membrane complex (CF₀) and an extrinsic complex (CF₁). CF₁ is a latent ATPase, activated additively by the high-energy state of the thylakoids, and by reduction of a disulfide bond on the gamma subunit. Once reduced, ATP synthesis occurs at lower energy levels. The search for an ‘intermediate’ linking electron flow and ATP synthesis led to the discovery of post-illumination ATP synthesis by thylakoids, where turnover occurs in the dark. Once interpreted by P.Mitchell's chemiosmotic hypothesis, this led to the discovery of light-driven proton uptake into the thylakoid lumen, with accompanying Cl⁻ intake and Mg²⁺ and K⁺ output. Chemiosmosis was confirmed in several ways, including ATP synthesis in the dark due to an acid-to-base transition of thylakoids, and photophosphorylation accomplished in artificial lipid vesicles containing both the proton-pumping bacterial rhodopsin and a mitochondrial ATPase complex. The now generally accepted chemiosmotic interpretation is able to clarify some other aspects of photosynthesis as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in the structure and the functional state of thylakoids under the conditions of osmotic shock

The effect of osmotic shock on the ultrastructure and functions of C-class pea chloroplasts has been examined. When incubated in a non-sucrose medium for 30 s or more, thylakoids were found to pass to a stable deformed state. This state was characterized by an altered orientation of thylakoids to each other with the lumen thickness remaining the same as in the normal state. Experiments with shorter incubation periods (10–20 s) revealed a swelling of thylakoids, which probably represented an intermediate stage. The deformation of the thylakoid system was accompanied by a decrease in the non-cyclic ATP synthesis but by an increase in the rate of cyclic photophosphorylation. Besides, the deformed thylakoids demonstrated an acceleration of the basal electron transport, as well a rise in the light-induced H⁺ and imidazol uptake. The data obtained are discussed in the light of membrane interactions fixing the configuration of a thylakoid.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers.

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.     

Effect of high KCl concentrations on membrane-localized metastable proton buffering domains in thylakoids

Recent work showed that chloroplast thylakoid membranes stored in 100 mM KCl-containing media have delocalized energy coupling consistent with a rapid equilibration of the proton gradient between the proton-producing redox steps and the lumen bulk phase (Beard and Dilley 1986). Thylakoids stored in low salt media showed localized energy coupling. A related thylakoid membrane property is the occurrence of sequestered, metastable, acidic domains, associated with pK _a 7.5 amine groups. For low salt-stored membranes the domain protons appear to be in the direct (localized) diffusion pathway of protons involved in energizing ATP formation, whereas in thylakoids stored in high KCl, domain protons equilibrated with the lumen during the development of the ATP energization threshold (Theg et al. 1988). This work tested whether the 100 mM KCl storage treatment did or did not cause the dissipation of the metastable acidic domain protons in the dark, storage period. By three criteria, it was found that the 100 mM KCl storage treatment had only a slight tendency to dissipate the acidic domain protons into alkaline media under dark conditions. Storage in KCl does not cause the dissipation of the acidic domains in the dark, but allows domain protons to equilibrate with the lumen after the redox system begins turning over, but before the ATP energization threshold pH is reached. These results must be considered in models of how the thylakoid structure can accommodate metastable acidic domains and how such domain protons diffuse to the CF₀-CF₁ complexes in energy coupling.Abbreviations PSII photosystem 2 - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholine) ethanesulfonic acid - Taps N-tris (hydroxymethyl)-methyl-3-amino-propanesulfonic acid - EPPS N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid - gram D gramicidin D This research supported in part by grants from the USDA and NSF.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

The effect of permeant buffers on initial ATP synthesis by chloroplasts using rapid mix-quench techniques

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KCl are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KCl pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post-illumination ATP synthesis are driven by distinct components of the proton motive potential.     

Acid-induced phosphorylation of adenosine 5'-diphosphate bound to coupling factor 1 in spinach chloroplast thylakoids.

Adenosine 5'-diphosphate, bound to coupling factor 1 (CF1) in spinach chloroplast thylakoids, is in part converted to adenosine 5'-triphosphate, upon injection of the thylakoids into strong acids in the dark. Bound phosphate serves as the phosphoryl donor for this uncoupler-insensitive conversion. Exposure of the thylakoids to heat or to urea prior to their injection into acid caused dissociation of ADP and prevents the apparent acid-induced synthesis of ATP. Conformational changes in CF1 may be elicited by acid denaturation which resemble those brought about by the proton electrochemical gradient across thylakoid membranes.     

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms.(2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists.(3) Various ions which permeate the thylakoid membrane (K⁺ or Rb⁺ in the presence of valinomycin, SCN^?, I^?, or ClO₄^?) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K⁺ with valinomycin and the effect of SCN^? are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination.(4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.     

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers

(1) The amounts of orthophosphate, bicarbonate and tris(hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid.(2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KCl are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl)aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KCl, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrpancies occur with all of the other buffers mentioned.(3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KCl, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase.(4) We suggest that the protons produced by electron transport may be used directly for phosophorylation without ever entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.