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Fatty acid composition of the membrane lipids in the mesophilic cyanobacterium Synechocystis sp. PCC 6803 was altered in earlier work by targeted mutagenesis of genes for fatty acid desaturases. In this work, cells of several mutant strains, depleted in the unsaturated fatty acids in membrane lipids, were grown at 34 degrees C. Spheroplasts (permeabilized cells) were prepared by lysozyme digestion of the cell wall followed by gentle osmotic shock. The bioenergetic parameters ATP formation, electron transport, and H+ uptake were measured at various temperatures. All three bioenergetic parameters for spheroplasts from wild-type cells (which had abundant polyunsaturated fatty acids) were active down to the lowest temperatures used (1 degrees - 2 degrees C). In two strains, which lacked the capacity to desaturate fatty acids at the A 12 position and at the A 12 and A6 positions (designated as desA- and desA-/desD-, respectively), the spheroplasts lost the capacity to form ATP (measured as phenazine methosulfate cyclic phosphorylation) at about 5 degrees C but retained electron transport (water oxidation-dependent ferricyanide reduction) and H+ uptake linked to phenazine methosulfate cyclic electron transport. It appears that the absence of the unsaturation of fatty acids in the A 12 and A6 positions blocks the ability of the photosynthetic membranes to couple a bioenergetically competent proton-motive force to the ATP formation mechanism at temperatures below 5 degrees C. It remains to be determined whether the loss of ATP formation in the mutant strains is the failure of available protons to properly flow into the CF0CF1-ATP synthase or a failure in the CF1 part of the complex in coupling the dissipative H+ flow to the enzyme mechanism of the synthase.

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

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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 (Delta[Formula: see text] (H+)) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca(2+) binding to the lumen side of the thylakoid membrane; in particular, Ca(2+) 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 Ca(2+) from the CF(o) H(+) channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation.

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The 8-kDa subunit c of the E. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mutants, Asp61-->Asn and Asp61-->Gly. The Ca++ binding is pH dependent in both the E. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation. The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.

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The high osmotic potential inhibition of photosynthetic electron transport was determined to be related to membrane compaction rather than to an effect of primary thylakoid volume changes. Osmotic inhibition of proton fluxes and phosphorylation were entirely due to osmotic inhibition of electron transport. The ATPase activity, the nature of coupling and the rate constant of proton efflux were not influenced by osmotic pressure, while the rate constant and the extent of proton influx were inhibited by osmotic pressure.

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Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.

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This work tested the hypothesis that thylakoid localized proton-binding domains, suggested to be involved in localized delta mu H(+)-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 f2, 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 delta mu H+ 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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Subunit III, the 8 kDa component of the chloroplast CFo H+ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca(2+)-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrene-divinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, the psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca(2+)-ligand blot assay known to detect high affinity Ca(2+)-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl2 in the presence of 10-fold excess of MgCl2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Ca2+ binding was maximum at pH 7.5-8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca(2+)-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca(2+)-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca(2+)-binding site can form on the lumenal side of the membrane in the functional CFo structure.

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In previous work, calcium ions, bound at the lumenal side of the CF0H+ channel, were suggested to keep a H+ flux gating site closed, favoring sequestered domain H+ ions flowing directly into the CF0-CF1 and driving ATP formation by a localized delta approximately mu H+ gradient. Treatments expected to displace Ca++ from binding sites had the effect of allowing H+ ions in the sequestered domains to equilibrate with the lumen, and energy coupling showed delocalized characteristics. The existence of such a gating function implies that a closed-gate configuration would block lumenal H+ ions from entering the CF0-CF1 complex. In this work that prediction was tested using as an assay the dark, acid-base jump ATP formation phenomenon driven by H+ ions derived from succinic acid loaded into the lumen. Chlorpromazine, a photoaffinity probe for many proteins having high-affinity Ca(++)-binding sites, covalently binds to the 8-kDa CF0 subunit in the largest amounts when there is sufficient Ca++ to favor the localized energy coupling mode, i.e., the "gate closed" configuration. Photoaffinity-bound chlorpromazine blocked 50% or more of the succinate-dependent acid-base jump ATP formation, provided that the ionic conditions during the UV photoaffinity treatment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CF0 subunit. Thylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CF0 subunit did not show any inhibition of acid-base jump ATP formation. Chlorpromazine and calmidazolium, another Ca(++)-binding site probe, were also shown to block redox-derived H+ initially released into sequestered domains from entering the lumen, at low levels of domain H+ accumulation, but not at higher H+ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observation that the inhibition of lumenal succinate-dependent ATP formation by photoaffinity-attached chlorpromazine can be reversed by lowering the pH of the acid stage from 5.5 to 4.5. The evidence is consistent with the concept that Ca++ bound at the lumenal side of the CF0 H+ channel can block H+ flux from either direction, consistent with the existence of a molecular structure in the CF0 complex having the properties of a gate for H+ flux across the inner boundary of the CF0. Such a gate could control the expression of localized or delocalized delta approximately mu H+ energy coupling gradients.

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The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.

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Earlier work suggested that Ca2+ ions in the chloroplast thylakoid lumen interact with thylakoid membrane proteins to produce a proton flux gating structure which functions to regulate the expression of the energy-coupling H+ gradient between localized and delocalized modes [Chiang, G., & Dilley, R. A. (1987) Biochemistry 26, 4911-4916]. In this work, one of the phenothiazine Ca2+ antagonists, chlorpromazine, was used as a photoaffinity probe to test for Ca(2+)-dependent binding of the probe to thylakoid proteins. [3H]Chlorpromazine photoaffinity-labels thylakoid polypeptides of Mr 8K and 6K, with generally much less label occurring in other proteins (some experiments showed labeled proteins at Mr 13K-15K). More label was incorporated in circumstances where it is expected that Ca2+ occupies the putative H+ flux gating site, compared to when the gating site is not occupied by calcium. The photoaffinity labeling of the 8-kDa protein was also influenced by the energization level of the thylakoids (less labeling under H+ uptake energization). The 8-kDa protein was identified by partial amino acid sequence data as subunit III of the thylakoid CF0 H+ channel complex. The partial amino acid sequence of the 6-kDa protein (19 residues were determined with some uncertainties) was compared to data in the GCG sequence analysis data base, and no clear identity to a known sequence was revealed. Neither the exact site of putative Ca2+ binding to the CF0 proteolipid nor the site of covalent attachment of the chlorpromazine to the CF0 component has been identified. Evidence for gating of energy-linked H+ fluxes by the hypothesized Ca(2+)-CF0 gating site came from the correlation between Ca(2+)-dependent binding of chlorpromazine to the CF0 8-kDa protein with inhibition of light-driven H+ uptake into the lumen but no inhibition of H+ uptake into sequestered membrane domains. When conditions favored a delocalized delta mu H+ coupling mode, less chlorpromazine was bound to the CF0 structure, and much larger amounts of H+ ions were accumulated in the lumen. The data support the hypothesis that Ca2+ ions act in concert with the 8-kDa CF0 protein (and perhaps another protein, the 6-kDa polypeptide?) in a gating mechanism for regulating the expression of the energy-coupling H+ gradient between localized or delocalized coupling modes.

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The effect of pretreatment with ultraviolet-B (UV-B) light (280-320 nanometers) on the enzymatic conversion of the diepoxyxanthophyll violaxanthin to the epoxy-free zeaxanthin occurring in thylakoid membranes was investigated. When isolated chloroplasts of pea (Pisum sativum) were exposed to UV-B, a biologically effective fluence of 7000 joules per square meter caused about 50% inhibition of the activity of the violaxanthin deepoxidase, measured as the first order rate constant of the absorbance change at 505 nanometers. The dose requirement for the inhibition of the deepoxidase in intact leaves, however, was about 2 orders of magnitude higher. The inhibition of the rate constant was observed for both the dark deepoxidation at pH 5, and for the light-driven deepoxidation induced by the lumen acidification due to electron transport from H(2)O to methylviologen or due to a photosystem I partial reaction with duroquinol as the electron donor. The availability of violaxanthin was not directly affected by UV-B radiation, as shown for UV-B-treated chloroplasts by the final extent of the 505 nanometer change measured in the dark at pH 5 or by the partial photosystem I reaction. A significant decrease in the violaxanthin availability was observed when lumen acidification was caused by electron transport from H(2)O to methylviologen. That effect was probably caused by the wellknown UV-B inhibition of photosystem II with a subsequent decreased ability to reduce the plastoquinone pool, the redox state of which is believed to regulate the final amount of converted violaxanthin.

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Energy transduction from proton gradients into ATP formation in chloroplast thylakoids has been hypothesized to be driven equally efficiently by localized domain delta mu H+ or by a delocalized delta mu H+ (Beard, W. A. and Dilley, R. A. (1988) J. Bioenerg. Biomembr. 20, 129-154). An important question is whether the apparent localized protonmotive force energy coupling mode can be observed only in the dark-to-light transient in the flash excitation protocol commonly used, or whether the localized energy coupling gradient can be maintained under conditions of continuous illumination ATP formation. The assay in the previous work was to use permeable amines, added to thylakoids in the dark, and observe the effect of the amine on the length of the energization lag (number of single-turnover flashes) required to initiate ATP formation in the dark-to-light transition. Amine buffers delayed the ATP onset in high-salt-stored membranes but did not delay the onset with low salt-stored membranes. This work tested whether permeable amines show the different effects in low- or high-salt-stored thylakoids which had attained a steady-state ATP formation rate (in continuous light) for 20-40 s prior to adding the amine. Hydroxyethylmorpholine was the preferred amine for such experiments, a suitable choice inasmuch as it behaves similarly to pyridine in the flash-induced ATP formation onset experiments, but it permeates more rapidly than pyridine and it has a higher pKa, which enhances its buffering effects. With high-salt-stored thylakoids, 0.5 or 1.0 mM hydroxyethylmorpholine added after 40 s of continuous illumination caused a marked, but transient, slowing of the ATP formation rate, but little or no slowing of the rate was observed with low-salt-stored thylakoids (at similar phosphorylation rates for the two thylakoid samples). Those data indicate that in continuous illumination conditions the proton gradient driving ATP formation in thylakoids from the low-salt-stored treatment did not equilibrate with the lumen, but in thylakoids stored in high-salt the delta mu H+ freely equilibrated with the lumen. That suggestion was supported by measurement of the luminal pH under coupling conditions by the [14C]methylamine distribution method using low- or high-salt-stored thylakoids. Further supportive evidence was obtained from measuring the effect of permeable amine buffers on H+ uptake under coupled and basal conditions with both types of thylakoid.(ABSTRACT TRUNCATED AT 400 WORDS)

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Thylakoid membrane proteins are organized so as to shield 30-50 nmol H+ (mg Chl)-1 from freely equilibrating with either the external or the lumen aqueous phases. Amine groups provide binding sites for this metastable buffering array and can be quantitatively measured by acetylation using [3H]acetic anhydride. The principle of the assay is that a metastable acidic domain will have relatively less of the reactive neutral form of the amine compared to the amount present after addition of an uncoupler. The extent of the acetylation reaction is strongly influenced by whether the lumen pH comes to complete equilibrium with the external pH prior to adding the acetic anhydride. Determination of the lumen pH by [14C]methylamine distribution after the standard 3 or 5 min equilibration in pH 8.6 buffer indicated that the lumen may have been 0.2 to 0.3 pH more acidic than the external phase. This effect was taken into account by determining the pH dependence, in the pH 8.2-8.6 range, of acetylation of the membrane proteins studied, and the labeling data were conservatively corrected for this possible contribution. Experiments were carried out to identify the thylakoid proteins that contribute such metastable domain amine groups, using the above conservative correction. Surprisingly, plastocyanin contributes buried amine groups, but cytochrome f did not give evidence for such a contribution, if the conservative correction in the labeling was applied. If the correction was too conservative, cytochrome f may contribute amines to the sequestered domains. The new methodology verified earlier results suggesting that three Tris-releasable photosystem II-associated proteins also contribute significantly to the sequestered amine-buffering array.

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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 Ca(2+)-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 CaCl(2) (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 CaCl(2) 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 CaCl(2) 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 Ca(2+) 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 Ca(2+)-regulated proton flux gating function.

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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 ΔpH 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.

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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 Pi 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 Pi 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 delta psi 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 (less than or equal to 5.7 when the external pH was 8, less than or equal to 4.7 at pH 7 external) to drive significant ATP formation when no further proton production occurs due to the redox turnovers.(ABSTRACT TRUNCATED AT 400 WORDS)

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When 100 mM KCl replaced sucrose in a chloroplast thylakoid stock suspension buffer, the membranes were converted from a localized proton gradient to a delocalized proton gradient energy coupling mode. The KCl-suspended but not the sucrose-suspended thylakoids showed pyridine-dependent extensions of the ATP onset lag and pyridine effects on post-illumination phosphorylation. The ATP formation assays were performed in a medium of identical composition, using about a 200-fold dilution of the stock thylakoid suspension; hence the different responses were due to the pretreatment, and not the conditions present in the phosphorylation assay. Such permeable buffer effects on ATP formation provide a clear indicator of delocalized proton gradients as the driving force for phosphorylation. The pyridine-dependent increases in the onset lags (and effects on post-illumination phosphorylation) were not due to different ionic conductivities of the membranes (measured by the 515 nm electrochromic absorption change), H+/e- ratios, or electron transport capacities for the two thylakoid preparations. Thylakoid volumes and [14C]pyridine equilibration were similar with both preparations. The KCl-induced shift toward a bulk-phase delocalized energy coupling mode was reversed when the thylakoids were placed back in a low-salt medium. Proton uptake, at the ATP-formation energization threshold flash number, was much larger in the KCl-treated thylakoids and they also had a longer ATP formation onset lag, when no pyridine was present. These results are consistent with the salt treatment exposing additional endogenous buffering groups for interaction with the proton gradient. The concomitant appearance of the pyridine buffer effects implies that the additional endogenous buffering groups must be located on proteins directly exposed in the aqueous lumen phase. Kinetic analysis of the decay of the post-illumination phosphorylation in the two thylakoid preparations showed different apparent first-order rate constants, consistent with there being two different compartments contributing to the proton reservoirs that energize ATP formation. We suggest that the two compartments are a membrane-phase localized compartment operative in the sucrose-treated thylakoids and the bulk lumen phase into which protons readily equilibrate in the KCl-treated thylakoids.

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