Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of Photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg²⁺. The reaction is inhibited by (i) an antibody to ferredoxin-NADP⁺ reductase (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q.It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Artificial increase of the light-harvesting ability of photosynthetic units in isolated chloroplasts

A synthetic fluorochromous lipid, rhodaminyl triglyceride (rhodaminyl TG), was intercalated into isolated thylakoid membranes of chloroplasts up to 30 molecules per 100 molecules of chlorophyll. As a result of fluorochrome presence, an absorption band appeared in a yellow-green spectrum region, its intensity being comparable with the red and blue chlorophyll bands. The energy absorbed by rhodaminyl TG was transferred through chlorophyll to the reaction centers of photosystems I and II, inducing an additional electron flow of about 30%. Therefore the exogenous fluorochrome dissolved in lipid matrix functions as an accessory pigment which significantly modifies the spectral sensitivity of the photosynthetic process. The energy transfer from rhodaminyl TG to chlorophyll occurs by mechanism of the inductive resonance type.Abbreviations rhodaminyl TG rhodaminyl triglyceride (rac-1,2-dioleoyl-3[11(3-rhodaminyl)amino-undelanoyl]glycerol) - Me₂SO dimethylsulfoxide - PS photosystem - PPC pigment-protein complex - F₀, F_m initial and maximal levels of chlorophyll fluorescence     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Changes in composition and function of thylakoid membranes as a result of photosynthetic adaptation of chloroplasts from pea plants grown under different light conditions

The hypothesis that chloroplasts having different light-saturated rates of photosynthesis will have different proportions of the intrinsic thylakoid complexes engaged in light-harvesting and electron transport (Anderson, J.M. (1982) Mol. Cell. Biochem. 46, 161–172) has been tested. Peas were grown in light regimes which varied in light intensity, quality and time of irradiance, and ranged from sunlight through red to blue-enriched light of very low radiation. The electron-transport capacity at saturating light of Photosystem I and Photosystem II of chloroplasts isolated from light-adapted peas was 2-fold and 5–6-fold lower, respectively, in the lowest radiation compared to sunlight. There was a marked increase in the amount of total chlorophyll associated with the main chlorophyll $\text{a}\text{b-}\text{proteins}$ (LHCP¹, LHCP² and LHCP³) and a 2-fold decrease in the core reaction centre complex of Photosystem II (CP a) as the radiation decreased; the $\text{LHCP}{}^{\mathrm{1–3}}\text{CP}$ a ratio changed from 3.5 to 9.0. The amount of chlorophyll associated with Photosystem I varied from 34% in sunlight to 27% in the lowest radiation, but the antenna size of Photosystem I was not markedly different; there was a 2-fold decrease in the amount of cytochrome f on a chlorophyll basis, which partly accounted for the decreased electron-transport capacity of Photosystem I. Since the increases or decreases in the levels of each of the components correlated with decreasing radiation, it is clear that the light-adaptation of both light-harvesting and electron-transport components is indeed closely co-ordinated.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

The effect of redox potential of the kinetics of fluorescence induction in pea chloroplasts I. Removal of the slow phase

The effect of alteration of redox potential on the kinetics of fluorescence induction in pea chloroplasts has been investigated. Potentiometric titration of the initial (F_i) level of fluorescence recorded upon shutter opening gave a two component curve, with E_m(7) at ?20 mV and ?275 mV, almost, identical to results obtained using continuous low intensity illumination (Horton, P. and Croze, E. (1979) Biochim. Biophys. Acta 545, 188–201). The slow or tail phase of induction observed in the presence of DCMU can be eliminated by poising the redox potential at approx. 0 to +50 mV. At this potential F_i was increased by less than 10% and the higher potential quencher described above was only marginally reduced. The disappearance of the slow phase titrated as an n = 1 component with an E_m(7) of +120 mV. Therefore it seems unlikely that the slow phase of fluorescence induction is due to photoreduction of the ?20 mV quencher. These results are discussed with reference to current ideas concerning heterogeneity on the acceptor side of Photosystem II.     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Energetics of photosynthesis in Zea mays. II. Studies of the flash-induced electrochromic shift and fluorescence induction in mesophyll chloroplasts

Patterns of electron transfer in isolated mesophyll chloroplasts of maize (Zea mays L.) were studied in the presence of the physiological substrates, oxaloacetate, 3-phosphoglycerate and pyruvate. Flash-induced absorbance changes due to the electrochromic pigment band-shift (P-518) were used to estimate relative electron flow rates through the cyclic and non-cyclic pathways of electron transport. Further information on the redox state of electron carriers and the activity of coupled electron flow was obtained from measurements of fluorescence induction and of actinic-light-induced fluorescence changes. The results demonstrate the importance of correct redox poising for optimal rates of photosynthesis and are discussed in relation to the operation and regulation of photosynthesis in the C₄ system.     

Oxidation-reduction properties of the electron acceptors of Photosystem II I. Redox titration of the flash-induced carotenoid band shift,of C550 and of the variable fluorescence yield in spinach chloroplasts

Redox titration of the electrochromic carotenoid band shift, detected at 50 μs after a saturating actinic flash, in spinach chloroplasts, shows that only one electron acceptor in Photosystem II participates in a transmembrane primary electron transfer. This species, the primary quinone acceptor, Q, shows only one midpoint potential (E_m,7.5) of approx. 0 V and is undoubtedly equivalent to the fluorescence quencher, Q_H. A second titration wave is observed at low potential ($\text{E}{}_{\mathrm{<mtext>m</mtext><mtext>,7.5</mtext>}}\text{? ? 240}\text{mV}$) and at greater than 3 ms after a saturating actinic flash. This wave has an action spectrum different from that of Photosystem II centers containing Q and could arise from a secondary but not primary electron transfer. A low-potential fluorescence quencher is observed in chloroplasts which largely disappears in a single saturating flash at ? 185 mV and which does not participate in a transmembrane electron transfer. This low-potential quencher (probably equivalent to fluorescence quencher, Q_L) and Q are altogether different species. Redox titration of C550 shows that if electron acceptor Q_β is indeed characterized by an E_m,7 of + 120 mV, then this acceptor does not give rise to a C550 signal upon reduction and does not participate in a transmembrane electron transfer. This titration also shows that C550 is not associated with Q_L.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: I. A 56 kilodalton manganese-containing protein,a probable component of the coupling factor enzyme

The binding of endogenous manganese (Mn) to proteins released from spinach grana-thylakoid membranes by 2% cholate detergent or by osmotic shock is investigated. A mixture of 15–20 proteins is released by cholate and has been separated by isoelectric focusing in a sucrose gradient or by chromatofocusing. Mn coelutes with several proteins, but is lost upon dialysis. A dramatic redistribution of this Mn occurs in proteins released by osmotic shock in the presence of hydrophobic and hydrophilic oxidants. Maintaining an oxidizing solution potential during extraction apparently precludes reduction of the higher oxidation states of Mn to the labile Mn(II) state by reducing agents released from the membranes during lysing. This allows proteins to be separated which bind non-labile Mn ions. Under these extraction conditions, a protein is isolated which has an apparent molecular weight (M_r) of 65 000 or 56 000 on SDS-polyacrylamide gel electrophoresis depending on the sample buffer system used. The nondissociated protein occurs as a monomer of 58 kDa (90%) and an apparent dimer of 112 kDa (10%) by gel filtration. This protein binds little Mn if extracted by cholate and separated by isoelectric focusing. However, extraction by osmotic shock in the presence of oxidants and separation by chromatofocusing results in the retention of 1.9 ± 0.3 Mn ions per monomer. This protein is identical to that reported by Spector and Winget (Spector, M., and Winget, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 957–959). Contrary to their result, this protein does not reconstitute O₂ evolution when added to depleted membranes. Rabbit antibody to this purified protein inhibits O₂ evolution by 20% when incubated with intact grana-thylakoid membranes or 10–20% with partially inverted, French-pressed thylakoids. This inhibition is completely removed by 10^?3 M NH₃Cl as an uncoupler of photophosphorylation. These results support a role in Phosphorylation and a location on the outer surface of the thylakoids. This antibody also selectively binds purified coupling factor, CF₁, the multisubunit phosphorylation enzyme which is located on the outer thylakoid surface and which is known to bind two Mn ions tightly (Hochman, Y. and Carmeli, C. (1981) Biochemistry 20, 6293–6297). Thus the β-subunit of CF₁, which has a molecular weight of 56 kDa, can be identified as the locus of Mn binding in CF₁ and as the Mn protein isolated by Spector and Winget. This protein plays no role on O₂ evolution.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

The effect of low concentrations of uncouplers on the detectability of proton deposition in thylakoids. Evidence for subcompartmentation and preexisting pH differences in the dark

Flash-induced pH changes inside thylakoids were measured with neutral red as an indicator in the presence and absence of low concentrations of uncouplers. We found that both carrier-type and pore-forming uncouplers caused the rapidly rising phase of the neutral red signal, previously attributed to proton deposition by water oxidation, to disappear. Gramicidin was particularly efficient in this respect, requiring only one molecule of uncoupler per 10⁴ chlorophyll molecules to render the rapid proton deposition undectectable. This suggests that gramicidin did not act on each water-oxidizing enzyme individually, but rather at the level of the thylakoid membrane. In contrast to the effect on water-derived protons, the appearance of protons from plastoquinol was unaffected by gramicidin. Nor did gramicidin affect the rise of the neutral red signal due to proton deposition during two Photosystem I partial reactions with artificial donors. At the low gramicidin concentrations used, its effect on the neutral red signal could not be attributed to a general increase in proton permeability of the thylakoid membrane (acceleration of half decay from 9 to 0.8 s). The extent of alkalinization of the external medium during the first few hundred milliseconds following a light flash was unaltered by gramicidin, and we did not observe a kinetic correlation between the disappearance of the water proton and the decay of the transmembrane electric field. The last two findings suggest that the undetected protons had not crossed the thylakoid membrane, but instead were buffered away by some gramicidin-induced extra buffering power. pH titration of this extra buffering power revealed an apparent pK ranging between 7.2 and 7.7 and a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{site}$. The rapid phase of the neutral red signal regained 90% of its original amplitude after seven flashes were applied at 6.7 Hz repetition rate to a sample containing gramicidin. This suggests limits to the extra buffering power. One possible interpretation of our experiments is the following: Protons derived from water oxidation are initially deposited into extended and highly buffering special domains, and only escape into the common internal phase when the buffering capacity of the domains is saturated. As an alternative one may consider that the thylakoid lumen is partitioned into at least two domains, each dominated by different photosystems and with slow proton equilibration between them. Either view requires internal subcompartmentation. The consequences of such subcompartmentation for chloroplast bioenergetics are still obscure.