High Fluorescence State Research Articles

Dual excited state deactivation pathways in TPZ2: A centrosymmetric dye with both high fluorescence and triplet state quantum yield

In this letter, excited state dynamics of TPZ2, a centrosymmetric PRODAN dye, has been studied by using several time-resolved spectroscopy techniques. Fluorescence quantum yield of TPZ2 is found to be 0.50 in both acetonitrile and ethanol solution. The radiative decay rate of the excited state of TPZ2 is determined to be 2.0×108 s−1. Meanwhile, highly efficient triplet state and singlet oxygen generation have been observed in TPZ2 and the intersystem crossing (ISC) rate is determined to be 2.0×108 s−1. The almost identical ISC and non-radiative decay rates indicate that ISC is the only non-radiative decay pathway in TPZ2. Thus, dual excited state (S1) deactivation mechanism (50/50, fluorescence/ISC) of TPZ2 is proposed. Because of this unique property, TPZ2 has the potential to be used as biocompatible imaging and photodynamic therapy agent in the same time.

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between photosystem I (PS I) and photosystem II (PS II) to provide balanced photosynthesis. Chlorophyll (Chl) a fluorescence induction (the so-called OJIPSMT transient) is a signature of several photosynthetic reactions. Here, we show that the slow (seconds to minutes) S to M fluorescence rise is reduced or absent in the stt7 mutant (which is locked in state 1) in C. reinhardtii. This suggests that the SM rise in wild type C. reinhardtii may be due to state 2 (low fluorescence state; larger antenna in PS I) to state 1 (high fluorescence state; larger antenna in PS II) transition, and thus, it can be used as an efficient and quick method to monitor state transitions in algae, as has already been shown in cyanobacteria (Papageorgiou et al. 1999, 2007; Kaňa et al. 2012). We also discuss our results on the effects of (1) 3-(3,4-dichlorophenyl)-1,4-dimethyl urea, an inhibitor of electron transport; (2) n-propyl gallate, an inhibitor of alternative oxidase (AOX) in mitochondria and of plastid terminal oxidase in chloroplasts; (3) salicylhydroxamic acid, an inhibitor of AOX in mitochondria; and (4) carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an uncoupler of phosphorylation, which dissipates proton gradient across membranes. Based on the data presented in this paper, we conclude that the slow PSMT fluorescence transient in C. reinhardtii is due to the superimposition of, at least, two phenomena: qE dependent non-photochemical quenching of the excited state of Chl, and state transitions.

Single-Molecule Measurements to Investigate the Negative Cooperativity in Na+/K+ -ATPase

The Na+/K+-ATPase, a cell membrane ion motive ATPase, uses energy from the hydrolysis of ATP to move Na+ out of and K+ into cells, thus maintaining the membrane resting potential and cellular volume. To investigate how this pump functions, we isolated ATPase from duck supraorbital salt glands and labeled it with Cy3-maleimide (Cy3-ATPase). In bulk experiments, we found that the fluorescence of Cy3-ATPase decreases in the presence of ATP (Biochim Biophys Acta 2009; 1794:1549-1557). The kinetics of this ATP-induced fluorescence decrease exhibited negative cooperativity and could be explained in terms of protein aggregation. To further explore the phenomenon of negative cooperativity on the level of individual monomers, we used single-molecule total internal reflection fluorescence (SM-TIRF) microscopy. Protein monomers were solubilized and reconstituted into lipid vesicles to investigate the effect of varying ATP concentration on the fluorescence.Data from SM-TIRF experiments, analyzed using a hidden Markov model (HMM), suggest that the Cy3-ATPase exists in dynamic equilibrium between a high fluorescence state (unquenched) and a low fluorescence state (partially quenched). These kinetics are characterized by either rapid or slow transitions between these states. Two subpopulations are observed, one where the transitions between the states occur rapidly and the other where the kinetics are slower. Preliminary analysis of the data suggests that ATP shifts the population distribution from those exhibiting rapid transitions to those exhibiting slow transitions. Here, we report on the analysis of these effects and the implications of the above observations on the working of the pump.

Effects of Deficiency and Overdose of Group 2 Sigma Factors in Triple Inactivation Strains of Synechocystis sp. Strain PCC 6803

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.

Effect of dibromothymoquinone on Chlorophyll a Fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium

The influence of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. The reagent affected differently control cells incubated in complete medium and S-starved cells. Thus, the fluorescence yield in the control essentially increased in the presence of dibromothymoquinone, which can be due to the inactivation of light-harvesting complex II protein kinase, followed by the suppression of membrane transition from high-fluorescence state 1 to low-fluorescence state 2. On the contrary, S-starved cells with membranes in state 2 showed a lower fluorescence yield in the presence of dibromothymoquinone than without it. The JIP test of OJIP fluorescence transients suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to control, while in starved cells, it acts predominantly as an electron acceptor.

The Mechanism of the Reverse Recovery Step, Phosphate Release, and Actin Activation of Dictyostelium Myosin II

The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation.

The Association of Actin and Myosin in the Presence of γ-Amido-ATP Proceeds Mainly via a Complex with Myosin in the Closed Conformation

The interaction of gamma-amido-ATP (ATPN) and its 2'(3')-O-methylanthraniloyl derivative (mantATPN) with skeletal myosin subfragment 1 (S1) and actomyosin (actoS1) was studied in stopped-flow experiments. Tryptophan fluorescence and fluorescence of the mant label or light scattering were measured simultaneously. Information about the binding of mant nucleotides was obtained from the quenching of tryptophan fluorescence by the mant label. The parameters of various kinetic models were fitted to the experimental traces. The high-fluorescence state of S1 forms with ATPN at a rate of 95 s-1 ("open-closed" transition); the transition is only slowly reversible, in contrast to the very fast equilibrium seen with its better known isomer AMPPNP [Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173]. The stabilization of the closed state of myosin by ATPN may be due to the formation of a complex with a pentacoordinated amido-gamma-phosphate, from which ATPN can dissociate at a rate of 0.005 s-1 or be hydrolyzed by cleavage of the beta-gamma bond at a rate of 2.5 x 10(-4) s-1. A corresponding actoS1-ATPN complex with myosin in the "closed" conformation is the first detectable intermediate in the association of actin and S1-ATPN, giving an experimental access to a state analogous to a key intermediate in the cross-bridge cycle.

Fluorescence induction in the phycobilisome-containing cyanobacterium Synechococcus sp PCC 7942: Analysis of the slow fluorescence transient

At room temperature, the chlorophyll (Chl) a fluorescence induction (FI) kinetics of plants, algae and cyanobacteria go through two maxima, P at ∼ 0.2–1 and M at ∼ 100–500 s, with a minimum S at ∼ 2–10 s in between. Thus, the whole FI kinetic pattern comprises a fast OPS transient (with O denoting origin) and a slower SMT transient (with T denoting terminal state). Here, we examined the phenomenology and the etiology of the SMT transient of the phycobilisome (PBS)-containing cyanobacterium Synechococcus sp PCC 7942 by modifying PBS → Photosystem (PS) II excitation transfer indirectly, either by blocking or by maximizing the PBS → PS I excitation transfer. Blocking the PBS → PS I excitation transfer route with N-ethyl-maleimide [NEM; A. N. Glazer, Y. Gindt, C. F. Chan, and K.Sauer, Photosynth. Research 40 (1994) 167–173] increases both the PBS excitation share of PS II and Chl a fluorescence. Maximizing it, on the other hand, by suspending cyanobactrial cells in hyper-osmotic media [G. C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1–4] diminishes both the PBS excitation share of PS II and Chl a fluorescence. Here, we show for the first time that, in either case, the slow SMT transient of FI disappears and is replaced by continuous P → T fluorescence decay, reminiscent of the typical P → T fluorescence decay of higher plants and algae. A similar P → T decay was also displayed by DCMU-treated Synechococcus cells at 2 °C. To interpret this phenomenology, we assume that after dark adaptation cyanobacteria exist in a low fluorescence state (state 2) and transit to a high fluorescence state (state 1) when, upon light acclimation, PS I is forced to run faster than PS II. In these organisms, a state 2 → 1 fluorescence increase plus electron transport-dependent dequenching processes dominate the SM rise and maximal fluorescence output is at M which lies above the P maximum of the fast FI transient. In contrast, dark-adapted plants and algae exist in state 1 and upon illumination they display an extended P → T decay that sometimes is interrupted by a shallow SMT transient, with M below P. This decay is dominated by a state 1 → 2 fluorescence lowering, as well as by electron transport-dependent quenching processes. When the regulation of the PBS → PS I electronic excitation transfer is eliminated (as for example in hyper-osmotic suspensions, after NEM treatment and at low temperature), the FI pattern of Synechococcus becomes plant-like.

Delineation of Positron Emission Tomography Imaging Agent Binding Sites on β-Amyloid Peptide Fibrils

A range of imaging agents for use in the positron emission tomography of Alzheimer's disease is currently under development. Each of the main compound classes, derived from thioflavin T (PIB), Congo Red (BSB), and aminonaphthalene (FDDNP) are believed to bind to mutually exclusive sites on the beta-amyloid (Abeta) peptide fibrils. We recently reported the presence of three classes of binding sites (BS1, BS2, BS3) on the Abeta fibrils for thioflavin T derivatives and now extend these findings to demonstrate that these sites are also able to accommodate ligands from the other chemotype classes. The results from competition assays using [3H]Me-BTA-1 (BS3 probe) indicated that both PIB and FDDNP were able to displace the radioligand with Ki values of 25 and 42 nM, respectively. BSB was unable to displace the radioligand tracer from the Abeta fibrils. In contrast, each of the compounds examined were able to displace thioflavin T (BS1 probe) from the Abeta fibrils when evaluated in a fluorescence competition assay with Ki values for PIB, FDDNP, and BSB of 1865, 335, and 600 nM, respectively. Finally, the Kd values for FDDNP and BSB binding to Abeta fibrils were directly determined by monitoring the increases in the ligand intrinsic fluorescence, which were 290 and 104 nM, respectively. The results from these assays indicate that (i) the three classes of thioflavin T binding sites are able to accommodate a wide range of chemotype structures, (ii) BSB binds to two sites on the Abeta fibrils, one of which is BS2, and the other is distinct from the thioflavin T derivative binding sites, and (iii) there is no independent binding site on the fibrils for FDDNP, and the ligand binds to both the BS1 and BS3 sites with significantly lower affinities than previously reported.

Estimation of the energetic connectivity of PS II centres in plants using the fluorescence rise O–J–I–P: Fitting of experimental data to three different PS II models

In presence of DCMU, the sigmoidicity of Chl a fluorescence induction curve is correlated to the energetic connectivity (grouping) between photosynthetic units PS II. We are using the experimental data of the fluorescence transients without DCMU (i.e. the O–J–I–P fast fluorescence induction curves) to calculate the overall probability ‘ p’ of PS II connectivity of the samples (e.g. leaves or chloroplasts). The values obtained using the same method on DCMU treated samples give very close results. Beside the practical advantage of the quantitative determination of the grouping probability on plants from O–J–I–P fluorescence transients, we also present a possible role of the regulation of the connectivity process in photosynthesis as a response to external perturbations. A theoretical discussion based on numerical simulation and fitting of the experimental data, shows three different possible approaches of the fluorescence signal. All are deduced from the same reaction scheme of the acceptor part of PS II centre, involving the primary acceptor pheophytine (Ph), the two bound quinones (Q A and Q B), and the plastoquinone pool. The fraction of closed reaction centres B( t), responsible for the fluorescence transient, is calculated using different definitions as distinct combinations of the redox components for every case. The fluorescence rise of two models is based on the conventional assumption, that the presence of Q A − determines the high fluorescence state, and one model is based solely on the photochemical charge separation in the reaction centre complex. The transformation of a fluorescence induction curve into a kinetic of the fraction of closed reaction centres is a precondition for the simulation and fitting of biochemical models with experimental data.

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.

Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate.

Crystal structures are presented for the A197C mutant of Escherichia coli phosphate binding protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both proteins are complexed with inorganic phosphate. The latter molecule, MDCC-PBP, exhibits a large increase in fluorescence on binding inorganic phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic phosphate binding are interpreted in terms of the specific interactions of MDCC with the protein. The structure helps to explain why this particular label gives a high-fluorescence state on binding inorganic phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on proteins.

Kinetics of Electron Transfer between QA and QB in Wild Type and Herbicide-Resistant Mutants of Chlamydomonas reinhardtii

Abstract We have investigated the electron transfer kinetics for reduction of plastoquinone by photo­ system II in six mutant strains of Chlamydomonas reinhardtii by following the decay of the high fluorescence state after flash activation, and compared the separate reactions of the two-electron gate with those of a wild type strain. By analysis of the electron transfer kinetics, and separate measurement of the equilibrium constant for stabilization of the bound semiquinone after one flash, we have been able to deconvolute the contributions of rate constants and equilibrium constants for plastoquinone binding and electron transfer to the overall process. Two mutations, S 264 A and A 251 V, led to a marked slowing of kinetics for reduction of plastoquinone to the bound semiquinone. In S 264 A , the second electron transfer was also slower, but was normal in A 251 V. In mutant G 256 D , the electron transfer kinetics were normal after the first flash, but slowed after the second. In mutants L 257 F , V 219 I, and F 255 Y , the electron transfer kinetics after both flashes were similar to those in wild type. We discuss the results in terms of a model which provides a description of the mechanism of the two-electron gate in terms of measured kinetic and equilibrium constants, and we give values for these parameters in all strains tested.

Photochemical and thermal phases in the short-term chlorophyll fluorescence induction kinetics of Chlorella fusca

The yield of chlorophyll fluorescence from photosystem II of Chlorella fusca was measured during a short xenon flash (half-width, 2 μs) and rectangular laser pulse (duration, 100 μs) of variable intensity. At the highest light intensity (above 10 5Wm −2), the photochemical rise and the decay attributed to the carotenoid triplet quenching could be well separated and a non-photochemical phase was observed which completed within 60 μs. If the algae are pre-excited with a saturating nitrogen laser pulse, the yield of fluorescence can exceed the maximum value measured routinely (stationary conditions and longer time range). It is argued that the unusually high fluorescence state can arise from a model of the reaction centre of two photoreactions separated by a thermal step. A numerical simulation of the model, based on the freely moving exciton approximation and competition between the carotenoid triplet traps and the different states of the reaction centre for exciton capture, gave reasonably good agreement with the experimental results.

Light Energy Distribution in the Brown Alga Macrocystis pyrifera (Giant Kelp)

The brown alga Macrocystis pyrifera (giant kelp) was studied by a combination of fluorescence spectroscopy at 77 kelvin, room temperature modulated fluorimetry, and photoacoustic techniques to determine how light energy is partitioned between photosystems I and II in states 1 and 2. Preillumination with farred light induced the high fluorescence state (state 1) as determined by fluorescence emission spectra measured at 77K and preillumination with green light produced a low fluorescence state (state 2). Upon transition from state 1 to state 2, there was an almost parallel decrease of all of the fluorescence bands at 693, 705, and 750 nanometers and not the expected decrease of fluorescence of photosystem II and increase of fluorescence in photosystem I. The momentary level of room temperature fluorescence (fluorescence in the steady state, F(s)), as well as the fluorescence levels corresponding to all closed (F(m)) or all open (F(o)) reaction-center states were measured following the kinetics of the transition between states 1 and 2. Calculation of the distribution of light 2 (540 nanometers) between the two photosystems was done assuming both the ;separate package' and ;spill-over' models. Unlike green plants, red algae, and cyanobacteria, the changes here of the light distribution were rather small in Macrocystis so that there was approximately an even distribution of the photosystem II light at 540 nanometers to photosystem I and photosystem II in both states 1 and 2. Photoacoustic measurements confirmed the conclusions reached as a result of fluorescence measurements, i.e. an almost equal distribution of light-2 quanta to both photosystems in each state. This conclusion was reached by analyzing the enhancement phenomenon by light 2 of the energy storage measured in far red light. The effect of light 1 in decreasing the energy storage measured in light 2 is also consistent with this conclusion. The photoacoustic experiments showed that there was a significant energy storage in light 1 which could be explained by cyclic electron transport around photosystem I. From a quantitative analysis of the enhancement effect of background light 2 (maximum enhancement of 1.4-1.5) it was shown that around 70% of light 1 was distributed to this cyclic photosystem I transport.

Kinetic studies on the association and dissociation of myosin subfragment 1 and actin.

The reactions of pyrene-labeled actin with myosin subfragment 1 (S1) and S1-ligand complexes at low ionic strength are described by the schemes [formula: see text] where M refers to a myosin head; A is actin; L is ligand; the asterisk refers to a high fluorescence state of actin; and K1 and K3 are association constants. K1 is reduced approximately 10-fold for M.ADP or M.pyrophosphate versus M alone. The rate constant of the isomerization step (k2) is 150-200 s-1 for A*M, A*M.ADP, and A*M-pyrophosphate (20 degrees C). The interaction between the ligand the actin binding sites reduces K2 from 2,000 for A*M to 50-100 for A*M.ADP and to approximately unity for A*M-pyrophosphate. The A*M.ADP state is equated with the AM'.ADP state of Sleep and Hutton (Sleep, J., A., and Hutton, R. L. (1980) Biochemistry 19, 1276-1283).

Participation of hydrogen ion in the calcium-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase

The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.

The involvement of a high potential acceptor in the acid-base induced reduction of the acceptor Q in chloroplasts

Study of the acid-based induced fluorescence transient, the so-called ‘reverse electron flow’, in chloroplasts revealed the following new properties: 1. (1) Experiments in which the acid-base transition was performed in the absence of the measuring beam showed that the high fluorescence state induced by a pH-jump was attained and decayed even in complete darkness. These results indicated that a non-photochemical electron transfer was affected by the pH transition. A pH-induced reverse electron transfer from some secondary electron acceptor to Q probably occurred during that process. 2. (2) This conclusion was supported by the effect of the Photosystem I electron acceptor methyl viologen. Methyl viologen accelerated the decay phase of the transient showing that this phase was controlled by the rate of electron flow to Photosystem I, but this acceptor did not diminish the size of the transient's initial rise, probably because this rise reflected a pH effect on a non-photochemical step located between Q and P-700. 3. (3) The size of the fluorescence transient was dependent upon the reduction state of both parts of the secondary pool of electron acceptors, A 2 and A 1. 4. (4) Redox potential measurements using ferricyanide-ferrocyanide mixtures showed that the size of the transient was directly dependent on a midpoint potential of +385 mV at pH 6.9 and with n = 1. This suggested the involvement of a high potential secondary electron acceptor in the acid-base induced reduction of Q. 5. (5) The restoration of the acid-base transient in long-time dark-adapted chloroplasts was demonstrated by using additional dark incubation with appropriate couples of reductants and lipophilic mediators. Most significant was the restoration of the full size of the transient by the couple ascorbate + diaminodurene which poised a potential of +100 mV, a potential at which the A 2 pool of 5 equivalents stayed completely oxidized in the dark. I conclude that the source of reducing equivalents for the ‘reverse reduction of Q’ was either in the high potential pool A 1 at the level of cytochrome f and plastocyanin or in a similarly high-potential side component.