Picosecond fluorescence kinetic studies of electron acceptor Q redox heterogeneity

We have investigated the influence of chloroplast organization on the nature of chemical reductive titrations of Photosystem II fluorescence decay kinetics in spinach chloroplasts. Structural changes of the chloroplast membrane system were induced by varying the ionic environment of the thylakoids. A single-photon timing system with picosecond resolution monitored the kinetics of the chlorophyll a fluorescence emission. At all ionic concentrations studied, we have observed biphasic potentiometric titration curves of fluorescence yield; these have been interpreted to be suggestive of electron acceptor Q heterogeneity (Karukstis, K.K. and Sauer, K. (1983) Biochim. Biophys. Acta 722, 364–371; Cramer, W.A. and Butler, W.L. (1969) Biochim. Biophys. Acta 172, 503–510). A direct relation is observed between the E_m value of the low-potential component of Q and the Mg²⁺ concentration of the chloroplast suspending medium. We have attributed these midpoint potential variations to the thylakoid structural rearrangements involved in cation-regulated grana stacking. Ionic effects on the fluorescence decay kinetics at the redox transitions are discussed in terms of the heterogeneity of Photosystem II units (α- and β-centers) and the mechanism of deexcitation at a closed reaction center (fluorescence or nonradiative decay).     

Theoretical fluorescence induction curves derived from coupled differential equations describing the primary photochemistry of photosystem II by an exciton-radical pair equilibrium

Fluorescence induction curves were calculated from a molecular model for the primary photophysical and photochemical processes of photosystem II that includes reversible exciton trapping by open (PHQ_A) and closed (PHQ^-_A) reaction centers (RCs), charge stabilization as well as quenching by oxidized (P⁺HQ^(-)_A) RCs. For the limiting case of perfectly connected photosynthetic units (“lake model”) and thermal equilibrium between the primary radical pair (P⁺H^-) and the excited singlet state, the primary reactions can be mathematically formulated by a set of coupled ordinary differential equations (ODE). These were numerically solved for weak flashes in a recursive way to simulate experiments with continuous illumination. Using recently published values for the molecular rate constants, this procedure yielded the time dependence of closed RCs as well as of the fluorescence yield (= fluorescence induction curves). The theoretical curves displayed the same sigmoidal shapes as experimental fluorescence induction curves. From the time development of closed RCs and the fluorescence yield, it was possible to check currently assumed proportionalities between the fraction of closed RCs and either (a) the variable fluorescence, (b) the complementary area above the fluorescence induction curve, or (c) the complementary area normalized to the variable fluorescence. By changing selected molecular rate constants, it is shown that, in contrast to current beliefs, none of these correlations obeys simple laws. The time dependence of these quantities is strongly nonexponential. In the presence of substances that quench the excited state, the model predicts straight lines in Stern-Volmer plots. We further conclude that it is impossible to estimate the degree of physical interunit energy transfer from the sigmoidicity of the fluorescence induction curve or from the curvature of the variable fluorescence plotted versus the fraction of closed RCs.     

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Theoretical assessment of alternative mechanisms for non-photochemical quenching of PS II fluorescence in barley leaves

The components of non-photochemical chlorophyll fluorescence quenching (qN) in barley leaves have been quantified by a combination of relaxation kinetics analysis and 77 K fluorescence measurements (Walters RG and Horton P 1991). Analysis of the behaviour of chlorophyll fluorescence parameters and oxygen evolution at low light (when only state transitions — measured as qN_t — are present) and at high light (when only photoinhibition — measured as qN_i — is increasing) showed that the parameter qN_t represents quenching processes located in the antenna and that qN_i measures quenching processes located in the reaction centre but which operate significantly only when those centres are closed. The theoretical predictions of a variety of models describing possible mechanisms for high-energy-state quenching, measured as the residual quenching, qN_e, were then tested against the experimental data for both fluorescence quenching and quantum yield of oxygen evolution. Only one model was found to agree with these data, one in which antennae exist in two states, efficient in either energy transfer or energy dissipation, and in which those photosynthetic units in a dissipative state are unable to exchange energy with non-dissipative units.Abbreviations: F_o, F_m room-temperature chlorophyll fluorescence yield with all centres open, closed - F_v variable fluorescence yield - LHC II light-harvesting chlorophyll-protein complex of PS II - PS I, PS II Photosystem I, II - P700, P680 primary donor in Photosystem I, II - Q_A primary electron acceptor of PS II - P_max maximum quantum yield of oxygen evolution - qN coefficient of non-photochemical quenching of variable fluorescence - qN_e, qN_t, qN_i coefficient of non-photochemical quenching due to high-energy-state, state transition, photoinhibition - qO coefficient of quenching of dark level fluorescence - qP coefficient of photochemical quenching of variable fluorescence - _P intrinsic quantum yield of open PS II reaction centres = _s/qP - _{PS 2} quantum yield of PS = qP × F_v/F_m - _S quantum yield of oxygen evolution = rate of oxygen evolution/light intensity     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Structure and organization of System II photosynthetic units during the greening of a dark-grown Chlorella mutant

The formation and the organization of Photosystem II photosynthetic units during the greening of a dark-grown Chlorella vulgaris, mutant 5/520, have been investigated by analysing the kinetics of the “activation” of oxygen evolution and of the fluorescence induction.

1. 1. The existence during the early stages of the greening of a stationary photosynthesis demonstrates the presence of active Photosystem II at these initial stages, which are integrated in a functional whole, leading to overall photosynthesis.

2. 2. The rise-time of oxygen evolution has been measured using far-red and green light in order to estimate the relative number of chlorophylls per unit. The amount of chlorophyll a remains relatively constant during the greening, while the progressive addition of chlorophyll b causes the size of the units to increase approx. 2-fold.

3. 3. The induction kinetics of the fluorescence are exponential during the early phases of greening and later become distinctly sigmoidal; this suggests that the first units synthesized on the surface of the membrane are isolated from each other by obstacles preventing electronic excitation transfers and that such obstacles which might correspond to some distance between such units, can disappear at later stages, allowing energy transfers to occur.

These observations suggest that the Photosystem II units represent organized functional entities. They apparently consist of a relatively constant number of chlorophyll a molecules, which during the greening is complemented progressively by the addition of chlorophyll b.     

A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events

The initial (F₀), maximal (F_M) and steady-state (F_S) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J₂p_2F/(1–G), Y=T/(1–G) and V, where V is the relative variable fluorescence, J₂ is the light absorption flux in PS II, p_2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F₀=X, F_M=X/(1–Y) and F_S=X(1+(YV/(1–Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, q_P and q_N, reflect, not just photochemical (q_P) or nonphotochemical (q_N) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient q_P is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-q_P is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-q_N equals the (normalized) ratio of the rate constant of photochemistry (k_2b) to the combined rate constant (k_N) of all the nonphotochemical deactivation processes excluding the rate constant k₂₂ of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, k_N-k₂₂ and k_2b, is provided by the fluorescence ratios 1/F_M and (1/F₀)–(1/F_M), respectively. Although, in theory, 41-5 is determined by the value of both k_2b and k_N-k₂₂, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in k _N, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.Abbreviations Chl chlorophyll - F₀, F_M and F_S initial, maximal and steady-state levels of modulated Chl fluorescence emitted by light-adapted leaves - PS I and II photosystem I and II - q_P and q_N (previously defined) photochemical and nonphotochemical components of Chl fluorescence quenching     

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.     

Energy transfer and quantum yield in Photosystem II

The photoreduction and dark reoxidation of Q_α and Q_β, the primary electron acceptors of Photosystems (PS) IIα and IIβ, respectively, in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) were studied in tobacco chloroplasts by means of fluorescence and absorbance measurements. The magnitude of a correction for an absorbance change by the oxidizing side of PS II needed in our previous study of the quantum yield of Q reduction (Biochim. Biophys. Acta 635 (1981), 111–120) has been determined. The absorbance change occurs in PS IIα mainly. The maximum fluorescence yield was found to be the same as in the mutant Su/su, which has a 3-fold higher reaction center concentration and a lower PS IIα to PS IIβ ratio. The kinetics of the light-induced fluorescence increase were measured after various pretreatments and the corresponding kinetics of the integrated fluorescence deficit were analyzed into their α and β components. From the results the contribution to the minimum fluorescence level, the degree of energy transfer between units, and the quantum efficiency of Q reduction were calculated for both types of PS II. This led to the following conclusions. The absence of energy between PS IIβ antennae is confirmed. Fluorescence quenching in PS IIα was adequately described by the matrix model, except for a decrease in the energy transfer between units during photoreduction of Q_α, possibly due to the formation of ‘islets’ of closed centers. PS II reaction centers in which Q is reduced do not significantly quench fluorescence. The ratio of variable to maximum fluorescence, 0.77 in PS IIα and 0.92 in PS IIβ, multiplied by the fraction of Q remaining in the reduced state after one saturating flash, 0.88 in PS IIα and greater than 0.95 in PS IIβ, leads to a net quantum efficiency of Q reduction in the presence of DCMU and NH₂OH of 0.68 in PS IIα and about 0.90 in PS IIβ. These values are in good agreement with the measured overall quantum efficiency of Q reduction.     

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented:

1. (1) MgCl₂ (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl₂ addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg²⁺.

2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.

3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl₂, the values of rate constants of Photosystem II deactivation were calculated.

4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl₂ addition. When this transfer is decreased by MgCl₂ the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.

A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl₂ decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.     

Preparation and characterization of homogeneous coupling factor 6 from bovine heart mitochondria.

Coupling factor 6 (F₆) and mitochondrial ATPase inhibitor were isolated from the rutamycin-sensitive ATPase complex of bovine heart mitochondria by heating and fractionation with ethanol. F₆ appeared in acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and urea as a single band corresponding to a molecular weight of 8,000. This protein which is required for the ³²P_i-ATP exchange in submitochondrial particles treated with silicotungstate was very sensitive to trypsin.     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

Comparison of ATP-induced and DCMU-induced increases of chlorophyll fluorescence

A comparative study of the ATP-induced and the DCMU-induced increases of dark chlorophyll fluorescence after activation of the latent ATPase gave the following results: (1) The ATP-induced fluorescence rise exceeds the DCMU-induced rise by an amount equivalent to the rapid component of the biphasic ATP-induced change. There is complementarity between the slow component and any preceding DCMU-induced fluorescence rise. (2) Up to 10^?4 M DCMU (3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea)), with the slow component being completely suppressed, the rapid ATP-induced phase is unaffected. It becomes eliminated, though, with an I₅₀ of about 3 · 10^?4 M. (3) No binary oscillations in dependence of the number of preilluminating flashes are observed for the rapid ATP-induced fluorescence increase. Under identical conditions such oscillations are found upon DCMU-addition. (4) The amplitude of the rapid ATP-induced fluorescence rise is unaffected by closure of Photosystem II reaction centers in presence of DCMU and NH₂OH by a single saturating flash (removal of about 50% of total quenching). With further flashes and gradual complete removal of quenching, the rapid ATP-induced change is eliminated with a two-step dependency. It is concluded that the rapid phase of the ATP-induced increase in fluorescence reflects reverse electron flow at non-B-type reaction centers, while the slow phase is linked to reverse electron flow at B type centers. On the basis of these results a model is proposed for heterogeneous interactions between the ATPase and B-type and non-B-type electron-transport chains. ‘Direct coupling’ appears to be possible between CF₀-CF₁ and those electron-transport chains which are located in the stroma-exposed margin region of the grana stacks (PS IIβ units with non-B-type properties).     

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

Single-photon timing with picosecond resolution is used to investigate the kinetics of the fluorescence emission of chlorophyll a in chloroplasts from spinach and pea and in the algae Chlorella pyrenoidosa and Chlamydomonas reinhardii. The fluorescence decay is best described by three exponential components in all species. At low light intensity and with open reaction centers of Photosystem II (F₀), we find lifetimes of approx. 100, 400 and 1100 ps for the three components. Closing the reaction centers by addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea plus hydroxylamine and by increasing light intensity produces only minor changes in the almost constant fast- and medium-lifetime components; however, there is a dramatic increase in the yield of the slow component, by a factor of about 20, accompanied by only a modest increase in the lifetime to 2200 ps (F_max). In good agreement with previous fluorescence lifetime measurements, we find an increase in the averaged lifetime of the three components from 0.5 to 2.0 ns, which is proportional to the 4-fold increase in the total fluorescence yield. Our time-resolved results are inconsistent with models which are based on the proportionality between lifetime and yield and which involve a homogeneous origin of fluorescence that is sensitive to the state of the reaction centers. We conclude that the variable part of the fluorescence, which is dominated by the slow phase, reflects the kinetics of charge recombination in the reaction center, as proposed previously (Klimov, V.V., Allakhverdiev, S.I. and Paschenko, V.Z. (1978) Dokl. Akad. Nauk S.S.S.R. 242, 1204–1207). The modest increase in lifetime of the slow phase indicates the presence of some energy transfer between photosynthetic units.     

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O₂-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680⁺ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced Q_A(Q_A ^-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated Q_A (Q_AH₂). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680^*Q_A P680⁺Q_A ^-), is nearly the same in O₂-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the Q_AH₂-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of Q_A leading to Q_AH₂ formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.     

Further evidence for closed, nonspherical aggregates in the cubic I1 phase of lysolecithin and water

Measurements of time-resolved fluorescence quenching have been performed in the binary lauroyllysophosphatidylcholine (LaLPC)/water system. The aggregation numbers, N, are determined for the micellar solution phase (N_micelle ≈ 80) and the cubic liquid crystalline I₁ phase (N_cub ≈ 90) at 298-303 K. When a quencher is present, the fluorescence decays for the hexagonal phase of the LaLPC/water system and for the bicontinuous cubic phase of monooleoylglycerol/water system are nonexponential, as expected for phase structures having long-range continuous apolar regions. Nuclear magnetic resonance (NMR) measurements of the lipid translational diffusion conclusively show that the cubic I₁ phase consists of closed micelles. NMR spectra of ³¹P obtained at 202.4 MHz of this cubic phase exhibit a characteristic line shape, which is compatible with a phase structure containing short nonspherical micelles. A comparison between electron spin resonance (ESR) spin-label spectra recorded for a micellar solution and the cubic phases of the LaLPC and monooleoylglycerol systems are also shown to support a structure of closed micelles in the cubic I₁ phase of the lysolecithin system.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

Studies on the slow fluorescence decline in isolated chloroplasts

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar.At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission.Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.