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).     

The influence of cytochrome b559 on the fluorescence yield of chloroplasts at low temperature

The maximum light-induced fluorescence yield, F_M, of spinach chloroplasts at − 196 °C was less when the chloroplasts were oxidized with ferricyanide prior to freezing; the minimum fluorescence yield, F₀, of the dark-adapted chloroplasts at − 196 °C was unaffected. The ratio of the fluorescence yields, F_M/F₀, measured at 695 nm at low temperature was 4.5–5.0 for normal chloroplasts and 2.0–2.5 in the presence of ferricyanide. The oxidative titration curve of F_M followed a 1 electron Nernst equation with a midpoint potential of 365 mV and followed closely to the oxidation of cytochrome b₅₅₉. The photoreduction of C−550 at low temperature was the same at all redox potentials over the range of 200–500 mV. It is suggested that a relatively strong oxidant associated with the water-splitting side of Photosystem II, possibly the primary electron donor, can chlorophyll fluorescence of Photosystem II as well as the primary electron acceptor.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

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.     

The relationship between Q, C-550 and cytochrome b 559 in photoreactions at −196° in chloroplasts

Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.     

Redox titration of the primary electron acceptor of Photosystem I in spinach chloroplasts

The midpoint potential of the primary electron acceptor of Photosystem I in spinach chloroplasts was titrated using the photooxidation of P₇₀₀ at −196 °C as an index of the amount of primary acceptor present in the oxidized state. The redox potential of the chloroplast suspension was established by the reducing power of hydrogen gas (mediated by clostridial hydrogenase and 1,1′-trimethylene-2,2′-dipyridylium dibromide) at specific pH values at 25 °C. Samples were frozen to −196 °C and the extent of the photooxidation of P₇₀₀ was determined from light-minus-dark difference spectra. This titration indicated a midpoint potential of −0.53 V for the primary electron acceptor of Photosystem I.     

Effects of α-benzyl-α-bromo-malodinitrile on the primary electron acceptor of Photosystem II in spinach chloroplasts

Reactions at the reducing side of Photosystem II in spinach chloroplasts are modified by α-benzyl-α-bromo-malodinitrile (BBMD).On addition of 50 μM BBMD to chloroplasts the following phenomena can be observed: (1) electron flow to an acceptor like 2,6-dichlorophenolindophenol is partly deflected to electron flow to oxygen; (2) the electron flow to oxygen is carbonyl cyanide m-chlorophenylhydrazone sensitive but 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive; (3) variable fluorescence is abolished but basal fluorescence is not altered; (4) a strong photobleaching of carotenoids is induced. BBMD seems a very efficient acceptor for electrons from the primary electron acceptor of Photosystem II, resulting in a BBMD-mediated electron transport from this primary acceptor to oxygen.On pretreatment of chloroplasts with 50 μM BBMD the effects are different; (1) electron flow to 2,6-dichlorophenolindophenol, ferricyanide, or NADP is almost completely inhibited and is not restored by addition of artificial electron donors: (2) no electron flow to oxygen is observable unless BBMD again is added to reaction media; (3) no variable fluorescence is observable but basal fluorescence is not affected; (4) there is no photobleaching of carotenoids unless BBMD again is added; (5) no reduction of C-550 can be recorded. Pretreatment of chloroplasts with BBMD seems to induce an intense cycling of electrons around Photosystem II and only anew added BBMD can interrupt this cycling.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of phosphorylation

We have used single-photon timing with picosecond resolution to investigate the effect of phosphorylation on the fluorescence decay from broken spinach chloroplasts. Phosphorylation of spinach thylakoids causes a quenching of the slow decay phase (equivalent to a quenching of variable fluorescence) and an increase in the yield of the middle phase decay component. In addition, phosphorylation alters the intensity dependence of fluorescence in a manner which indicates a decreased antenna size of Photosystem II. The observed changes are indicative of a State 1-State 2 transition and show a clear reversal when the membranes are dephosphorylated.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

The emission yields of delayed and prompt fluorescence from chloroplasts.

1. The decay of delayed fluorescence from chloroplasts blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and uncoupled with gramicidin has been measured in the time range 0.75--45 ms by use of a laser phosphoroscope. 2. The decays have been analysed as the sum of three first-order components of approximate half-lives 0.2, 2.5 and 300 ms by a computer-assisted least-squares fit procedure. 3. The prompt fluorescence yield of the chloroplasts was manipulated by changing the cation concentration of the chloroplast-suspending medium. 4. Analysis of the concentration dependence of the components of the delayed fluorescence decay and of the prompt fluorescence inductions indicates that the emission yield of the intermediate (tau approximately 2.5 ms) component of the decay is equal to the fluorescence yield of a Photosystem II photosynthetic unit with an open trap, and that for the slow (tau approximately 300 ms) component the emission yield is equal to the total Photosystem II prompt fluorescence yield. 5. It is concluded that the delayed fluorescence yield in the time range studied is a complex function of time, which may be due to there being different mechanisms leading to delayed fluorescence production at short and long times after cessation of illumination.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

EPR-detectable magnetically interacting manganese ions in the photosynthetic oxygen-evolving system after continuous illumination

Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.     

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.     

Primary reactions of Photosystem II at low pH. I. Prompt and delayed fluorescence

Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 μs, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 μs and was practically irreversible. The back reaction had a pH dependent half time: about 270 μs at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.     

Salts and chloroplast fluorescence

Chloroplast fluorescence was excited by a weak measuring beam. A time-separated actinic light was used to modify the redox states of Q which in turn induced a change in the fluorescence yield. In salt-depleted chloroplasts, fluorescence saturated at a low actinic light intensity. CaCl₂ increased the “variable” fluorescence as well as the rate of ferricyanide-Hill reaction. With Tris-washed chloroplasts, Photosystem II donor couple, phenylenediamine and ascorbate, did not increase the fluorescence to a large extent without the presence of CaCl₂. It is suggested that salt-depletion inactivates the Photosystem II reaction center of chloroplasts.     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F₀, a much higher yield, up to F_max, was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F₀. This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q^? to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.