Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 Of photosystem II

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P+ are fluorescence quenchers, and Q- is a weak quencher, and that the reduction time for P+ is 20-35 ns. 2. The P+ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P+ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.     

Delayed fluorescence from Rhodopseudomonas sphaeroides reaction centers. Enthalpy and free energy changes accompanying electron transfer from P-870 to quinones

Delayed fluorescence from isolated reaction centers of Rhodopseudomonas sphaeroides was measured to study the energetics of electron transfer from the bacteriochlorophyll complex (P-870, or P) to the primary and secondary quinones (Q_A and Q_B). The analysis was based on the assumption that electron transfer between P and Q reaches equilibrium quickly after flash excitation, and stays in equilibrium during the lifetime of the P⁺Q⁻ radical pair. Delayed fluorescence of 1Q reaction centers (reaction centers that contain only Q_A) has a lifetime of about 0.1 s, which corresponds to the decay of P⁺Q⁻_A. 2Q reaction centers (which contain both Q_A and Q_B) have a much weaker delayed fluorescence, with a lifetime that corresponds to that of P⁺Q⁻_B (about 1 s). In the presence of o-phenanthroline, the delayed fluorescence of 2Q reaction centers becomes similar in intensity and decay kinetics to that of 1Q reaction centers. From comparisons of the intensities of the delayed fluorescence from P⁺Q⁻_A and P⁺Q⁻_B, the standard free energy difference between P⁺Q⁻_A and P⁺Q⁻_B is calculated to be 78 ± 8 meV. From a comparison of the intensity of the delayed fluorescence with that of prompt fluorescence, we calculate that P⁺Q⁻_A is 0.86 ± 0.02 eV below the excited singlet state of P in free energy, or about 0.52 eV above the ground state PQ_A. The temperature dependence of the delayed fluorescence indicates that P⁺Q⁻_A is about 0.75 eV below the excited singlet state in enthalpy, or about 0.63 eV above the ground state.     

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.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Determination of free energies in reaction centers of Rb. sphaeroides

Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG(P⁺H⁻ − ³P*) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state ³P* is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG(P⁺H⁻ − ³P*) ΔH(P⁺H⁻ − ³P*) and is consistent with ΔG(¹P* − P⁺H⁻) and ΔH(¹P* − ³P*) from previous delayed fluorescence and phosphorescence data, implying ΔG ΔH for all combinations of these states.     

Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs

The fluorescence induction and other fluorescence properties of spinach chloroplasts at room temperature were probed utilizing two 30-ps wide laser pulses (530 nm) spaced Δt (s) apart in time (Δt = 5–110 ns). The energy of the first pulse (P₁) was varied (10¹²–10¹⁶ photons · cm⁻²), while the energy of the second (probe) pulse (P₂) was held constant (5 · 10¹³ photons · cm⁻²). A gated (10 ns) optical multichannel analyzer-spectrograph system allowed for the detection of the fluorescence generated either by P₁ alone, or by P₂ alone (preceded by P₁). The dominant effect observed for the fluorescence yield generated by P₁ alone is the usual singlet-singlet exciton annihilation which gives rise to a decrease in the yield at high energies. However, when the fluorescence yield of dark-adapted chloroplasts is measured utilizing P₂ (preceded by pulse P₁) an increase in this yield is observed. The magnitude of this increase depends on Δt, and is characterized by a time constant of 28 ± 4 ns. This rise in the fluorescence yield is attributed to a reduction of the oxidized (by P₁) reaction center P-680⁺ by a primary donor. At high pulse energies (P₁ = 4 · 10¹⁴ photons · cm⁻²) the magnitude of this fluorescence induction is diminished by another quenching effect which is attributed to triplet excited states generated by intense P₁ pulses. Assuming that the P₁ pulse energy dependence of the fluorescence yield rise reflects the closing of the reaction centers, it is estimated that about 3–4 photon hits per reaction center are required to close completely the reaction centers, and that there are 185–210 chlorophyll molecules per Photosystem II reaction center.     

Carotenoid triplet states in reaction centers from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Purified photochemical reaction centers from three strains of Rhodopseudomonas sphaeroides and two of Rhodospirillum rubrum were reduced with Na₂S₂O₄ so as to block their photochemical electron transfer reactions. They then were excited with flashes lasting 5–30 ns. In all cases, absorbance measurements showed that the flash caused the immediate formation of a transient state (P^F) which had been detected previously in reaction centers from Rps. sphaeroides strain R26. Previous work has shown that state P^F is an intermediate in the photochemical electron transfer reaction in the reaction centers of that particular strain, and the present work generalizes that conclusion.

In the reaction centers from two strains that lack carotenoids (Rps. sphaeroides R26 and R. rubrum G9), the decay of P^F yields a longer-lived state (P^R) which is probably a triplet state of the bacteriochlorophyll of the reaction center. In the R26 preparation, the decay of P^F was found to have a half-time of 10±2 ns. The decay kinetics rule out the identification of P^F as the fluorescent excited singlet state of the reaction center.

In the reaction centers from three strains that contain carotenoids (Rps sphaeroides 2.4.1 and Ga, and R. rubrum S1), state P^R was not detected, and the decay of P^F generated triplet states of carotenoids. The efficiency of the coupling between the decay of P^F and the formation of the carotenoid triplet appeared to be close to 100% at room temperature, but somewhat lower at 77 °K. Taken with previous results, this suggests that the coupling is direct and does not require the intermediate formation of state P^R. This conclusion would be consistent with the view that P^F is a biradical which can be triplet in character.     

Coupling of nuclear wavepacket motion and charge separation in bacterial reaction centers

The mechanism of the charge separation and stabilization of separated charges was studied using the femtosecond absorption spectroscopy. It was found that nuclear wavepacket motions on potential energy surface of the excited state of the primary electron donor P* leads to a coherent formation of the charge separated states P⁺B_A⁻, P⁺H_A⁻ and P⁺H_B⁻ (where B_A, H_B and H_A are the primary and secondary electron acceptors, respectively) in native, pheophytin-modified and mutant reaction centers (RCs) of Rhodobacter sphaeroides R-26 and in Chloroflexus aurantiacus RCs. The processes were studied by measurements of coherent oscillations in kinetics at 890 and 935 nm (the stimulated emission bands of P*), at 800 nm (the absorption band of B_A) and at 1020 nm (the absorption band of B_A⁻) as well as at 760 nm (the absorption band of H_A) and at 750 nm (the absorption band of H_B). It was found that wavepacket motion on the 130–150 cm⁻¹ potential surface of P* is accompanied by approaches to the intercrossing region between P* and P⁺B_A⁻ surfaces at 120 and 380 fs delays emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A⁻). In the presence of Tyr M210 (Rb. sphaeroides) or M195 (C. aurantiacus) the stabilization of P⁺B_A⁻ is observed within a few picosseconds in contrast to YM210W. At even earlier delay (40 fs) the emission at 895 nm and bleaching at 748 nm are observed in C. aurantiacus RCs showing the wavepacket approach to the intercrossing between the P* and P⁺H_B⁻ surfaces at that time. The 32 cm⁻¹ rotation mode of HOH was found to modulate the electron transfer rate probably due to including of this molecule in polar chain connecting P_B and B_A and participating in the charge separation. The mechanism of the charge separation and stabilization of separated charges is discussed in terms of the role of nuclear motions, of polar groups connecting P and acceptors and of proton of OH group of TyrM210.     

Evidence for slow turnover in a fraction of Photosystem II complexes in thylakoid membranes

We used two different techniques to measure the recovery time of Photosystem II following the transfer of a single electron from P-680 to Q_A in thylakoid membranes isolated from spinach. Electron transfer in Photosystem II reaction centers was probed first by spectroscopic measurements of the electrochromic shift at 518 nm due to charge separation within the reaction centers. Using two short actinic flashes separated by a variable time interval we determined the time required after the first flash for the electrochromic shift at 518 nm to recover to the full extent on the second flash. In the second technique the redox state of Q_A at variable times after a saturating flash was monitored by measurement of the fluorescence induction in the absence of an inhibitor and in the presence of ferricyanide. The objective was to determine the time required after the actinic flash for the fluorescence induction to recover to the value observed after a 60 s dark period. Measurements were done under conditions in which (1) the electron donor for Photosystem II was water and the acceptor was the endogenous plastoquinone pool, and (2) Q₄₀₀, the Fe²⁺ near Q_A, remained reduced and therefore was not a participant in the flash-induced electron-transfer reactions. The electrochromic shift at 518 nm and the fluorescence induction revealed a prominent biphasic recovery time for Photosystem II reaction centers. The majority of the Photosystem II reaction centers recovered in less than 50 ms. However, approx. one-third of the Photosystem II reaction centers required a half-time of 2–3 s to recover. Our interpretation of these data is that Photosystem II reaction centers consist of at least two distinct populations. One population, typically 68% of the total amount of Photosystem II as determined by the electrochromic shift, has a steady-state turnover rate for the electron-transfer reaction from water to the plastoquinone pool of approx. 250 e⁻ / s, sufficiently rapid to account for measured rates of steady-state electron transport. The other population, typically 32%, has a turnover rate of approx. 0.2 e⁻ / s. Since this turnover rate is over 1000-times slower than normally active Photosystem II complexes, we conclude that the slowly turning over Photosystem II complexes are inconsequential in contributing to energy transduction. The slowly turning over Photosystem II complexes are able to transfer an electron from P-680 to Q_A rapidly, but the reoxidation of Q⁻_A is slow (t1/2 = 2 s). The fluorescence induction measurements lead us to conclude that there is significant overlap between the slowly turning over fraction of Photosystem II complexes and PS II_β reaction centers. One corollary of this conclusion is that electron transfer from P-680 to Q_A in PS II_β reaction centers results in charge separation across the membrane and gives rise to an electrochromic shift.     

The study of the state [P870 B800] in bacterial reaction centers by selective picosecond and low-temperature spectroscopies

The selective picosecond excitation of Rhodopseudomonas sphaeroides (R-26) reaction centers (RCs) at 870 nm induces the formation of the transient state within <1 ps followed by the conversion into the state P^F (P^± Bph^±− during 7 ± 2 ps at both 293 K and 110 K. The transient state including the intense bleaching at 800 nm has been shown not to be due: (a) to photon excitation at 870 nm; (b) the excitation of P⁺; (c) photoselection effects. The transient state is interpreted as the state ¹[P⁺B⁻] in agreement with earlier works. The primary formation of the state ¹P⁺B⁻] and the big effective singlet-triplet splitting in this state correspond to the spectral splitting of the P band at 900 nm in R-26 RCs and at 1000 nm in Rhodopseudomonas viridis RCs found at 4.2 K and attributed to the optical transition to both ¹P and ¹[P⁺B⁻] states.     

Magnetic field-stimulated luminescence and a matrix model for energy transfer. A new method for determining the redox state of the first quinone acceptor in the reaction center of whole cells of Rhodospirillum rubrum

In whole cells of Rhodospirillum rubrum the light-induced absorbance difference spectrum of the reduction of the first quinone electron acceptor Q₁ was determined in order to relate the emission yield ф and the magnetic field-induced emission increase Δф to the redox state of Q₁. It was found that Δф/ф² is a linear function of the number of reaction centers, in which Q₁ is reduced, independent of the fraction of reaction centers in the oxidized state. The emission yield is a hyperbolic function of the fraction of reaction centers closed, either by reduction of the acceptor Q₁ or by oxidation of the primary electron donor P. Apparently, in whole cells of R. rubrum a matrix model for energy transfer between various photosynthetic units can be applied. A model is presented, which is a generalization of theoretical considerations reported before (Duysens, L.N.M. (1978) in Chlorophyll Organization and Energy Transfer in Photosynthesis, Ciba Found. Symp. 61 (New Series), pp. 323–340, Elsevier/North-Holland, Amsterdam) and which is in excellent agreement with the experiments. From simultaneous measurements of Δф and ф the redox state of the reaction center can relatively easily be determined. So far, this is the only method for simultaneously measuring the fractions P⁺ and Q⁻₁ in intact cells under steady-state conditions.     

The deactivation of Photosystem II in Chlorella as a second-order process

The kinetics of deactivation of the S₃ state in Chlorella have been observed under a variety of conditions. The S₃ state appears to decline in a dark period coming after a sequence of 30 saturating flashes in a second-order reaction, the rate constant of which is 0.132/[S*₃] s⁻¹ and which involves an electron donor, D₁, of concentration 1.25[S*₃] where [S*₃] is the concentration of the S₃ state when the oxygen yield of the light flashes is constant. If a 1 min period of 650 nm illumination is employed after the sequence of flashes, the subsequent S₃ state deactivation kinetics are more complex. There is an initial phase of S₃ state deactivation, accounting for about 35% of the original S₃ state, which is complete in less than 100 ms. The remaining 65% of the S₃ state appears to deactivate in a second-order reaction, the rate constant of which is 1.36/[S*₃] s⁻¹ and which involves an electron donor of initial concentration 0.58[S*₃]. If a 1 min period of 710 nm illumination comes after the 30 flashes, at least 98% of the S₃ state deactivates according to first-order kinetics. It is shown that this can be explained using a second-order model if there is an electron donor present of which the concentration is large compared with [S*₃]. However, S₃ state deactivation observed after 5 min of dark and two saturating flashes can be described neither by a first-order model nor a second-order model. Deactivation of the S₂ state after a 5 min dark period and one saturating flash follows second-order kinetics with a rate constant of 0.2/[S*₃] s⁻¹ and appears to involve an electron donor of initial concentration 1.3[S*₃]. Arguments are presented which tend to rule out the primary electron acceptor to Photosystem II as being any of the electron donors but it appears quite possible that the large plastoquinone pool is involved.     

Photo-accumulation of the P+QB- radical pair state in purple bacterial reaction centres that lack the QA ubiquinone

Photo-excitation of membrane-bound Rhodobacter sphaeroides reaction centres containing the mutation Ala M260 to Trp (AM260W) resulted in the accumulation of a radical pair state involving the photo-oxidised primary electron donor (P). This state had a lifetime of hundreds of milliseconds and its formation was inhibited by stigmatellin. The absence of the Q_A ubiquinone in the AM260W reaction centre suggests that this long-lived radical pair state is P⁺Q_B⁻, although the exact reduction/protonation state of the Q_B quinone remains to be confirmed. The blockage of active branch (A-branch) electron transfer by the AM260W mutation implies that this P⁺Q_B⁻ state is formed by electron transfer along the so-called inactive branch (B-branch) of reaction centre cofactors. We discuss how further mutations may affect the yield of the P⁺Q_B⁻ state, including a double alanine mutation (EL212A/DL213A) that probably has a direct effect on the efficiency of the low yield electron transfer step from the anion of the B-branch bacteriopheophytin (H_B⁻) to the Q_B ubiquinone.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q^-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q^- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption.

For Cyanidium caldarium the zero fluorescence yield Ф₀ and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups.

2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation.

3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C^T) and the measurement of the number of C^T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C^T is calculated to be k_T = 3.3 · 10¹¹ s⁻¹ which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340).

4. The fluorescence quenching by C^T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k_T is used as given in point 3.

5. The observations mentioned under point 1 indicate that the fluorescence yield Ф₀ for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

Delayed fluorescence induction in chloroplasts. Irradiance dependence

We have studied the delayed fluorescence in spinach chloroplasts produced 0.5 ms after each of a pair of (sub)-microsecond flashes. We observe an increase in the delayed fluorescence from the second flash relative to that produced by the first. This increase is proportional to the product of the first and second flash irradiances, appearing as an I² dependence if both flashes are increased together. The enhancement is observable at very weak flash levels (roughly 1 photon absorbed/100 PS II centers). If the irradiance of the first flash is increased, but the irradiance of the second held constant, the delayed fluorescence from the second flash is observed to increase, but then to saturate well below the first flash irradiance at which the delayed fluorescence from the first flash itself saturates. For most experiments, the dark time between flashes was 30 ms. If the dark time is varied, the enhancement changes, reaching a half-maximal value for a dark time of approx. 300 μs. The enhancement is stopped by hydroxylamine, but not by gramicidin, valinomycin, DCMU, or mild heating. These experiments are consistent with the notion that there are two different types of Photosystem II centers if we assume that only one type is responsible for the induction we see and has an optical cross-section about 4-times the size of the other type of center.     

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t_1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component (t_1/2 = 50–400 ms) reflecting the recombination between P-700⁺ and center X⁻ occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700⁺ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A⁻₁ as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.     

Reactions between primary and secondary acceptors of photosystem II in Chlorella pyrenoidosa under anaerobic conditions as studied by chlorophyll fluorescence.

The kinetics of the fluorescence yield phi of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 mus to several minutes after short (t 1/2 = 30 ns or 5 mus) saturating flashes. The fluorescence yield "in the dark" increased from phi = 1 at the beginning to phi approximately 5 in about 3 h when single flashes separated by dark intervals of about 3 min were given. After one saturating flash, phi increased to a maximum value (4-5) at 50 mus, then phi decreased to about 3 with a half time of about 10 ms and to the initial value with a half time of about 2 s. When two flashes separated by 0.2 s were given, the first phase of the decrease after the second flash occurred within 2 ms. After one flash given at high initial fluorescence yield, the 10-ms decay was followed by a 10 s increase to the initial value. After the two flashes 0.2 s apart, the rapid decay was not followed by a slow increase. These and other experiments provided additional evidence for and extend an earlier hypothesis concerning the acceptor complex of Photosystem II (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250-256; Velthuys, B. R. and Amesz. J. (1974) Biochim. Biophys. Acta 333, 85-94): reaction center 2 contains an acceptor complex QR consisting of an electron-transferring primary acceptor molecule Q, and a secondary electron acceptor R, which can accept two electrons in succession, but transfers two electrons simultaneously to a molecule of the tertiary acceptor pool, containing plastoquinone (A). Furthermore, the kinetics indicate that 2 reactions centers of System I, excited by a short flash, cooperate directly or indirectly in oxidizing a plastohydroquinone molecule (A2-). If initially all components between photoreaction 1 and 2 are in the reduced state the following sequence of reactions occurs after a flash has oxidised A2- via System I: Q-R2- + A leads to Q-R + A2- leads to QR- + A2-. During anaerobiosis two slow reactions manifest themselves: the reduction of R (and A) within 1 s, presumably by an endogenous electron donor D1, and the reduction of Q in about 10 s when R is in the state R- and A in the state A2-. An endogenous electron donor, D2, and Q- complete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.     

Fluorescence induction and activity of ferredoxin-NADP reductase in Bryopsis chloroplasts

Effects of medium osmolarity on the rate of CO₂ fixation, the rate of the NADP⁺-Hill reaction, and the DPS₁ transient of chlorophyll fluorescence were measured in intact Bryopsis chloroplasts. Upon decreasing the sorbitol concentration from 1.0 M (the isoosmotic conditions) to 0.25 M, the envelopes of the chloroplasts became leaky to small molecules, resulting in a considerable depression of the CO₂-fixation rate and a higher rate of the NADP⁺-Hill reaction whereas the DPS₁ transient was unaffected. This DPS₁ transient of chlorophyll fluorescence is thought to be caused by the photoactivation of electron flow on the reducing side of Photosystem I at a site occurring after ferredoxin and probably before the reduction of NADP⁺ (Satoh, K. and Katoh, S. (1980) Plant and Cell Physiol. 21, 907–916). Little effect of NADP⁺ on the DPS₁ transient and a marked lag in NADP⁺ photo-reduction in dark-adapted (inactivated) chloroplasts support the hypothesis that the site of dark inactivation is prior to the reduction site of NADP⁺, and therefore, that ferredoxin-NADP⁺ reductase is inactivated in the dark and activated in the light. Moreover, at 0.25 M sorbitol, the activity of ferredoxin-NADP⁺ reductase itself (2,6-dichlorophenolindophenol reduction by NADPH) was shown to increase according to dark-light transition of the chloroplasts. At low osmolarities (below 0.1 M sorbitol), the difference in the diaphorase activity between dark-and light-adapted chloroplasts and the lag time observed in the NADP⁺ photoreduction were lowered. This may correspond to a less pronounced DPS₁ transient at low concentrations of sorbitol. The mechanism of the photo-activation is discussed.