The existence of a high photochemical turnover rate at the reaction centers of system II in Tris-washed chloroplasts.

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of approximately 1 mus. It has been found that X 320 is reduced by a flash in less than or equal to 1 mus. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100-200 mus kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Turnover kinetics of Photosystem I measured by the electrochromic effect in Chlorella

The rise kinetics of the absorption changes induced at 515 nm and 480 nm by a flash were studied using two types of xenon flashes of different durations. The ‘slow’ rise of the absorption change ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15–20 μ}\text{s}$) observed by Cox and Delosme (1978 C.R. Acad. Sci. (Paris) Sér. D 282, 775–778) and Joliot P., Delosme, R. and Joliot, A. ((1977) Biochim. Biophys. Acta 459, 47–57) was found to be due to double hits occurring in the reaction centers of System I during the flash.The turnover kinetics of the reaction centers of System I after a short flash were studied by a double flash method. They are in agreement with a second order reaction between P⁺-700 and its electron donor.     

Flash-induced carotenoid radical cation formation in Photosystem II

Light excitation of chloroplasts at low temperature produces absorption changes (ΔA) with a large positive peak at 990 nm and a bleaching around 480 nm. ΔA at 990 nm rises with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.6}\text{ms}$ at 20–77 K and remains largely stable. This signal is not observed when Photosystem II (PS II) photochemistry is blocked by reduction of the primary plastoquinone. It is observed also in purified PS II particles, in which case it could be shown that during a sequence of short flashes, the absorption at 990 nm rises in parallel with plastoquinone reduction measured at 320 nm. In chloroplasts the light-induced 990-nm ΔA at 77 K is increased under oxidizing conditions (addition of ferricyanide) and upon addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p). At 21°C, flash excitation of chloroplasts or of PS II particles induces only a very small ΔA at 990 nm, even when this is measured with a 100-ns time resolution or when the material is preilluminated. In both materials, however, a large flash-induced ΔA takes place when various lipophilic anions are added. After a flash the signal rises in less than 100 μs and its decay varies with experimental conditions; the decay is strongly accelerated by benzidine. The difference spectrum measured in PS II particles includes a broad peak around 990 nm and a bleaching around 490 nm. These absorption changes are attributed to a carotenoid radical cation formed at the PS II reaction center. It is estimated that in the presence of lipophilic anions at room temperature, one cation can be formed by a single flash in 80% of the reaction centers. At cryogenic temperature approx. 8% of the PS II reaction centers can oxidize a carotenoid after a single flash.     

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.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Precursors to microsecond delayed luminescence in oxygenevolving and inhibited chloroplasts

We have investigated submillisecond delayed luminescence in spinach chloroplasts under a variety of conditions. In Tris-washed chloroplasts, which are inhibited on the oxidizing side of P-680, the delayed light emission in the 7–200 μs time-range decayed with biphasic behavior. In fully dark-adapted samples illuminated by a single saturating laser pulse, the fast phase of delayed luminescence followed a nearly identical pH-dependent time-course as that observed optically and by ESR for P⁺-680 reduction, thus verifying the recombination hypothesis for the origin of delayed light. The observed slower phase of delayed luminescence was also pH dependent, but unlike the fast phase, could not be ascribed to specific electron transfer events of PS II. This phase could be rationalized by a heterogeneity in the population of P-680. While kinetic parameters were found to be insensitive to changes in ionic strength, the overall luminescence intensity was quite sensitive to the electrical parameters, thus indicating the role of ionic strength and local charges in delayed luminescence modulation. A similar series of experiments was performed on untreated chloroplasts. The pH-dependent delayed luminescence behavior in both untreated chloroplasts and Tris-washed chloroplasts was similar despite significantly faster kinetics associated with the reduction of P⁺-680 by the secondary PS II electron donor, Z, in the former preparation (e.g., Van Best, J.A. and Mathis, P. (1978) Biochim. Biophys. Acta 503, 178–188). Thus, it was concluded that, in untreated samples, microsecond delayed luminescence emanates primarily from centers which are not competent in oxygen evolution. The nearly identical delayed luminescence intensity in untreated chloroplasts and in Tris-washed chloroplasts was rationalized by a model which predicts modulations in delayed luminescence yield by the exciton-quenching effect of P⁺-680. Computer simulations demonstrate the feasibility of this model. The previously documented flash oscillations in microsecond delayed luminescence intensity in untreated chloroplasts (Bowes, J.M. and Crofts, A.R. (1979) Biochim. Biophys. Acta 547, 336–346), which we readily observed, were attributed to alterations in delayed luminescence yield (in nonfunctional centers) by variations in charge density stored at the oxygen-evolving complex of functional centers. Taken together, our results emphasize the dependence of delayed luminescence kinetics upon electron-transfer kinetics and the dependence of delayed luminescence amplitude upon the photochemical parameters, the exciton yield and the emission yield.     

A rapid vectorial back reaction at the reaction centers of photosystem II in tris-washed chloroplasts induced by repetitive flash excitation.

In Tris-washed chloroplasts, completely lacking the oxygen-evolving capacity, absorption changes in the range of 420--560 nm induced by repetitive flash excitation have been measured in the presence and absence of electron donors. It was found: (1) At 520 nm flash-induced absorption changes are observed, which predominantly decay via a 100--200-mus exponential kinetics corresponding to that of the back reaction between the primary electron donor and acceptor of Photosystem II (Haveman, J. and Mathis, P. (1976) Biochim. Biophys. Acta 440, 346--355; Renger, G. and Wolff, Ch. (1976) Biochim. Biophys. Acta 423, 610--614). In the presence of hydroquinone/ascorbate as donor couple the amplitude is nearly doubled and the decay becomes significantly slowed down. (2) The difference spectrum of the absorption changes obtained in the presence of hydroquinone/ascorbate, which are sensitive to ionophores, is nearly identical with that of normal chloroplasts in the range of 460--560 nm (Emrich, H.M., Junge, W. and Witt, H.T. (1969) Z. Naturforsch. 24b, 114--1146). In the absence of hydroquinone/ascorbate the difference spectrum of the absorption changes, characterized by a 100--200-mus decay kinetics, differs in the range of 460--500 nm and by a hump in the range of 530--560 nm. The hump is shown to be attributable to the socalled C550 absorption change, which reflects the turnover of the primary acceptor of Photosystem II (van Gorkom, H.J.(1976) Thesis, Leiden), while the deviations in the range of 460--500 nm are understandable as to be due to the overlapping absorption changes of chlorphyll alpha II+. The problems arising with the latter explanation are discussed. (3) The electron transfer due to the rapid turnover at Photosystem II, which can be induced by flash groups with a short dark time between the flashes, is not able to energize the ATPase and to drive photophosphorylation. On the basis of the present results it is inferred, that in Tris-washed chloroplasts under repetitive flash excitation a rapid transmembrane vectorial electron shuttle takes place between the primary acceptor (X320) and donor (Chl alpha II) of Photosystem II, which is not able to energize the photophosphorylation. Furthermore, the data are shown to confirm the localization of X320 and Chl alpha II within the thylakoid membrane at the outer and inner side, respectively.     

Reactions between primary and secondary acceptors of Photosystem II in Chlorella Pyrenoidosa under anaerobic conditions as studied by chlorophyll a fluorescence

The kinetics of the fluorescence yield Ф of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 μs to several minutes after short ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{ns}$ or 5 μs) saturating flashes. The fluorescence yield “in the dark” increased from $\text{Ф = 1}$ at the beginning to $\text{Ф ≈ 5}$ in about 3 h when single flashes separated by dark intervals of about 3 min were given.After one saturating flash, Ф increased to a maximum value (4–5) at 50 μs, then Ф 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 follewed 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 (A^2?). 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 A^2? via System I: Q^?R^2? + A → Q^?R + A^2? → QR^? + A^2?. 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 A^2?. An endogenous electron donor, D₂, and Q^? compete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

A flash photolysis ESR study of Photosystem II Signal IIvf, the physiological donor to P-680+

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II ($\text{g ≈ 2.0045}$, $\text{ΔH}{}_{\mathrm{<mtext>pp</mtext>}}\text{≈ 19}\text{G}$). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination ($\text{λ < 680}\text{nm}$ and occurs stoichiometrically in a 1 : 1 ratio with $\text{P-700}{}^{\mathrm{+}}$. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol^?1. Inhibition of electron transport through Photosystem II by $\text{o-}\text{phenanthroline}$, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, $\text{P-680}{}^{\mathrm{+}}$.     

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 μs to 20 μs after a short saturating flash ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25}\text{ns}$) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 μs luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ^? before the flash, about one third for centers in the state S⁺PQ^? or SPQ before the flash, and about one tenth for centers in the state S⁺PQ before the flash. S is the secondary donor complex with zero charge; S⁺ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q^? is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two.The 1 μs decay of the luminescence is probably caused by the disappearance of P⁺ formed in the laser flash according to the reaction ZP⁺ → Z⁺P in which Z is the molecule which donates an electron to P⁺ and which is part of S. After addition of hydroxylamine, the 1 μs luminescence component changed with the incubation time exponentially (τ = 27 s) into a 30 μs component; during the same time, the variable fluorescence yield, measured 9 μs after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 μs luminescence increased about 50% with the same time constant (τ = 7 min). Heat treatment abolished both luminescence components.The 1 μs luminescence component saturated at about the same energy as the System II fluorescence yield 60 μs after the laser flash and as the slower luminescence components. From the observation that the amplitude is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ^?), we conclude that the 1 μs luminescence is probably caused by the reaction

$\text{PWQ}{}^{\mathrm{?}}\text{+hv →}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→}\text{P}{}^{\mathrm{+}}\text{W}{}^{\mathrm{?}}\text{Q}{}^{\mathrm{?}}\text{→}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→ PWQ}{}^{\mathrm{?}}\text{+hv}$

in which W is an acceptor different from Q. The presence of S⁺ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.     

Electron transfer between the two photosystems. I. Flash excitation under oxidizing conditions

The redox changes of cytochrome b-563 (cytochrome b), cytochrome f, plastocyanin and P-700 were measured on dark-adapted chloroplasts after illumination by a series of flashes in oxidizing conditions (0.1 mM ferricyanide). In these conditions, the plastoquinone pool is fully oxidized and the only available plastoquinol are those formed by Photosystem (PS) II reaction. According to the two-electron gate mechanism proposed by Bouges-Bocquet (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256), plastoquinol is mainly formed after the second and the fourth flashes. After the second flash, the reoxidation of plastoquinol occurs by a concerted reaction which reduces most of the cytochrome b present and a fraction of PS I donors. Most of these electrons are stored on P-700, which implies a large equilibrium constant between the secondary PS I donors and P-700. One electron is stored on cytochrome b during a time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{s}$) much longer than the dark interval between flashes. After the fourth flash, a new plastoquinol molecule is formed, which induces the reduction of PS I donors with no corresponding further reduction of cytochrome b. The number of electrons transferred after the fourth flash is larger than that transferred after the second flash although the rate of transfer is lower. To interpret these data, we assume that the plastoquinol formed after the fourth flash is reoxidized by a second concerted reaction: one electron is directly transferred to PS I donors while the other cooperates with the electron stored on cytochrome b to reduce a plastoquinone molecule localized on a site close to the outer face of the membrane. This newly formed plastoquinol crosses the membrane and transfers a second electron to PS I donors. This interpretation resembles a model proposed by Velthuys (Velthuys, B.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2765?2769) and which belongs to the modified Q-cycle class of models.     

Internal pH of human neutrophil lysosomes

We focus this report on the relationship between signal II fast and slow during a flash sequence for Tris-washed chloroplasts at different pH-values. The pH influences both the redox state and spectral form of signal II slow in dark-adapted chloroplasts. At pH 6.0, signal II slow is oxidized and does not influence the kinetics of signal II fast equally formed on each flash. At pH 8.5, signal II slow in mainly reduced in the dark and the first flash produces signal II slow and no signal fast. Signal II fast appears on the following flashes only. Signals II fast and slow are connected to the same center and signal II fast is observed only if signal II slow is oxidized.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.