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.     

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.     

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.     

Sub-microsecond chlorophyll A delayed fluorescence from Photosystem I Magnetic field-induced increase of the emission yield

(1) In photosystem I (PS I) particles in the presence of dithionite and intense background illumination at 290 K, an external magnetic field (0–0.22 T) induced an increase, ΔF, of the low chlorophyll a emission yield, F ($\text{ΔF}\text{F}\text{? 1–1.5\%}$). Half the effect was obtained at about 35–60 mT and saturation occurred for magnetic fields higher than about 0.15 T. In the absence of dithionite, no field-induced increase was observed. Cooling to 77 K decreased ΔF at 685 nm, but not at 735 nm, to zero. Measuring the emission spectra of F and ΔF, using continuous excitation light, at 82, 167 and 278 K indicated that the spectra of F and ΔF have about the same maximum at about 730, 725 and 700 nm, respectively. However, the spectra of ΔF show more long-wavelength emission than the corresponding spectra of F. (2) Only in the presence of dithionite and with (or after) background illumination, was a luminescence (delayed fluorescence) component observed at 735 nm, after a 15 ns laser flash (530 nm), that decayed in about 0.1 μs at room temperature and in approx. 0.2 μs at 77 K. A magnetic field of 0.22 T caused an appreciable increase in luminescence intensity after 250 ns, probably mainly caused by an increase in decay time. The emission spectra of the magnetic field-induced increase of luminescence, ΔL, at 82, 167 and 278 K coincided within experimental error with those of ΔF mentioned above. The temperature dependence of ΔF and ΔL was found to be nearly the same, both at 685 and at 735 nm. (3) Analogously to the proposal concerning the 0.15 μs luminescence in photosystem II (Sonneveld, A., Duysens, L.N.M. and Moerdijk, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5889–5893), we propose that recombination of the oxidized primary donor P-700⁺ and the reduced acceptor A^?, probably A^?₁, of PS I causes the observed fast luminescence. The effect of an external magnetic field on this emission may be explained by the radical pair mechanism. The field-induced increase of the 0.1–0.2 μs luminescence seems to be at least in large part responsible for the observed increase of the total (prompt + delayed) emission measured during continuous illumination in the presence of a magnetic field.     

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA₈₂₀) in the microsecond time range. The extent of ΔA₈₂₀ induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg²⁺ free medium (plus 15 mM K⁺) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg²⁺ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.     

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.     

Analysis of absorption changes in the ultraviolet related to charge-accumulating electron carriers in Photosystem II of chloroplasts

Spinach chloroplasts were dark adapted and then submitted to a sequence of short saturating flashes. The resulting absorption changes in the near ultraviolet were analyzed and attributed to the donor and acceptor sides of Photosystem II. Our results provide a spectroscopic support to some current models of these parts of the photosynthetic electron transport.In Tris-treated chloroplasts (supplied with artificial donors) the absorption changes are largely due to the acceptor side. After each flash the signal decays with a fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms at}\text{9 °}\text{C}$) leaving a stationary level (on a 100-ms time scale). The fast phase has a small amplitude after odd-numbered flashes, whereas the stationary level behaves in a complementary fashion. The non-decaying signal is attributed mostly to the reduced secondary acceptor (A₂^?) and the fast phase to the simultaneous reoxidation of A₂^? and of the reduced primary acceptor (A₁^?). The effect of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and of redox mediators (ascorbate, ferricyanide) also support this assignment. A fraction of A₂ is shown to be reduced in dark-adapted chloroplasts, as proposed by Velthuys and Amesz (Biochim. Biophys. Acta (1974) 333, 85–94). The difference spectra support the view that A₁^? and A₂^? are plastoquinone radical anions. There are also some absorption changes that we cannot identify.In untreated chloroplasts a non-decaying absorption change (“slow phase”) occurs with a 4-flash periodicity. It is attributed to the transitions among the S states associated with the O₂-evolving complex. A fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms}$) in the decay following the first two flashes behaves like in Tris-treated chloroplasts, so that the assignment is tentatively the same. After the third flash, however, the magnitude of this fast phase is too large according to the hypothesis, so that there may be some contribution from the donor side. The fast phases become slower at lower pH (5.5 instead of 7.6), although there is no evidence for a protonation A₁^? or A₂^?.     

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP⁺. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the $\text{P}\text{e}{}_2$ ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg2+

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll $\text{a}\text{b}$ light-harvesting antenna.     

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.     

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.     

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.     

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 $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs 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.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

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.