Flash-induced Absorption Changes Research Articles

Light-dependent trans to cis isomerization of the retinal in halorhodopsin

Flash-induced absorption changes in the near UV were determined for bacteriorhodopsin and halorhodopsin on a millisecond time scale. The difference spectrum obtained for bacteriorhodopsin was comparable to model difference spectra of tyrosine (aromatic OH deprotonated vs protonated), as found by others. The flash-induced difference spectrum for halorhodopsin, in contrast, resembled a model spectrum opbtained for trans to 13- cis isomerization of retinal in bacteriorhodopsin. A model for chloride translocation by halorhodopsin is presented, in which the retinal isomerization moves positive charges, which in turn modulate the affinity of a site to chloride.

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane.

Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W X m-2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W X m-2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W X m-2, the ratio was approximately 35% lower than that of plants grown at 60 W X m-2. The amount of both cytochrome b563 and cytochrome f was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.

Absorption changes of Photosystem II donors and acceptors in algal cells

Flash-induced absorption changes were measured in the 390–450 nm region, using whole cells of a double mutant strain of Chlorella sorokiniana, lacking PS I centers and part of the pigment antenna. Spectral changes associated with PS II acceptors (Q a and Q b) and donors (p 680, Z and L) are described. In this region, the spectrum obtained for Q b −/Q b differs markedly from that of Q a −/Q a. Different spectra were also obtained for the secondary donor (Z +/Z) when measured after one or three flashes.

Nature of the principal photointermediate of halorhodopsin

Two alternative hypotheses have been presented as to the nature of the principal halorhodopsin photointermediate: a) it is a form whose its absorption band is shifted from the 575 nm position to 500 or 520 nm, and b) it is a form whose absorption band is shifted to only about 565 nm, but with an altered band shape so it exhibits a fortuitous difference peak near 500 nm. Such a shift with a maximum near 500 nm is also obtained in the dark when chloride is removed from the sample, suggesting the hypothesis that the spectral changes reflect the transient detachment of chloride from a binding site (Ogurusu et al, J. Biochem. Tokyo 95 , 1073–1082, 1984) . Comparison of the quantum yields of flash-induced absorption changes in halorhodopsin and bacteriorhodopsin strongly suggests, however, that hypothesis b) is untenable.

Effect of dibromothymoquinone (DBMIB) on reduction rates of photosystem I donors in intact chloroplasts

Dual effect of dibromothymoquinone (DBMIB), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700 + by DBMIB proceeds at a very low rate (half-time of ∼10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.

Effect of partial removal and readdition of a 23 kilodalton protein on oxygen yield and flash-induced absorbance changes at 320 nm of inside-out thylakoids

Washing inside-out spinach thylakoids with 250 mM NaCl at pH 7.4 causes up to 75% inhibition of oxygen evolution which has been shown by reconstitution experiments to be due mainly to the removal of a 23 kDa protein (Åkerlund, H.E., Jansson, C. and Andersson, B. (1982) Biochim. Biophys. Acta 681, 1–10). Here we have found the same degree of inhibition and reconstitution of oxygen evolution measured either in flash light or in continuous light at different light intensities, which suggests that the salt-washing causes a complete block of electron transport in certain chains rather than a decreased rate in all chains. Flash-induced absorbance changes at 320 nm, reflecting mainly the electron transfer at the Photosystem-II acceptor side, from the primary to the secondary plastoquinone in their semiquinone form (PQ − APQ − B → PQ APQ 2− B) and to minor extent the donor side of Photosystem II (Renger, G. and Weiss, W. (1983) Biochim. Biophys. Acta 722, 1–11), were measured under repetitive excitation conditions. Salt-washing the inside-out thylakoids caused a change in decay rate from 500–600 μs down to 200 μs, interpreted as an increased proportion of recombination between PQ − A and chlorophyll- a + II. Addition of the 23 kDa protein restored the kinetics up to some 400 μs. Measurements made on dark-adapted material gave evidence that salt-washing also affected S-state turnover. We conclude that the 23 kDa protein functions on the water-splitting side of Photosystem II and is indispensible for the photosynthetic water oxidation.

Evidence for a sulfhydryl group near the retinal-binding site of halorhodopsin.

Amino acid analysis of the halorhodopsin chromoprotein shows that this protein contains a cysteine residue. Such a residue is absent in bacteriorhodopsin. Low concentrations (micromolar) of HgCl2 inhibit light-dependent chloride transport by halorhodopsin in envelope vesicles prepared from Halobacterium halobium strain L-33 and increase the Km for chloride. The decay rate of the flash-induced absorption change of halorhodopsin, measured at 570 nm, is considerably slowed by HgCl2, and this effect is reversed at higher concentrations of chloride. In addition, the magnitude of the absorption changes is diminished by HgCl2. These effects of the mercurial are also seen with the purified, solubilized chromoprotein. Upon addition of HgCl2 to the chromoprotein at low chloride concentrations in the dark, a decrease of absorption at 580 nm and an increase at 380 nm occur, as well as a blue shift of the chromophore by about 20 nm. Sustained illumination of halorhodopsin results in a 410 nm photoproduct. The reconversion of this species to 580 nm in the dark is strongly inhibited by HgCl2. These results show that a thiol group is essential for the stability of the halorhodopsin chromophore and for its photochemical reactions and suggest that this group is in the vicinity of both the retinal Schiff's base and the chloride-binding site.

Evidence for a halide-binding site in halorhodopsin.

In attempting to describe a halide-binding site in halorhodopsin (P580), a light-driven chloride pump in halobacterial membranes, we have investigated the effects of chloride and bromide on flash-induced absorption changes in this pigment, and studied the effects of a diuretic drug, MK-473, on the photochemistry and the transport. We find that at high sulfate or phosphate concentrations, but in the absence of halide, the principal photointermediate is P660, whose half-life is about 1.5 ms. When chloride or bromide are added, the production of P660 is depressed and its half-life becomes longer (up to approximately 10 ms). With increasing halide concentration, the cycle proceeds more and more via the alternative photointermediate, P520, whose half-life varies with the halide concentration in a fashion similar to that of P660. Transport activity, measured during sustained illumination, increases in a manner parallel to the accumulation of P520 up to about 400 mM halide, but declines at concentrations above this value. The transport is inhibited by MK-473 with competitive kinetics, and the effects of this inhibitor on the photocycle are also consistent with displacement of halide ions from their binding site. The observations reported here suggest that chloride and bromide bind to P580, P660, and P520, and that this binding is to a distinct site on the protein.

Energetics of photosynthesis in Zea mays. II. Studies of the flash-induced electrochromic shift and fluorescence induction in mesophyll chloroplasts

Patterns of electron transfer in isolated mesophyll chloroplasts of maize ( Zea mays L.) were studied in the presence of the physiological substrates, oxaloacetate, 3-phosphoglycerate and pyruvate. Flash-induced absorbance changes due to the electrochromic pigment band-shift (P-518) were used to estimate relative electron flow rates through the cyclic and non-cyclic pathways of electron transport. Further information on the redox state of electron carriers and the activity of coupled electron flow was obtained from measurements of fluorescence induction and of actinic-light-induced fluorescence changes. The results demonstrate the importance of correct redox poising for optimal rates of photosynthesis and are discussed in relation to the operation and regulation of photosynthesis in the C 4 system.

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b 1, -b 2, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b 1, -b 2, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A 2. The cytochrome photooxidation and P430 and A 2 photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A 2 was least active in the cytochrome photooxidation. A 1, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes ( Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys. 219, 209–218 ). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A 1, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A 2. The 13,000-dalton polypeptide which is associated with CP1-a, -b 1, and -b 2 is not required for the functioning of the reaction center.

Flash-induced absorption changes in Photosystem I, Radical pair or triplet state formation?

Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700 + ... A − 1 (A 1 being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700 +. A saturation curve ( ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700 +. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700 + and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.

ANALYSIS OF THE COMPLEX BAND SPECTRUM OF P700 BASED ON PHOTOSELECTION STUDIES WITH PHOTOSYSTEM I PARTICLES

— The linear dichroism of the flash induced absorption changes of immobilized photosystem I reaction center particles from spinach chloroplasts has been investigated by means of the photoselection technique. Under flash excitation of predominantly β-carotenes at 500 nm, the photoinduced linear dichroism of the absorption changes has been measured in the spectral region from 620–720 nm. The most prominent feature in the spectrum of the dichroic ratio is the symmetrical pole at 687 nm. In parallel to all photoselection experiments, we recorded the light induced absorption changes of P700 under saturating flash excitation. A Gaussian deconvolution of this well-known difference spectrum of P700 has been attempted taking the additional features of the linear dichroism spectrum into account. From 670-720 nm, the best fit for both spectra was obtained by the following components: 1 a disappearing wide band at 695.5 nm (σ= 200.0 cm−1) attributed to the reduced special pair of chlorophyll a (Ch1-a); 2 an appearing narrower band at 690 nm (σ= 120.0 cm−1) with half the oscillatory strength of the former tentatively attributed to the non-oxidized moiety in the special pair; and 3 a bathochromic bandshift centered at 688.4 nm attributed to the local electrochromic response of certain antennae Ch1-a molecules close to the primary electron donor. The linear dichroism gives no evidence for any substructure within the absorption band of the reduced special pair.

Acceleration of the Decay of Membrane Potential after Energization of Chloroplasts in Intact <italic>Zea mays</italic> Leaves

The relationship between dissipation of the flash-induced membrane potential across the thylakoid membrane and the high energy state was studied in Zea mays leaves. The dark decay of the flash-induced 515-nm absorbance change was accelerated by short preillumination of the leaf. No acceleration of the decay by preillumination was observed when leaves were incubated in argon or COa gas or treated with DCMU. These effects of preillumination and incubation were reversible. The delayed fluorescence from chlorophyll a was reversibly decreased by incubating leaves in argon or CO2 gas, though the modes of depression were somewhat different from each other. In leaves incubated in argon or CO2 gas, the phase of slow decrease of the intensity of prompt fluorescence during illumination reversibly disappeared. The results suggested that the dissipation of membrane potential generated by a flash was accelerated after the energization of chloroplasts in leaves, probably by increased H+ permeability of the thylakoid membrane. O2 was important in maintaining (in darkness) and forming (under illumination) the high energy state in chloroplasts in intact leaves.

The primary charge separation, cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations. Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex. The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840 + was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.

The oxidation-reduction potential of P-700 in chloroplast lamellae and subchloroplast particles

The oxidation-reduction potential of P-700 has been determined in chloroplast lamellae and in subchloroplast particles by measuring the magnitude of flash-induced absorption changes at 820 or at 703 nm (due to the oxidation of P-700) in the presence of known concentrations of potassium ferro- and ferricyanide. A midpoint potential of about +490 mV was determined in chloroplast lamellae and in particles prepared with digitonin (D-144) or Triton (TSF-1). A lower potential was determined with Photosystem I particles obtained after harsher treatments with Triton or a mixture of detergents. The potential is even lower in chlorophyll-protein complex I particles prepared with sodium dodecyl sulfate (about +430 mV). Very similar values were determined from oxidized minus reduced spectra measured with a double-beam spectrophotometer. Titrations were also made with D-144 and TSF-I particles, with 66% glyeerol in the buffer, at 21 °C and at 77 °K. P-700 was found to be half-oxidized at ferricyanide/ferrocyanide ratios of about 60 at 21 °C and of about 1 at 77 °K. This shows that the redox equilibrium is largely perturbed by the cooling process.

Flash-induced charge separation and dark recombination in a Photosystem-II subchloroplast particle

The decay time of flash-induced absorption changes in a Photosystem-II subchloroplast fragment is very temperature sensitive down to 210 K, below which it remains constant at 1.25 ± 0.05 ms. The difference spectrum from the near-infra-red to the ultraviolet regions indicates that the monophasic decay represents charge recombination between P-680 + and the reduced primary acceptor. The charge recombination proceeds by electron tunneling. The P-680 concentration in the TSF-IIa fragment was estimated to be one in 30 ± 5 total chlorophyll molecules.

331 - Studies on the structural and functional organization of water cleavage by visible light in photosynthesis

Photosynthetics water cleavage by visible light into molecular oxygen and metabolically bound hydrogen is supplied by exciton dissociation into strongly oxidizing defect electrons and moderately reducing electrons at a special chlorophyll- a-complex (Chl- a II) associated with acceptor component(s). This device is referred to as the reaction center of system II (RC-II). Water oxidation by the defect electrons originally localized at Chl- a II +, is catalyzed by a manganoprotein, the so-called water-splitting enzyme system Y. Spectroscopic measurements of flash induced absorption changes in the range of 250–850 nm as well as polarographic detection of oxygen formation lead to the following conclusions about the functional and structural organization scheme of photosynthetic water cleavage: 1. Exciton dissociation leading to water oxidation and electric field formation across the thylakoid membrane requires a special plastoquinone molecule (X-320) as electron acceptor. Under conditions of X-320 remaining functionally blocked in its reduced state, exciton dissociation under the formation of Chl- a II + is still possible, but this reaction is highly dissipative. 2. The electron transfer from system Y to Chl- a II +, which reflects the functional coupling of these units, occurs via a multiphasic kinetics in the micro- and submicrosecond time scale. These kinetics are dependent on the charge accumulation state of system Y and the pH of the inner thylakoid space. 3. A postulated model for the reaction sequence in system Y is discussed. 4. The life times of the intermediary stages of water oxidation in system Y can selectively be modified by special chemical (ADRY-reagents). 5. The reaction centers RC-II nearly totally span the impermeable core of the thylakoid membrane, with Chl- a II located towards the inner side and X-320 attached near the outer side. The redox component X a, which is assumed to be the primary electron acceptor, is inferred to be located rather close to Chl- a II. 6. X-320 is enwrapped by a proteinaceous component, which provides — via another special plastoquinone molecule (B) — the functional connection to the plastoquinone pool. This trypsin digestible component probably functions as an allosteric regulator for the electron flux from X-320 to the pool and its blockage by DCMU-type inhibitors. On the basis of the experimental results and theoretical considerations a preliminary model is proposed for the functional and structural organization of water oxidation by visible light in photosynthesizing organisms.

Studies on the structural and functional organization of water cleavage by visible light in photosynthesis

Photosynthetics water cleavage by visible light into molecular oxygen and metabolically bound hydrogen is supplied by excition dissociation into strongly oxidizing defect electrons and moderately reducing electrons at a special chlorophyll- a -complex (Chl- a II ) associated with acceptor component(s). This device is referred to as the reaction center of system II (RC-II). Water oxidation by the defect electrons originally localized at Chl- a II + , is catalyzed by a manganoprotein, the so-called water-splitting enzyme system Y. Spectroscopic measurements of flash induced absorption changes in the range of 250–850 nm as well as polarographic detection of oxygen formation lead to the following conclusions about the functional and structural organization scheme of photosynthetic water cleavage: o 1. Exciton dissociation leading to water oxidation and electric field formation across the thylakoid membrane requires a special plastoquinone molecule (X-320) as electron acceptor. Under conditions of X-320 remaining functionally blocked in its reduced state, exciton dissociation under the formation of Chl- a II + is still possible, but this reaction is highly dissipative. 2. The electron transfer from system Y to Chl- a II + , which reflects the functional coupling of these units, occurs via a multiphasic kinetics in the micro- and submicrosecond time scale. These kinetics are dependent on the charge accumulation state of system Y and the pH of the inner thylakoid space. 3. A postulated model for the reaction sequence in system Y is discussed. 4. The life times of the intermediary stages of water oxidation in system Y can selectively be modified by special chemical (ADRY-reagents). 5. The reaction centers RC-II nearly totally span the impermeable core of the thylakoid membrane, with Chl- a II located towards the inner side and X-320 attached near the outer side. The redox component X a , which is assumed to be the primary electron acceptor, is inferred to be located rather close to Chl- a II . 6. X-320 is enwrapped by a proteinaceous component, which provides — via another special plastoquinone molecule (B) — the functional connection to the plastoquinone pool. This trypsin digestible component probably functions as an allosteric regulator for the electron flux from X-320 to the pool and its blockage by DCMU-type inhibitors. On the basis of the experimental results and theoretical considerations a preliminary model is proposed for the functional and structural organization of water oxidation by visible light in photosynthesizing organisms.

Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome ( E m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.

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. (1) At 520 nm flash-induced absorption changes are observed, which predominantly decay via a 100–200-μs 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. (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, 1144–1146). In the absence of hydroquinone/ascorbate the difference spectrum of the absorption changes, characterized by a 100–200-μs 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 chlorophyll a + II. The problems arising with the latter explanation are discussed. 3. (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 a 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 a II within the thylakoid membrane at the outer and inner side, respectively.