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Solar energy exploitation by photosynthetic water cleavage is of central relevance for the development and sustenance of all higher forms of living matter in the biosphere. The key steps of this process take place within an integral protein complex referred to as Photosystem II (PS II) which is anisotropically incorporated into the thylakoid membrane. This minireview concentrates on mechanistic questions related to i) the generation of strongly oxidizing equivalents (holes) at a special chlorophyll a complex (designated as P680) and ii) the cooperative reaction of four holes with two water molecules at a manganese containing unit WOC (water oxidizing complex) resulting in the release of molecular oxygen and four protons. The classical work of Pierre Joliot and Bessel Kok and their coworkers revealed that water oxidation occurs via a sequence of univalent oxidation steps including intermediary redox states Si (i = number of accumulated holes within the WOC). Based on our current stage of knowledge, an attempt is made a) to identify the nature of the redox states Si, b) to describe the structural arrangement of the (four) manganese centers and their presumed coordination and ligation within the protein matrix, and c) to propose a mechanism of photosynthetic water oxidation with special emphasis on the key step, i.e. oxygen-oxygen bond formation. It is assumed that there exists a dynamic equilibrium in S3 with one state attaining the nuclear geometry and electronic configuration of a complexed peroxide. This state is postulated to undergo direct oxidation to complexed dioxygen by univalent electron abstraction with YZ (ox) and simultaneous internal ligand to metal charge transfer.Key questions on the mechanism will be raised. The still fragmentary answers to these questions not only reflect our limited knowledge but also illustrate the challenges for future research.

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy γ-rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the QB site: H2O → (Mn)4 → Yz → P680 → Pheophytin → Q → phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the α and β subunits of cytochrome b 559and the 4.5 kDa psbI gene product.

The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which roughly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in delta 1 (I265-F268), delta 2 (T271-K277), delta 4 (T304-L309), delta 5 (F311-N317), and delta 12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains delta 3 (K277-E283) and delta 11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain delta 8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains delta 6 (G333-I336), delta 7 (K347-R352), delta 9 (V392-Q394), and delta 10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (delta 6, delta 7, delta 8, delta 9, delta 10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.

The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O2-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A ('free peroxide') peaking around 0.2-0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O2 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 μg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C ('bound peroxide') detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S2 or formed at S2 upon acid shock treatment. Signal D ('slowly released peroxide') peaking around 2-3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl2, Cl(-)-depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H2O2 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of 'bound' peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.

Photoinhibition was analyzed in O2-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680(+) formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a 'stable' charge separation between P680(+) and QA (-) within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from YZ to P680(+). The quantum yield of site I photoinhibition (2-3×10(-7) inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O2-evolving samples, the quantum yield of site II photoinhibition is about 10(-7) inhibited RC/quantum. After selective elimination of the O2-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (3 W/m(2)), the quantum yield is 10(-4) inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.

Oxygen-evolving photosystem II core complexes were prepared from spinach by solubilizing photosystem II membrane fragments with dodecyl-beta-D-maltoside. The core complexes consist of the intrinsic 47-kDa, 43-kDa, D1 and D2 polypeptides, the two subunits of cytochrome b559 and the extrinsic 33-kDa protein. In the presence of 50 mM CaCl2 they exhibit a high oxygen evolution rate of 1.3 +/- 0.2 mmol O2.mg chlorophyll-1.h-1 with either 2,6-dichloro-benzoquinone or K3[Fe(CN)6] as acceptor. Electron micrographs of these complexes reveal an obtuse triangular structure in when viewed from the top measuring 15.3 nm on one side and 10.6 nm on the other two sides. An average height of 7.3 nm was determined from the side view position. These data are in good agreement with previously reported dimensions for photosystem II core complexes [Irrgang, K.-D., Boekema, E. J., Vater, J. and Renger, G. (1988) Eur. J. Biochem. 178, 209-217]. In contrast to previous reports the extrinsic 33-kDa subunit could be resolved for the first time. It appears as a small protrusion when the complex is viewed from the side and seems to cover the lumenal side of the core complex appearing as a disk with a thickness of 1.5-3.3 nm.

Prolonged illumination of higher plants with intense visible light leads to photoinhibition as observed by diminution of O2 evolution and electron transport rate through photosystem II (PS II) and by a decrease of variable fluorescence yield (for review see Ref. 1 and 2). The molecular mechanism of photoinhibition is not yet clear and there is still some discussion about the exact site(s) of the primary events. Most authors localized inhibition within the PS II reaction center or at the QB-binding site. In addition for chloroplats with already inhibited oxygen evolution capacity, the involvement of PS II donor side components has been reported.

It has been widely accepted that in higher plants all the pigments that are involved in the photosynthetic primary processes are bound to thylakoid membrane polypeptides. Among these pigment-binding proteins about the half belong to the so-called Chl a/b light-harvesting antennae. With respect to photosystem II we know at least four complexes with relative molecular masses of 29 (CP 29), 27 (CP27), 25 (major LHC II) and 24 (CP24) kDa (1–4).

Photosynthetic water oxidation to dioxygen takes place via a sequence of four univalent redox steps at a manganese containing catalytic site (for review see Ref. 1). This process is coupled with the release of four protons (H+). It is now widely accepted that the redox sequence S0 → S1 → S2 → S3 → S0 + O2 is coupled with a stoichiometric H+-release pattern of 1:0:1:2 (2). This pattern is often interpreted as the deprotonation pattern of water substrate at the catalytic site of water oxidation, referred to as the intrinsic H+-release pattern (3). However the experimentally detected l:0:l:2-stoichiometry does not necessarily reflect the deprotonation steps of substrate water because of the possible participation of deprotonizable groups of the protein matrix surrounding the catalytic site of water oxidation. A participation of the protein matrix in protolytic reactions induced by redox transitions of functional groups has been experimentally confirmed for the H+-uptake coupled with QA reduction in bacterial reaction centers (4). In this study, the H+-release pattern coupled with water oxidation in higher plants has been further analyzed.

The mechanism of atrazine binding and its modification by Chelex-100-induced Ca2+ depletion and proteolytic degradation by trypsin, was analyzed in PS II membrane fragments from spinach. It was found: 1) Chelex-100 treatment leads in a comparatively slow process (t1/2 = 5 - 10 min) to Ca2+ re­ moval from a site that is characterized by a high affinity as reflected by KD values of the order of 10-7 M. The number of these binding sites was found to be almost one per PS II in samples washed twice with Ca2+ -free buffer. 2) Chelex-100 treatment does not affect the affinity of atrazine binding but increases the susceptibility to proteolytic attack by trypsin. 3) The electron transport activity is only slightly affected by Chelex-100 treatment. 4) The atrazine binding exhibits a rather small T-dependence within the physiological range of 7 °C to 27 °C. The implications of these findings for herbicide binding are discussed.

Photosynthetic water oxidation into dioxygen occurs at a manganese containing catalytic site of photosystem II (PS II) in higher plants, algae and cyanobacteria [1]. The reaction sequence is energetically driven by photooxidation of a special chlorophyll a (P680) and subsequent electron transfer to a specially bound plastoquinone (QA) via pheophytin a as intermediate [1]. There is strong evidence that P680, Pheo a and QA are all bound to a heterodimer of two transmembrane polypeptides (D1/D2) which exhibit remarkable analogies with the subunits of purple bacteria reaction centers [2,3]. The D1/D2 heterodimer could be considered not only as reaction center of PS II but also as protein matrix for water oxidation. However, the smallest photoactive protein complex of PS II so far isolated containing D1, D2 and at least the two subunits cytochrome b559 is not only deprived of QA but also of the manganese cluster indispensable for water oxidation [4]. Functional and structural investigations of the catalytic site of water oxidation nevertheless require the isolation of a PS II subcomplex with oxygen evolution activity. In this work we report on the isolation of a PS II core complex with high oxygen evolution capacity consisting of at least seven polypeptides. The biochemical properties of this PS II core complex were characterized and functional studies were performed in parallel to the structural investigation by electron microscopy.

Photosynthetic water cleavage into dioxygen and hydrogen bound as plastoquinol takes place in a polypeptide complex referred to as photosystem II that is anisotropically incorporated into the thylakoid membrane. With QA − as reductant plastoquinol is formed in a specific site (referred to as QB −binding-site in polypeptide D-1) by a sequence of two univalent electron transfer steps via the semiquinone form of a tightly bound special plastoquinone QB − as intermediate. The plastoquinone QB is stably associated with the QB −site only in the semiquinone redox state QB − white QB is loosely bound and QBH2 rapidly exchanges with the plastoquinone of the thylakoid membrane thereby renewing the substrate for the transfer of the next two electrons. A large number of chemicals acting as PS II herbicides interfere with the QB −site in preventing binding and reduction of PQ by QA − (for review see Ref. 1). The mechanism of plastoquinol formation suggests that structural modifications at the QB−site could affect the electron transfer rate from QA − to QB/ QB − Therefore temperature induced dynamical changes of structural properties of the protein matrix are expected to modify the reactivity at the QB −site. In this communication the temperature dependence of the QA −-reoxidation is analyzed by measuring the relaxation kinetics of the flash induced enhancement of fluorescence quantum yield in thylakoids, PS II membrane fragments and PS II core particles with high oxygen evolution rate.

Water cleavage into dioxygen and four protons requires the cooperation of four redox equivalents of sufficient oxidizing power. In photosynthesis the 1-electron oxidant is generated by electron ejection from the excited singlet state of a special chlorophyll a complex (P680) with pheophytin a (Pheo) as primary acceptor and the indispensible stabilization through Pheo− reoxidation by a specifically bound plastoquinone (QA). The oxidizing equivalents are transferred from P680+ via a tyrosine residue (Yz) into a manganese containing hole storage unit referred to as HSU(Mn). After accumulation of four holes by a sequential univalent storage process (described by S0→S1→S2→S3→S4) oxygen is evolved and HSU(Mn) returns to state S0 (for review see ref. 1,2).

There is a large body of literature to suggest that chloride ions are a necessary cofactor in the O2-evolving reactions by Photosystem II (PS II) (1). Current hypotheses imply that chloride ions bind at (2) or near (3) the functional manganese center. Such hypotheses are based on measurements of samples in which the chloride ions are believed to be competitively removed from the functional site by such treatments as alkalization of the sample, and/or the addition of sulfate, amines or ammonia. However, more recent evidence shows that chloride interactions with PS II also involves secondary, structural reorganizations of the protein subunits as well (4). During our investigations of 35Cl NMR linewidth measurements of PS II samples, we observed that a strong chloride demand in O2 evolultion can be generated simply by slow ageing of the samples only in the absence of added chloride ions at pH 6.3. The results imply that the protein structural integrity of PS II plays a major, if not the primary, role in the chloride demand in O2 evolution.

The electron acceptors QA and QB in Photosystem II are known to be two specially bound plastoquinone molecules assumed to be linked by a histidine-liganded Fe2+ ion whose function is unknown. EPR/ENDOR spectroscopy (1) can, in principle, be applied to the quinone radical anions formed in the charge separation process, in order to gain information about their electronic structure. Due to the strong magnetic coupling between the semiquinones and the high spin Fe2+, ENDOR spectra are difficult to obtain. The removal of Fe, as shown in the work on bacterial reaction centers (2), removes this magnetic coupling, allowing the investigation of the semiquinone states. Removal of Fe from Photosystem II has already been described (3).

Measurements of time-resolved fluorescence decay, laser-flash-induced absorption changes in the UV and at 820 nm and of the relative fluorescence quantum yield in different preparations (thylakoids, photosystem II (PSII) membrane fragments and PSII core complexes) from spinach led to a number of conclusions. (1) Light is transformed into Gibbs energy with trapping times of 250 ps and 130 ps in open reaction centres of PSII membrane fragments and PSII core complexes, respectively. Assuming rapid Boltzmann distribution of excitation energy and taking into account the antenna properties (size and spectral distribution), the molecular rate constant of primary charge separation is estimated to be about (3 ps)-1. (2) The electron transfer from Pheo- to QA is characterised by a rate constant of (300 ps)-1. (3) The QA- reoxidation kinetics are significantly retarded in D2O suspensions. These H/D isotope effects are interpreted as to reflect hydrogen-bond dependent changes in the protein dynamics that are relevant to electron transfer. (4) In PSII reaction centres closed for photochemical trapping the yield of a primary radical pair with lifetimes exceeding 1 ns is comparatively small (c 30%) at room temperature. Short illumination in the presence of Na2S2O4 changes the radical pair dynamics. (5) Photoinhibition under aerobic conditions impairs the primary charge separation and leads to formation of quencher(s) of excitation energy.

Oxygen flash yield patterns of dark adapted thylakoid membranes as measured with a Joliot-type O2-electrode indicate that inhibitors that block the oxidation of the reduced primary quinone Q−A of Photosystem II vary greatly in the rate of binding to and release from the inhibitor / QB binding environment. The ‘classical’ Photosystem-II herbicides like diuron and atrazine exhibit slow binding and release kinetics, whereas, for example, phenolic inhibitors, o-phenanthroline and synthetic quinones are exchanging quite rapidly with QB (about once per second or faster at inhibitor concentrations causing about 50% inhibition of O2 evolution). No general relationship between the efficiency of the inhibitor and the exchange rate is observed; it depends mainly on the type of inhibitor. Based on the classical Kok model, equations are derived in order to calculate oxygen yields evolved by thylakoids in single-turnover flashes as a function of the rate constants of inhibitor binding to and release from the inhibitor / QB binding environment in the presence of an oxidized or semireduced QA · QB or QA · inhibitor complex. Fitting of theoretical and experimental values yields that o-phenanthroline binds much faster to an oxidized than to a semireduced QA · QB complex. This fits very well with the hypothesis that the Q−B affinity to the site is much higher than that of QB. In the case of i-dinoseb, however, inhibitor / quinone exchange seems to occur mainly in the semiquinone state. Possibilities to explain this result are discussed.

EPR signals from components functioning on the electron donor side of Photosystem II (PS II) have been monitored in PS II membranes isolated from spinach chloroplasts after treatment with trypsin at pH 7.5 and pH 6.0. The following information has been obtained. (1) The multiline manganese signal, the g = 4.1 signal and Signal IIslow are lost with trypsin treatment at pH 7.5, but not at pH 6.0. (2) At pH 7.5 the multiline S2 signal and the g = 4.1 signal are lost with approximately the same dependency on the incubation time with trypsin. At pH 6.0 trypsin treatment is known to block electron transfer between QA and QB (the first and the second quinone electron acceptors, respectively) allowing only a single turnover to occur. Under these conditions both the g = 4.1 signal and the multiline signal are induced by illumination at 200 K and their amplitudes are almost the same as in untreated samples. These results are interpreted as indicating that the g = 4.1 signal arises from a side path donor or from S2 itself rather than a carrier functioning between the S states and the reaction center as previously suggested. (3) Cytochrome b-559 is converted to its oxidized low-potential form by trypsin treatment at both values of pH. At pH 6.0 the S-state turnover still occurs indicating that the presence of reduced high-potential cytochrome b-559 is not necessary for this process.

The effect of mild trypsination on the system-II reaction pattern of inside-out thylakoids has been analyzed by measurements of oxygen yield, fluorescence induction and laser-pulse-induced absorption changes at 320 and 830 nm. The following was found. (1) The average oxygen yield per flash drastically declines after trypsination at pH 7.4, while at pH 6.5 only small effects are observed. (2) The area over the fluorescence induction curves becomes reduced by 30–40% after trypsination at either pH 6.5 or 7.4, but in the latter case the maximum level is attained only after addition of hydroxylamine as PS-II donor. On the other hand, the area over the induction curve in the presence of DCMU, which is 10–12 times smaller than without DCMU, remains unaffected by trypsin treatment. (3) The oscillation pattern of the oxygen yield induced by a flash train in dark-adapted inside-out thylakoids is not markedly affected by trypsin treatment, even at more than 80% inhibition of the oxygen-evolving capacity. (4) After trypsination of inside-out thylakoids, a large 10 μs decay arises in the relaxation kinetics of the 830 nm absorption changes, whereas the 320 nm absorption changes are dominated by a rather slow decay. (5) The half-life time of the microsecond kinetics at 830 nm elicited by trypsination of inside-out thylakoids reveals almost the same pH dependence as the corresponding relaxation kinetics in Tris-washed inside-out thylakoids. (6) The relaxation kinetics of the absorption changes at 320 and 830 nm in Tris-washed inside-out thylakoids become significantly modified after trypsin treatment. Based upon these findings, it is concluded that beyond the well-characterized polypeptides with 16, 23 and 33 kDa there exists a further protein that is exposed to the inner side of the thylakoid and that affects the electron transport in system II. The nature and the physiological role of this polypeptide still remain to be elucidated.

Based on the functional organization scheme of system-II-electron transport and its modification by different procedures a proteinaceous component enwrapping the redox components (plastoquinone molecules) of the acceptor side (thereby acting as regulatory element) is inferred to be the unique target for herbicidal interaction with system II. This proteinaceous component, which is attacked by trypsin, provides the receptor sites for the herbicides. Studies of the release kinetics in trypsinated chloroplasts of the inhibition of oxygen evolution with K3 [Fe (CN)6] as electron acceptor indicates, that there exists a binding area with different specific subreceptor sites rather than a unique binding site for the various types of inhibitors. Furthermore, trypsination of the proteinaceous component enhances the efficiency of the plastoquinone pool to act as a non-photochemical quencher for excitation energy.

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to be the most powerful compound among ADRY agents (Renger, G. (1972) Biochim. Biophys. Acta 256, 42–439), on the decay kinetics of the redox states S2 and S3 of the water-oxidizing enzyme system and on the donor component D1 in Tris-washed chloroplasts has been investigated. The following was found. (a) In isolated spinach chloroplasts without ANT 2p, the decay of S3 and S2 can be completely described by a sequence of one-electron-transfer steps: S3→S2→S1. (b) In the dark state, S0 is slowly auto-oxidizable with a half-life of more than 5 min in isolated spinach chloroplasts without ANT 2p. (c) Only one ANT 2p molecule per 40 reaction centers of system II is sufficient to induce a significant acceleration of the S2 and S3 decay. (d) The dependence on ANT 2p concentration of S2- and S3-decay rates can be consistently described by a simple kinetic scheme, assuming that ANT 2p acts as a very mobile catalyst within the thylakoid membrane reacting with the water-oxidizing enzyme system independent of its redox state forming a transient complex. The S2 and S3 decay induced by ANT 2p occurs via a one-electron-transfer sequence in normal chloroplasts. (e) The numerical evaluation of the scheme reveals a high ANT 2p affinity for the water-oxidizing enzyme system, with a dissociation constant of the order of 1 · 10−7 M−1. This value closely corresponds with an estimation based on thermoluminescence measurements (Renger, G. and Inoue, Y. (1983) Biochim. Biophys. Acta 725, 146−154). The kinetic parameters also suggest a rapid exchange of ANT 2p between the binding sites. (f) Fluorescence measurements obtained in normal chloroplasts in the presence of DCMU led to rate constants for the S2 decay which correspond to values derived from oxygen yield data. The ANT-2p-induced reduction of DOX1, the donor to P-680+, was inferred to occur with a larger rate constant than the S2 decay. Based on these data, ANT 2p is confirmed to be the most efficient reactant with the water-oxidizing enzyme system known so far which allows a very specific kinetic labeling of redox states S2 and S3.

The possibility to determine the difference spectra Δεi+1jλ of each univalent redox step Si→Si+1(i=0,...3) of the water-oxidizing enzyme system was analyzed by theoretical calculations and by measurements of 320 nm absorption changes induced by a train of saturating laser flashes (FWHM:7 ns) in PS II membrane fragments. It was found: a) Lipophilic quinones complicate the experimental determination of optical changes due the Si-state transitions because they lead to an additional binary oscillation probably caused by a reductant-induced oxidation of the Fe(2+) at the PS II acceptor side. b) In principle, a proper separation can be achieved at sufficiently high K3[Fe(CN)6] concentrations. c) An unequivocal deconvolution into the difference spectra Δεi+1jλ of flash train-induced optical changes which are exclusively due to Si-state transitions is impossible unless the Kok parameters α, β and [Si]0 can be determined by an independent method.Measurements of the oxygen yield induced by a flash train reveals, that in thylakoids and PS II membrane fragments Si is the stable state of dark adapted samples even at alkaline pH (up to pH=9). However, in PS II membrane fragments at pH7.7 the misses probability α markedly increases, in contrast to the properties of intact thylakoids. Based on these data the possibility is discussed that an equilibrium exists of two types of S2-states with different properties.

Absorption changes at 830 nm, ΔA 830 , induced by repetitive (1 Hz) strong actinic 35 ps laser pulses ( λ = 532 nm; E = 4 · 10 15 photons / cm 2 per pulse) were detected in oxygen-evolving Photosystem (PS) II membrane fragments with a time resolution of about 500 ps. The following results were obtained: (a) in the presence of 2 mM K 3 (Fe(CN) 6 ) the relaxation of ΔA 830 is dominated by the multiphasic P-680 + -reduction kinetics (ns and μs range) and the fast I ⨪ reoxidation, (b) if the reaction centers are kept largely in the reduced state P-680IQ ⨪ A , the initial amplitude of ΔA 830 remains almost unaffected, but the decay mainly occurs via a kinetics of at most 500 ps, (c) if the reaction centers are transformed into the state P-680I ⨪ Q ⨪ A by strong illumination in the presence of Na 2 S 2 O 4 , then the initial amplitude of ΔA 830 becomes drastically diminished, (d) after a few cycles of strong continuous actinic light and dark recovery (4 min) ΔA 830 is dominated by a 3 ns relaxation kinetics. Comparative measurements of the fluorescence decay after excitation with weak 620 nm pulses ( E = 2 · 10 10 photons / cm 2 per pulse) exhibited the following dependencies of the kinetics of the redox state of the reaction center: up to 200 ps kinetics dominate for the states P-680IQ A and for P-680I ⨪ Q ⨪ A , whereas for the state P-680IQ ⨪ A the fluorescence relaxes predominantly via 1.3 ns kinetics. The analysis of these results led to the following conclusions. (i) The PS II reaction center provides a rather shallow trap for excitons. (ii) The fluorescence emission in the state P-680IQ ⨪ a is probably not a recombination fast delayed light emission but represents a prompt fluorescence of the antenna. (iii) If the yield of flash-induced radical pair formation in reaction centers of the state P-680IQ ⨪ A is significant (at least 25%), then the recombination reaction P-680 + I • Q • A → P-680IQ • A takes place with t 1 2 ⩽ 500 ps .

The influence of UV-B irradiation on photosystem II activities has been investigated using isolated photosystem II membrane fragments from spinach. It was found: (a) The average amount of DCIP reduced per flash declined drastically with increasing irradiation time in the absence of DPC but remained almost unaffected in its presence, (b) After UV-B irradiation, the maximum amplitude of laser flash induced 830 nm absorption changes decreases only slightly; whereas the relaxation kinetics exhibit marked effects: the (JLS components dominate the decay at the expense of ns components. The γ.s kinetics already arise after illumination with a single flash of dark adapted samples, (c) The manganese content decreases only partly at irradiation times where the oxygen evolution capacity is almost completely lost, (d) The polypeptide pattern is hardly affected; the number of atrazine binding sites markedly decreases. Based on the results of this study, UV-B irradiation is inferred to deteriorate primarily the function of water oxidation. The action spectrum of the UV-B effect does not reveal a specific target molecule. It is assumed that structural changes of the D-l/D-2 polypeptide matrix are responsible for the modification by UV-B irradiation of the capacity of water oxidation and atrazine binding.

Mild trypsinization of PS II particles at pH 6.0, which almost completely blocks reoxidation of the primary plastoquinone acceptor, Q A − ·Fe 2+ , by endogenous plastoquinone (PQ) as well as by exogenous ( p -BQ) quinones, does not affect the shape or the amplitude of the EPR signal attributed to Q A − ·Fe 2+ . The effect of DCMU on the EPR signal [(1984) FEBS Lett. 105, 156-162] is completely eliminated in mildly trypsinized PS II particles. The lack of effect of mild trypsin treatment on the Q A − ·Fe 2+ microenvironment is briefly discussed in relation to the functional and structural organization of the PS II acceptor side. Photosystem II Semiquinone-iron complex Trypsin Herbicide Photosythesis

The temperature dependence of S-state transition kinetics was measured by spectroscopy in the ultraviolet region with O 2 -evolving Photosystem II particles from a thermophilic cyanobacterium. By proper selection of ferricyanide concentration and the measuring wavelength, the absorption changes due to S-state transitions were explicitly measured with minimized superposition of binary absorption changes due to acceptor side reactions. The half-times of S 1 → S 2 , S 2 → S 3 and S 3 → S 0 transitions were 60, 60 and 800 μs, respectively, at 50°C, the optimal temperature for oxygen evolution by the particles, but were slowed down to 70, 120–150 and 1300 μs at 25°C, and 106, 300 and 5500 μs at 1°C, respectively. In the whole range of 1–50°C the Arrhenius plots showed no break or discontinuity for S 1 → S 2 and S 2 → S 3 transitions, with apparent activation energies of 9.6 and 26.8 kJ/mol, respectively. The Arrhenius plot for S 3 → S 0 transition, however, was composed of two straight lines with a clear break at 16°C, and the apparent activation energies above and below the break temperature were 15.5 and 59.4 kJ/mol, respectively. The implications of these data and especially of the break temperature for the S 3 → S 0 transition were discussed.

As was described previously, an antiserum to polypeptide 11000 inhibited photosynthetic elec­tron transport on the oxygen evolving side of photosystem II. The effect of the antiserum on chloro­plasts from two tobacco mutants also clearly showed that the inhibition site is on the photosystem II-side of the electron transport chain. One of the two tobacco mutants lades the oxygen evolving capacity but exhibits some electron transport with tetramethyl benzidine, an artificial donor to PS II. In this mutant electron transport was barely inhibited. The effect of the antiserum on the primary photoevents showed that the initial amplitude of the absorption change of chlorophyll an at 690 nm and that of the primary electron acceptor X320 at 334 nm both diminished in the presence of the antiserum. Both signals were restored upon addition of diphenylcarbazide another artificial donor to photosystem II. Comparison of the degree of inhibition on the amplitudes of the fast and slow components of the 690 nm absorption change with the manometrically measured inhibition of electron transport shows that besides a full inactivation of a part of the reaction centers of photosystem II another part apparently mediates a fast cyclic electron flow around photosystem II as reported by Renger and Wolff earlier for tris-treated chloroplasts. Moreover, the antiserum affects the low temperature fluorescence in a way which is opposite to Murata’s effect of the Mg2+ -ion induced inhibition of energy spill-over from photosystem II to photosystem I. The antiserum under the condition in which the Hill reaction is inhibited lowered the 686 nm emission and enhanced the 732 nm emission which indicates an enhanced energy spill-over to photosystem I.

The effect of a-bromo-α-benzylmalodinitril (BBMD) on the oxygen evolution and on the absorption changes at 515 nm and 704 nm has been investigated in spinach chloroplasts. It has been found: 1. Under repetitive flash excitation conditions, where the back reaction around system II is practically excluded for kinetical reasons, BBMD does not restore the 515 nm absorption change in DCMU poisoned chloroplasts. 2. Under single flash excitation conditions, where the back reaction around system II becomes prominent in the presence of DCMU, BBMD moderately inhibits this back reaction. The deleterious effect is pronounced by preillumination with short flashes during the BBMD incubation period of the chloroplasts in the absence of DCMU. 3. Incubation of the chloroplasts with BBMD leads to an activity loss of oxygen evolution which increases with the time td between the repetitive short excitation flashes and with the dark incubation time. Preillumination during the incubation period with BBMD significantly enhances the effect. 4. In the absence of artificial electron acceptors BBMD suppresses in DCMU poisoned chloroplasts the 704 nm absorption change reflecting an internal cyclic electron flow around system I. On the other hand the linear electron transport at system I mediated by DCIP plus ascorbate as electron donor couple and benzylviologen as electron acceptor is not disturbed by BBMD. 5. BBMD incubation of chloroplasts accelerates the decay rate of the field indicating 515 nm absorption change. Based on these experimental findings the conclusion has been drawn, that - in contrast to the assumption of Brandon and Elgersma (Biochim. Biophys. Acta 292, 753 - 762 [1973]) - BBMD does not accept electrons from the primary electron acceptor X 320 of system II in DCMU poisoned chloroplasts. BBMD rather acts as a system I electron acceptor. Furthermore, BBMD exerts deleterious effects on the center of photosystem II, accompanied by a weak ADRY-effect on the water-splitting enzyme system Y. As a tentative explanation of the BBMD-action on system II it is assumed that BBMD transforms the photochemical centers of system II into dissipative energy sinks

The 688 nm absorption changes (ΔA688), indicating the photochemical turnover of chlorophyll aII (Chl aII) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a+II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a+II becomes re-reduced in the microsecond range.

Die Spaltung von Wasser durch Sonnenlicht in Sauerstoff und metabolisch gebundenen Wasserstoff bei der Photosynthese ist von zentraler Bedeutung fur die Existenz hoher organisierter Lebewesen auf der Erde. Die Realisierung dieses Prozesses in biologischen Organismen ermoglichte es nicht nur, das riesige Wasserreservoir unseres Planeten als Substrat fur die Sonnenenergie zu nutzen, sondern fuhrte zugleich auch zur Bildung einer aeroben Atmosphare. Der so bereitgestellte Sauerstoff ist ein Reagens, das einen energetisch auserst effizienten Nahrstoffumsatz bewirkt. In den letzten Jahren sind betrachtliche Fortschritte im Verstandnis der funktionellen und strukturellen Organisation der photosynthetischen Wasserspaltung erzielt worden. Dieser Beitrag versucht, einen Uberblick uber unsere heutigen Vorstellungen zu geben, wobei der Schwerpunkt auf Aspekten der Realisierung eines Teilschrittes der Wasserspaltung in biologischen Systemen liegt: der Wasseroxidation zu O2.

The interaction of halogenated p-benzoquinones with PS II has been analyzed by measurements of fluorescence induction curves and the average oxygen yield per flash in isolated class II chloroplasts. It was found: 1)The normalized area over the fluorescence induction curve in the presence of DCMU. A/Fmax, markedly increases if halogenated p-benzoquinones are added before DCMU. The effect is eliminated by DCMU addition prior to that of the quinones. 2)The extent of A/Fmax increases with increasing dark time between the additions of 2,3.5-tri- bromo-6-methyl-1,4-benzoquinone (TBTO) and DCMU. respectively. 3) Some of the halogenated p-benzoquinones were found to act as efficient electron acceptors under repetitive excitation at low flash frequency (2 Hz). In the case of TBTO and 2,3-di-chloro-5-t-butyl-1,4-benzoquinone (TBU 13) the sensitivity of the average oxygen yield per flash was shifted towards higher concentrations of DCMU. 4) Halogenated p-benzoquinones can also affect the stability of oxidizing redox equivalents in the water oxidizing enzyme system Y. This effect depends on the nature of the substituents. The present results are interpreted by the assumption of a covalent binding of halogenated p-benzoquinones in the vicinity of Qᴀ. This binding is prevented by DCMU. The possibility of allosteric interaction between the donor and acceptor side of PS II is discussed.

The applicability of fluorescence measurements for the detection of herbicide effects in whole leaves was analyzed. Based on the results known for isolated chloroplasts, normalized variable fluorescence of the initial rise was shown to be an appropriate tool for monitoring effects of photosystem II (PS II) herbicides. Equipment is described for monitoring the degree of inhibition by fluorescence induction measurements and microcomputer data analysis. The method is used to study the effect of pyrazon [5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone], BAY DRW 1139 [4-amino-3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one], and phenmedipham {3-[(methoxycarbonyl)amino] phenyl (3-methyl-phenyl)carbamate} after foliar application to different species. A rapid decrease of normalized variable fluorescence indicates penetration into leaf cells of all species tested. During a 5- to 7-day experiment, the apparent variable fluorescence decreased continuously in herbicide-susceptible plants, while it recovered in resistant plants due to an internal detoxification mechanism. The described method provides a rapid, simple, and nondestructive tool for analyzing the kinetics of penetration and detoxification of PS II herbicides in whole leaves.

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the QB-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of QA (possibly also at the QB-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.

Photosystem II particles have been isolated and purified from the thermophilic cyanobacterium Mastigocladus laminosus after sulfobetaine treatment. The light-induced steady-state electron transport, flash-induced absorption changes at 830 nm and 325 nm, reflecting the turnover of P-680 and of the acceptor side, respectively, have been measured. In the presence of artificial electron acceptors the preparations showed high O2-evolution activities (e.g., 3000–4000 μmol O2 per mg Chl a per h with phenyl-p-benzoquinone) which were DCMU-sensitive. O2-evolution activity was stable in the presence of various cations (10 mM) and of glycerol (25% (v/v) in the suspending medium. Furthermore, the activity was stable up to 60°C and maximum rate of the electron transport was observed at 50°C. The spectrum of the particles showed a chlorophyll a maximum at approx. 673 nm, some carotenoids were still present but phycocyanin was completely removed. After SDS-polyacrylamide gel electrophoresis the preparations contained mainly nine protein bands at 110, 105, 60, 55, 35, 33, 22, 18 and 17 kDa. Under repetitive laser flash excitation the P-680+-reduction kinetics contained components in the nanosecond and microsecond range with an absorption rate of approx. 1:1. When PS-II particles were purified at room temperature by anion-exchange chromatography nanosecond relaxation kinetics were observed even though the particles did not evolve oxygen. The gel analysis for this preparation showed mainly four protein bands at 60, 55, 35 and 18 kDa.

The protonization pattern of the endogenous donor component D1 which feeds electrons directly into chl-a+II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D1-oxidation, depending on the time between the flashes. The kinetics of D1-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D1 is protonized in the reduced state with pK(Dox1) < 5 and becomes deprotonized in the oxidized state with pK(Dred1) ≳ 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.

The 690 nm absorption change reflecting the turnover of the reaction center chlorophyll of System II, chlorophyll a II , (see Doring, G., Renger, G., Vater, J. and Witt, H. T. (1969) Z. Naturforsch. 31c, 712–721 and Glaser, M., Wolff, Ch., Buchwald, H. E. and Witt, H. T. (1974) FEBS. Lett. 42, 81–85) has been investigated as a function of pH of the chloroplast suspension. It was found: 1. (1) In the range 6.0 ⩽ pH ⩽ 8.0 the amplitude of the measured chlorophyll a II absorption change, ΔA 0 (chlorophyll- a II ), markedly declines with increasing pH, whereas the average oxygen yield per flash remains nearly unaffected. 2. (2) The dependency on actinic flash intensity of the amplitudes ΔA 0 (chlorophyll- a II ) at pH 6.0 closely resembles that for the amplitudes of the chlorophyll a I absorption change. On the contrary, at pH 7.5 a higher flash intensity is required for half saturation of ΔA 0 (chlorophyll- a II ), whereas the corresponding curve for ΔA 0 (chlorophyll- a I ) is practically invariant to pH. 3. (3) Addition of ionophores at concentrations sufficiently high for a complete collaps of proton gradients across the thylakoid membrane shifts the pH-dependence of ΔA 0 (chlorophyll- a II ) by more than 2 units towards the acidic range. On the basis of a hypothesis presented very recently (Glaser, M., Wolff, Ch. and Renger, G. (1976) Z. Naturforsch. 31c, 712–721) it is assumed, that the reduction in the dark of photooxidized chlorophyll a II + is regulated by the proton concentration in the inner thylakoid space. At lower pH the reaction proceeds predominantly via the well known 35 μs kinetics, whereas at higher pH a very rapid kinetics (⩽ 1 μs ), too fast to be resolvable by our measuring device, additionally contributes to a significant degree to the overall reduction of chlorophyll a II + . Furthermore, on the basis of Lavorels model (Lavorel, J. (1975) in Bioenergetics of Photosysthesis (Govindjee, ed.), pp. 223–317, Academic Press, New York) the close interrelationship between ΔA 0 (chlorophyll- a II ) and delayed fluorescence intensity in the μs range is shown to be interpretable mainly by kinetical rather than by energetical effects.

Many inhibitors prevent the oxidation of the primary electron-accepting quinone (QA) by the secondary quinone (Qв) in photosystem II by displacement of Qв from its binding site. On the other hand, plastoquinone-1 and 6-azido-5-decyl-2,3-dim ethoxy-p-benzoquinone displace herbicides. Binding studies show the herbicide/quinone interaction to be (apparently) competitive. The herbicide binding is influenced differentially by various treatments. In this paper it is shown that the affinity of, for example, bromoxynil is decreased by thylakoid unstacking or by light-or reductant-induced reduction of certain thylakoid components, whereas atrazine affinity remains unchanged. Furthermore, absence of HCO- 3 in the presence of form ate leads to an affinity decrease of bromoxynil and atrazine, but to an increase in i-dinoseb affinity. Other differential photosystem II herbicide effects are known from the literature. Since different and unrelated groups of Q- A oxidation inhibitors have been found, and because of the above-mentioned dissimilarities in binding characteristics for different inhibitor groups, the hypothesis of non-identical, but “overlapping” binding sites for different herbicide groups and the native quinone must be more extensively defined. In this manuscript we evaluate both the competitive herbicide/quinone binding model, and a model in which binding of one ligand alters the protein conformation resulting in a dramatic decrease in the binding affinity of ligands from other chemical groups; in this model ligands from the same or related chemical groups bind competitively. Thus, the latter model proposes that only one herbicide or quinone molecule can be bound with high affinity to the herbicide/quinone binding environment, but it depends on the chemical structure of the ligands whether the binding interaction between two ligands is truly competitive or more indirect (allosteric), mediated through the protein conformation.

In the present study the effect of TBTQ on PS II and its mutual interaction with DCMU was analyzed by measurements of the oxygen yield oscillation pattern and of DCMU binding. It was found: 1)TBTQ in its reduced form is able to induce the reduction of Dox which gives rise to an accelerated decay of S2 and S3 of the wateroxidizing complex. 2) Triton X-100 treatment used for isolation of PS II membrane fragments does not significantly affect the lateral mobility of p lastoquinone within the membrane. TBTQ bound to the thylakoid membrane does not enhance the electron pool capacity in PS II membrane fragments. 3) Preincubation of thylakoids with TBTQ diminishes the blockage of O2-evolution by DCMU significantly. In correspondence with previous findings [18, 19] the effect strongly depends on the order of addition of TBTQ and DCMU . 4) Excitation with a single saturating flash causes enhanced DCMU binding in TBTQ pretreated samples leading to the inhibition of flash induced oxygen evolution. The rate of the latter process depends on the DCMU concentration. 5) In thylakoids pretreated in the d ark with TBTQ the oxygen yield of the 3rd flash slowly declines as a function o f dark incubation time at constant DCMU concentration. Based on the above mentioned findings it is inferred that a mutual interaction between TBTQ and DCMU takes place at the PS II acceptor side. Two alternative mechanisms are discussed: i) TBTQ tightly (covalently?) bound at the QB-site (or very close to it) is modified in its function by DCMU via structural effects (allosteric type), or ii) there occurs a TBTQ /DCMU exchange that is fast in the light and slow in the dark.

Spinach (Spinacia oleracea L. cv. Matador) chloroplasts were irradiated with several levels of UV-B radiation. Measurements which reflect characteristic steps of photosynthetic electron transport were made to localize the site of impairment of photosynthesis by UV-B radiation. Variable fluorescence, the μs-kinetics of the 320 nm absorption changes and also oxygen evolution were substantially reduced in chloroplasts irradiated with UV-B. It was not possible to restore the amplitude of the 320 nm absorption changes nor the signal of the transmembrane electric field measured at 520 nm by adding the photo-system II donor couple hydroquinone/ascorbate to UV-B treated chloroplast samples. This indicates that impairment of photosystem II activity is not caused by selective inhibition of the water-splitting enzyme system Y, but rather is due to blockage of photosystem II reaction centers. Photosystem 1 is inferred to be highly resistant to UV-B radiation. These results suggest that the reaction centers of photosystem II are transformed into dissipative sinks for excitation energy by action of UV-B radiation.

Excitation of isolated thylakoids with sufficiently strong actinic light increases the fluorescence quantum yield up to a maximum level, Fmax, followed by a slower decline under certain experimental conditions. In this study the latter effect was analyzed as a function of the ambient redox potential and the actinic light intensity. Two different types of fluorescence decrease were found. (a) In the presence of specific quinones widely used as redox mediators a fast and comparatively small decrease (30% of Fmax), referred to as ΔFSQ, was observed at moderate redox potentials (−300

Laser-flash-induced absorption changes at 830 nm, fluorescence-induction curves and the average oxygen yield per flash have been measured in spinach Photosystem II membrane fragments as a function of trypsin treatment and its modification by CaCl2. The following was found. (i) The relative contribution of the nanosecond relaxation to the overall decay kinetics of 830 nm absorption changes reflecting the P-680+-reduction decreases as a function of incubation time with trypsin. Simultaneously, mild treatment at pH = 6.0 markedly increases the extent of 200 μs kinetics that highly revert back to nanosecond kinetics by CaCl2 addition. After harsher trypsin treatment (pH = 7.5) pH-dependent 2–20 μs kinetics appear that cannot be reverted to nanosecond kinetics by CaCl2. (ii) The CaCl2-induced restoration of nanosecond kinetics is mainly due to a Ca2+-induced effect rather than to a functional role of Cl−. Sr2+ can substantially substitute for Ca2+, whereas Mg2+, Mn2+ and monovalent ions are almost inefficient. (iii) A quantitative correlation between the extent of the nanosecond kinetics and the average oxygen yield per flash was not observed. (iv) If CaCl2 is present in the assay medium for trypsin treatment the samples are markedly protected to proteolytic degradation. This effect mainly refers to the reaction pattern of the acceptor side. Other bivalent cations can substitute Ca2+ for its protective function. (v) The CaCl2-induced protection to proteolytic attack is extremely sensitive to a very short trypsin pretreatment that does hardly affect the shape of the fluorescence induction curve. The results are discussed in relation to the functional and structural organization of Photosystem II.