Gernot Renger

ON THE MECHANISM OF PHOTOSYSTEM II DETERIORATION BY UV-B IRRADIATION

Abstract 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.

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Abstract Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y4) is lower compared to Y3 than at lower flash frequency. At 4 Hz, the calculated S0 concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent S 0 ( S 0 + S 1 ) ratio increases to about 0.2. This is explained by a relatively fast donation ( t 1 2 = 1.0–1.5 s ) of one electron by an electron donor to S2 and S3 in 15–25% of the Photosystem II reaction chains. The one-electron donor to S2 and S3 appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal IIs. (ii) The probability for the fast one-electron donation to S2 and S3 has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S2 decay becomes 10-fold faster ( t 1 2 = 5–6 s ) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S2 decay was extremely slow. The S3 decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S2 is reduced mainly by Q−A, whereas S3 is not. (iii) In the absence of CO2/HCO−A and in the presence of formate, the fast one-electron donation to S2 and S3 does not occur. Addition of HCO−3 restores the fast decay of part of S2 and S3 to almost the same extent as in control thylakoids. The slow phase of S2 and S3 decay is not influenced significantly by CO2/HCO−3. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q−A oxidation without interference of QB, were 2.3-fold slower in the absence of CO2/HCO−3 than in its presence. (iv) An almost 3-fold decrease in decay rate of S2 is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q−A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q−B is responsible for this shift of equilibrium Q−A concentration, these data would suggest that the pKa value for Q−B protonation is somewhat higher than 7.6, assuming that the protonated form of Q−B cannot reduce QA.

Studies on the structural and functional organization of system II of photosynthesis. The use of trypsin as a structurally selective inhibitor at the outer surface of the thylakoid membrane

The effect of trypsin on the photosynthetic electron transport has been investigated in the presence of various electron acceptors (benzyl viologen, p-benzo-quinone, K3[Fe(CN)6]) by measurements of flash-induced oxygen evolution and of the absorption changes at 334 nm, indicating the primary electron acceptor of System II, X 320, and at 515 nm, indicating via electrochromism the electrical potential gradient across the thylakoid membrane. It was found that the effect of trypsin is strongly dependent on the nature of the electron acceptor: (1) Oxygen evolution is completely inhibited in the presence of p-benzo-quinone, but remains nearly unaffected by K3[Fe(CN)]6. (2) The initial amplitude deltaAO of the 334 nm absorption change is insensitive to trypsin in the presence of K3[Fe(CN)6], but the absorption change is abolished if benzyl viologen is used as acceptor. (3) The initial amplitude deltaAO of the 515 nm absorption change decreases by trypsin down to 50% with K3[Fe(CN)6] and is completely suppressed with benzyl viologen. (4) In trypsinated chloroplasts, the above-mentioned activities appear to be rather insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in contrast to normal chloroplasts. On the basis of these results it is inferred that the primary electron acceptor of System II, X 320, is covered by a proteinaceous component susceptible to tryptic digestion. In addition, it is postulated that this component acts as well as an allosteric protein responsible for the regulation of the electronic interaction between X 320 and the plastoquinone pool, as for the inhibitory effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Various other possible effects caused by the proteinaceous shield and its modification by trypsin are discussed. The present results are in complete agreement with asymmetric membrane models postulating a zig-zag arrangement of the electron transport chain with the reducing side located towards the outer phase and the oxidizing side near the inner phase of the thylakoids.

Manganese Compounds as Water-Oxidizing Catalysts: From the Natural Water-Oxidizing Complex to Nanosized Manganese Oxide Structures

All cyanobacteria, algae, and plants use a similar water-oxidizing catalyst for water oxidation. This catalyst is housed in Photosystem II, a membrane-protein complex that functions as a light-driven water oxidase in oxygenic photosynthesis. Water oxidation is also an important reaction in artificial photosynthesis because it has the potential to provide cheap electrons from water for hydrogen production or for the reduction of carbon dioxide on an industrial scale. The water-oxidizing complex of Photosystem II is a Mn-Ca cluster that oxidizes water with a low overpotential and high turnover frequency number of up to 25-90 molecules of O2 released per second. In this Review, we discuss the atomic structure of the Mn-Ca cluster of the Photosystem II water-oxidizing complex from the viewpoint that the underlying mechanism can be informative when designing artificial water-oxidizing catalysts. This is followed by consideration of functional Mn-based model complexes for water oxidation and the issue of Mn complexes decomposing to Mn oxide. We then provide a detailed assessment of the chemistry of Mn oxides by considering how their bulk and nanoscale properties contribute to their effectiveness as water-oxidizing catalysts.

Effect of trypsin on PS-II particles. Correlation between Hill-activity, Mn-abundance and peptide pattern

The effect of trypsin treatment on Photosystem-II particles has been investigated by measurements of oxygen evolution, 2,6-dichlorophenolindophenol (DCIP)-reduction and Mn-abundance and by analyzing the peptide pattern. The following results were obtained. (1) Trypsin modifies both the acceptor and donor side of PS II, but striking differences are observed for the pH dependence: whereas the acceptor side is severely attacked between pH 5.5 and 9.0, the destruction of the donor side (oxygen-evolving capacity) by trypsin becomes significant only at pH values higher than 7.25. (2) The pH-dependence of the susceptibility of oxygen evolution to trypsin closely resembles that observed in inside-out thylakoids (Renger, G., Volker, M. and Weiss, W. (1984) Biochim. Biophys. Acta 766, 582–591). (3) The effect of trypsin on the functional integrity of water oxidation cannot be due to an attack on the surface exposed 16 kDa, 24 kDa and 33 kDa polypeptides, because they are digested rapidly even at pH 6.5, where the oxygen-evolving capacity remains almost unaffected. (4) Trypsination of PS-II particles as well as of the isolated 33 kDa protein leads to a 15 kDa fragment. In trypsinized PS-II particles this fragment remains membrane-bound. The amount of the 15 kDa fragment and Mn content are correlated with the oxygen-evolving capcity. These results indicate pH-dependent structural modifications at the donor side of System II which make target proteins accessible to trypsin. The 33 kDa protein is inferred to play a regulatory role in photosynthetic oxygen evolution and this function is realized by only a part of the protein, i.e., the 15 kDa fragment, that remains resistant to mild trypsination.

The action of 2-anilinothiophenes as accelerators of the deactivation reactions in the watersplitting enzyme system of photosynthesis.

Abstract The effect of a number of 2-anilinothiophene derivates on the O2 yield per flash obtained by excitation with repetitive flashes and repetitive double flash groups has been investigated. It was found that: 1. 1. The decrease of the average O2 yield per flash at long times (td) between the flashes caused by the natural deactivation reactions of the watersplitting enzyme system is strongly accelerated by various derivatives of 2-anilinothiophene. 2. 2. The action of these agents as Acceleration of the Deactivation Reactions of the watersplitting Enzyme System Y (ADRY) substances is unequivocally proved. 3. 3. The ADRY effect is dependent on the acidity of the imino group, present in all ADRY agents so far known. These observations are discussed in connection with the mechanism of the activation—deactivation reactions of the watersplitting Enzyme System Y. A new class of photosynthesis effectors is established: the class of the ADRY agents. These substances provide a tool both to label and to modify the oxidizing equivalents which are responsible for the oxidation of water, in a similar way as the uncouplers can be used for the characterization of the high energy state in the phosphorylation process.

Photosystem II: Structure and mechanism of the water:plastoquinone oxidoreductase

This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680+•QA−•; (b) reduction of plastoquinone to plastoquinol at the QB site via a two-step reaction sequence with QA−• as reductant and (c) oxidative water splitting into O2 and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680+• as oxidant and a redox active tyrosine YZ acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of the catalytic sites of PQH2 formation (QB-site) and oxidative water splitting (Mn4OxCa cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative water splitting and the structure of the catalytic sites are far from being satisfactorily answered.

Reactive oxygen species: Re-evaluation of generation, monitoring and role in stress-signaling in phototrophic organisms

This review provides an overview about recent developments and current knowledge about monitoring, generation and the functional role of reactive oxygen species (ROS) - H2O2, HO2, HO, OH(-), (1)O2 and O2(-) - in both oxidative degradation and signal transduction in photosynthetic organisms including microscopic techniques for ROS detection and controlled generation. Reaction schemes elucidating formation, decay and signaling of ROS in cyanobacteria as well as from chloroplasts to the nuclear genome in eukaryotes during exposure of oxygen-evolving photosynthetic organisms to oxidative stress are discussed that target the rapidly growing field of regulatory effects of ROS on nuclear gene expression.

Two sites of photoinhibition of the electron transfer in oxygen evolving and tris-treated PS II membrane fragments from spinach

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/m2), 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.

The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 μM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 μM. 3.) The photoproduction of O2− is coupled with H+-uptake. This effect is diminished by the addition of the O2−-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2− in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.