Storage of abnormal oxidants ‘∑1’, ‘∑2’ and ‘∑3’ in photosynthetic water oxidases inhibited by Cl−-removal

The Cl⁻ requirement of photosynthetic O₂ evolution was studied by thermoluminescence measurements with purified Photosystem II-containing membrane particles from chloroplast thylakoids. When Cl anions had been removed from the particles either by an alkaline shock in a Cl⁻-free medium, or by treatment with SO²⁻₄, the pattern of the thermoluminescence emission after illumination with increasing numbers of flashes suggested that the oxidant storage in the water oxidase could only proceed up to the final step. The final step itself. i.e., the advance to the water-oxidizing S₄ state, apparently was blocked. An upward shift of the emission temperatures of the thermoluminescence bands was seen both in the absence and in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea, indicating that the stored oxidants had redox properties different from those of normal, Cl⁻-sufficient preparations. These properties were readily interconvertible by addition or removal of Cl⁻. We postulate that in Cl⁻-deficient water oxidases abnormal S₁, S₂ and S₃ states, symbolized as ∑₁, ∑₂ and ∑₃, respectively, are formed which are in a Cl⁻-dependent equilibrium with the corresponding normal S states. An oxidation of ∑₃ to a ∑₄ state is not possible. It is proposed that Cl⁻ controls the oxidation potential of the stored oxidants by regulating events associated with the binding and/or oxidative modification of water molecules at the water oxidase.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Chloride relations of photosystem II membrane preparations depleted of,and resupplied with,their 17 and 23 kDa extrinsic polypeptides

Photosystem II membranes prepared from thylakoids of Phytolacca americana chloroplasts were depleted of their intrinsic 17 and 23 kDa polypeptides, and the effects of a reconstitution of these polypeptides on the Cl^– requirements of O₂ evolution activity were analyzed. It was found that the activating effectiveness of limiting amounts of added Cl^– was increased several fold by an addition of the 23 kDa polypeptide. When it was supplemented by the 17 kDa species, only a small additional increase occurred, but Cl^– retention in Cl^– free media was enhanced greatly. Addition of the 17 kDa polypeptide alone was without effect because it is known that it cannot bind to its native site unless the 23 kDa polypeptide is in place.Optimal enhancements of the effectiveness of activating added Cl^– were observed when the assays were done in the presence of the reconstituting polypeptides. When the reconstituting treatment with the polypeptides, and the assay of the Cl^– relations, were separated, it was advantageous to have Cl^– present in the reconstituting medium, and not to add Ca²⁺, another cofactor of photosynthetic water oxidation. Those requirements are attributed to the labilizing effects Cl^– free conditions and divalent cations have on the association of especially the 23 kDa polypeptides with the water oxidizing complex, and to a possible aggregation of the membranes under the influence of Ca²⁺ which might have impeded proper polypeptide binding.Abbreviations Chl Chlorophyll a+b - Mes 4-morpholineethanesulfonic acid - PSII photosystem II - SDS and LDS sodium or lithium dodecylsulfate     

Inhibitors affecting the oxidizing side of Photosystem II at the Ca2+- and Cl−-sensitive sites

In addition to compounds which inhibit the function of calmodulin (a ubiquitous calcium regularity protein found in plants and mammalian tissues), Ca²⁺ or Cl⁻ channel blockers in mammalian tissues were also found to inhibit electron transport in Photosystem II submembrane preparations. Their inhibition was overcome by electron donation to P-680 by diphenylcarbazide (for all of the compounds used) and by H₂O₂ (except with trifluoperazine). Addition of Ca²⁺ and/or Cl⁻ also partially prevented the inhibitory action. We postulate that the inhibitory action occurs at the level of the water-splitting system at the site of Ca²⁺ modulation of the Cl⁻ cofactor requirement for O₂ evolution (as hypothesized in Nakatani, H.Y. (1984) Biochem. Biophys. Res. Comm. 120, 299–304).     

Effects of phosphonium-based ionic liquids on the lipase activity evaluated by experimental results and molecular docking

In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P₄₄₄₄]⁺, [P_444(14)]⁺, [P_666(14)]⁺) and anions (Cl⁻, Br⁻, [Deca]⁻, [Phosp]⁻, [NTf₂]⁻) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]⁻ and [NTf₂]⁻ increase the BCL activity, while the remaining [P_666(14)]-based ILs with the Cl⁻, Br⁻, and [Deca]⁻ anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P_666(14)][NTf₂] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L⁻¹). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]⁻ has a higher binding affinity compared to Cl⁻ and Br⁻, and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]⁻ and [NTf₂]⁻ have high binding energies (−6.2 and −5.6 kcal·mol⁻¹, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

Metastable proton pools in thylakoids and their importance for the stability of Photosystem II

After isolated chloroplast thylakoids have been transferred to a medium which is more alkaline than their storage medium, they retain considerable amounts of unequilibrated protons for often longer than 10 min. Essentially all of these protons are released upon uncoupler addition when the thylakoids are osmotically swollen, but only a portion of them when they are in a shrunken state. Osmotic swelling also greatly accelerates the inactivation of the water-oxidizing system enzyme of Photosystem II, and its depletion of functional Cl^?, at alkaline pH. Analyses of the mestable proton gradient in terms of stoichiometry, temperature dependence, and effect on fluorescent amine probes, suggest that most of the protons involved are bound and exchange readily with the bulk phases only when the thylakoids are swollen. It is concluded that, in shrunken thylakoids, the water-oxidizing enzymes are buried in special H⁺-sequestering domains which probably are formed by cavities in the inner surface of the thylakoid membrane. An observed cooperative action of alkaline pH and divalent cations during Cl^?-extraction from Photosystem II is interpreted as revealing an involvement of both a negatively charged surface region and positively charged groups in maintaining the functional integrity of the site of water oxidization.     

The state of manganese in the photosynthetic apparatus: 5. The chloride effect in photosynthetic oxygen evolution. Is halide coordinated to the EPR-active manganese in the O2-evolving complex? Studies of the substructure of the low-temperature multiline EPR signal

The role of chloride in the manganese-containing oxygen-evolving complex of Photosystem II has been studied by observing the amplitude of the multiline EPR signal as a function of Cl⁻ concentration or when Cl⁻ is replaced by Br⁻ or F⁻. The correlation of the multiline EPR signal intensity and O₂ activity with the concentration of Cl⁻ shows that chloride is involved in oxygen evolution at the S₂ or earlier S states, and that it is necessary for the production of an EPR-detectable S₂ state. We have developed a new method for the preparation of subchloroplast PS II particles containing Br⁻ and F⁻) and have used these particles for studying the EPR fine structure at high resolution. The fine structure shows a multiplet of 4–6 lines with 10–15 G spacing; at the resolution of our experiment there are no significant differences between the Cl⁻-and Br⁻-containing samples, suggesting that the halide is not a ligand of the EPR-active Mn. Various structural possibilities for the Mn complex, which would account for the observed fine structure of the multiline EPR spectrum are discussed.     

Photoreduction of pheophytin as a result of electron donation from the water-splitting system to Photosystem-II reaction centers

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease of chlorophyll-fluorescence yield is observed in subchloroplast oxygen-evolving preparations of Photosystem II (PS II) under anaerobic conditions. This photoreaction is activated at addition of CCCP, inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and reactivated upon subsequent addition of ascorbate. Benzyl viologen as well as methyl viologen accelerates dark oxidation of reduced pheophytin, indicating that they are able to accept an electron from Pheo^⨪. The data on both the photoreduction of pheophytin in the absence of exogenous reductants - when electron donation to reaction centers of PS II occurs only from water - and the inhibition of this photoreaction by DCMU show that the pheophytin photoreduction is sensitized by reaction centers of PS II, and it probably occurs as a result of electron donation from the water-splitting system being in the sate S₃ to P^⨥-680Pheo^⨪Q⁻, producing the long-lived state S₀ P-680Pheo^⨪Q⁻ and O₂. Photoreduction of pheophytin in the presence of ascorbate (and dithionite) evidently occurs as a result of donation of its electrons to P^⨥-680Pheo^⨪Q⁻ by means of the S-states of the water-oxidizing system. It is shown that the photoinduced decrease of fluorescence in chloroplasts under anaerobic conditions is due to two processes: photoreduction of pheophytin in Photosystem II and photooxidation of Q⁻ by Photosystem I. It is suggested that photoreduction of pheophytin takes also place under aerobic conditions when Q is reduced. It may contribute to the P−S fluorescence decrease during fluorescence induction in leaves.     

The sensitivity of Photosystem II to damage by UV-B radiation depends on the oxidation state of the water-splitting complex

The water-oxidizing complex of Photosystem II is an important target of ultraviolet-B (280-320 nm) radiation, but the mechanistic background of the UV-B induced damage is not well understood. Here we studied the UV-B sensitivity of Photosystem II in different oxidation states, called S-states of the water-oxidizing complex. Photosystem II centers of isolated spinach thylakoids were synchronized to different distributions of the S₀, S₁, S₂ and S₃ states by using packages of visible light flashes and were exposed to UV-B flashes from an excimer laser (λ = 308 nm). The loss of oxygen evolving activity showed that the extent of UV-B damage is S-state-dependent. Analysis of the data obtained from different synchronizing flash protocols indicated that the UV-sensitivity of Photosystem II is significantly higher in the S₃ and S₂ states than in the S₁ and S₀ states. The data are discussed in terms of a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.     

The function of 33-kDa protein in the photosynthetic oxygen-evolution system studied by reconstitution experiments

Treatment of Photosystem II particles with 1.2 M CaCl₂ released three proteins of 33, 24 and 18 kDa of the photosynthetic oxygen evolution system, but left Mn bound to the particles as demonstrated by Ono and Inoue (Ono, T. and Inoue, Y. (1983) FEBS Lett. 164, 252–260). Oxygen-evolution activity of the CaCl₂-treated particles was very low in a medium containing 10 mM NaCl as a salt, but could be restored by the 33-kDa protein. When the particles were incubated in 10 mM NaCl at 0°C, two of the four Mn atoms per oxygen-evolution system were released with concomitant loss of oxygen-evolution activity. The 33-kDa protein suppressed the release of Mn and the inactivation during the incubation. These findings from reconstitution experiments suggest that the 33-kDa protein acts to preserve Mn atoms in the oxygen-evolution system. The 33-kDa protein could be partially substituted by 100 or 150 mM Cl⁻ for the preservation of the Mn and oxygen-evolution activity. The Mn in Photosystem II particles enhanced rebinding of the 33-kDa protein to the particles.     

The role of chloride in O2 evolution by thylakoids from salt-tolerant higher plants

(1) Thylakoids isolated from leaves of two salt-tolerant higher plant species were found to require high (greater than 250 mM) concentrations of Cl⁻ for maximal rates of photosynthetic O₂ evolution and maximum variable chlorophyll a fluorescence yield. These activities were also tolerant to extremely high (2–3 M) salt concentrations. Their pH dependence was markedly different in the absence and presence of sufficient salt levels. (2) When Cl⁻ was provided as CaCl₂, as opposed to MgCl₂, KCl or NaCl, higher rates of O₂ evolution were obtained, suggesting that Ca²⁺ has an important role in Photosystem II reactions. (3) The site of Cl⁻ action was located on the electron donor side of Photosystem II. (4) O₂ evolution in the presence of optimal Cl⁻ concentrations showed a pH dependence closely matched by that of ³⁵Cl-NMR line broadening, which is indicative of Cl⁻ binding. This pH-dependent ³⁵Cl-NMR line-width broadening was not altered significantly by treatment of the thylakoids with EDTA; it was, however, abolished by heat treatment. (5) Only anions with similar ionic radii (Br⁻, NO⁻₃) were effective in replacing Cl⁻. Small anions such as F⁻ and OH⁻ were inhibitory; larger ions had no effect. The inhibition by F⁻ is due, at least in part, to displacement of Cl⁻. The selectivity is attributed to a combination of steric and ionic field effects. (6) It is proposed that Cl⁻ facilitates Photosystem II electron transport by reversible ionic binding to the O₂-evolving complex itself or to the thylakoid membrane in close proximity to it.     

Interaction of molecular oxygen with the donor side of photosystem II after destruction of the water-oxidizing complex

Photosystem II (PSII) is a pigment-protein complex of thylakoid membrane of higher plants, algae, and cyanobacteria where light energy is used for oxidation of water and reduction of plastoquinone. Light-dependent reactions (generation of excited states of pigments, electron transfer, water oxidation) taking place in PSII can lead to the formation of reactive oxygen species. In this review attention is focused on the problem of interaction of molecular oxygen with the donor site of PSII, where after the removal of manganese from the water-oxidizing complex illumination induces formation of long-lived states (P680^+· and TyrZ^·) capable of oxidizing surrounding organic molecules to form radicals.     

Interactions between permeant and blocking anions inside the CFTR chloride channel pore

Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is antagonized by extracellular Cl⁻. In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl⁻ (and other anions) and cytoplasmic Pt(NO₂)₄^2 − ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO₂)₄^2 − binding affinity also greatly strengthened antagonistic Cl⁻:blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO₂)₄^2 − ions but also with extracellular Cl⁻, and that altered blocker Cl⁻- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl⁻ binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl⁻ and Pt(NO₂)₄^2 − to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl⁻ binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl⁻ permeation, are discussed.     

The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii

Light-activated hydrogen and oxygen evolution as a function of CO₂ concentration in helium were measured for the unicellular green alga Chlamydomonas reinhardtii. The concentrations were 58, 30, 0.8 and 0 ppm CO₂. The objective of these experiments was to study the differential affinity of CO₂/HCO ₃ ^- for their respective Photosystem II and Calvin cycle binding sites vis-à-vis photoevolution of molecular oxygen and the competitive pathways of hydrogen photoevolution and CO₂ photoassimilation. The maximum rate of hydrogen evolution occurred at 0.8 ppm CO₂, whereas the maximum rate of oxygen evolution occurred at 58 ppm CO₂. The key result of this work is that the rate of photosynthetic hydrogen evolution can be increased by, at least partially, satisfying the Photosystem II CO₂/HCO ₃ ^- binding site requirement without fully activating the Calvin-Benson CO₂ reduction pathway. Data are presented which plot the rates of hydrogen and oxygen evolution as functions of atmospheric CO₂ concentration in helium and light intensity. The stoichiometric ratio of hydrogen to oxygen changed from 0.1 at 58 ppm to approximately 2.5 at 0.8 ppm. A discussion of partitioning of photosynthetic reductant between the hydrogen/hydrogenase and Calvin-Benson cycle pathways is presented.Abbreviations PET photosynthetic electron transport - PS Photosystem     

Stability and oscillation properties of thermoluminescent charge pairs in the O2-evolving system depleted of Cl− or the 33 kDa extrinsic protein

The effects of Cl⁻ depletion and removal of the 33 kDa extrinsic protein on the charge stabilization in O₂-evolving Photosystem II (PS II) particles were studied by curve fitting and deconvolution of thermoluminescence bands. The following results were obtained. (1) Cl⁻ depletion reversibly decreases the redox potential of the S₂ state by 60–80 mV, and thereby elevates the recombination temperature of both S₂Q⁻_B and S₂Q⁻_A charge pairs. (2) Removal of the 33 kDa extrinsic protein specifically elevates the recombination temperature of the S₂Q⁻_A charge pair, with practically no effect on the S₂Q⁻_B pair. This was tentatively interpreted as showing that the protein removal decreases the redox potential of both S₂ and Q⁻_B, but not of Q⁻_A, and, thus, the effects are mutually cancelled for the S₂Q⁻_B pair, but are manifested for the S₂Q⁻_A pair. (3) Deconvolution of glow curves demonstrated that S₃ is not formed in Cl⁻-depleted PS II, but is formed in 33 kDa protein-depleted PS II even at a low (20 mM) Cl⁻ concentration. Analysis of thermoluminescence oscillations confirmed that Cl⁻ depletion interrupts S₂-S₃ transition, whereas the protein removal interrupts S₃-(S₄)-S₀ transition at mM Cl⁻. (4) Cl⁻ depletion by SO²⁻₄ replacement in the absence of 33 kDa protein affected thermoluminescence in a different way from that in the presence of the protein. Based on these findings, the properties of charge pairs in the Cl⁻-depleted PS II particles were discussed in relation to the role of the 33 kDa extrinsic protein.     

Inhibition of the catalase reaction of Photosystem II by anions

A new binding site for anions which inhibit the water oxidizing complex (WOC) of Photosystem II in spinach has been identified. Anions which bind to this site inhibit the flash-induced S₂/S₀ catalase reaction (2H₂O₂2H₂O+O₂) of the WOC by displacing hydrogen peroxide. Using a mass spectrometer and gas permeable membrane to detect the ³²O₂ product, the yield and lifetime of the active state of the flash-induced catalase (to be referred to simply as flash-catalase) reaction were measured after forming the S₂ or S₀-states by a short flash. The increase in flash-catalase activity with H₂O₂ concentration exhibits a K_m=10–20 mM, and originates from an increase in the lifetime by 20-fold of the active state. The increased lifetime in the presence of peroxide is ascribed to formation of the long-lived S₀-state at the expense of the unstable S₂-state. The anion inhibition site differs from the chloride site involved in stimulating the photolytic water oxidation reaction (2H₂OO₂+4e^-+4H⁺). Whereas water oxidation requires Cl^- and is inhibited with increasing effectiveness by F^-CN^-N₃ ^-, the flash-catalase reaction is weakly inhibited by Cl^-, and with increasing effectiveness by F^-CN^-, N₃ ^-. Unlike water oxidation, chloride is unable to suppress or reverse inhibition of the flash-catalase reaction caused by these anions. The inhibitor effectiveness correlates with the pK_a of the conjugate acid, suggesting that the protonated species may be the active inhibitor. The reduced activity arises from a shortening of the lifetime of the flash-induced catalase active state by 3–10 fold owing to stronger anion binding in the flash-induced states, S₂ and S₀, than in the dark S-states, S₁ and S_-1. To account for the paradoxical result that higher anion concentrations are required to inhibit at lower H₂O₂ concentrations, where S₂ forms initially after the flash, than at higher H₂O₂ concentrations, where S₀ forms initially after the flash, stronger anion binding to the S₀-state than to the S₂-state is proposed. A kinetic model is given which accounts for these equilibria with anions and H₂O₂. The rate constant for the formation/release of O₂ by reduction of S₂ in the WOC is <0.4 s^-1.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - BTP bis [tris(hydroxymethyl)methylamino]-propane - CCCP carbonylcyanide m-chlorophenylhyrazone - DCBQ 2,5-dichlorobenzoquinone - DMBQ 2,3-dimethylbenzoquinone - WOC water oxidizing complex