Fluoride inhibition of photosystem II and the effect of removal of the PsbQ subunit

Photosystem II (PSII), the light-absorbing complex of photosynthesis that evolves oxygen, requires chloride for activation of the oxygen evolving complex (OEC). In this study, fluoride was characterized as an inhibitor of Cl⁻-activated oxygen evolution in higher plant PSII. It was confirmed to be primarily a competitive inhibitor in intact PSII, with Cl⁻-competitive inhibition constant K_i = 2 mM and uncompetitive inhibition constant \textK_\texti^￠ {\text{K}}_{\text{i}}^{\prime }  = 79 mM. A pH dependence study showed that fluoride inhibition was more pronounced at lower pH values. In order to determine the location of the fluoride effect, PSII preparations lacking various amounts of the PsbQ subunit were prepared. The competitive F⁻ inhibition constant and the Michaelis constant for Cl⁻ activation increased with loss of the PsbQ subunit, while the uncompetitive F⁻ inhibition constant was relatively insensitive to loss of PsbQ. The S₂ state EPR signals from PSII lacking PsbQ responded to Ca²⁺ and Cl⁻ removal and to F⁻ treatment similar to intact PSII, with enhancement of the g = 4.1 signal and suppression of the multiline signal, but the effects were more pronounced in PSII lacking PsbQ. Together, these results support the interpretation that the PsbQ subunit has a role in retaining anions within the OEC.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

Effect of the 17- and 23-kilodalton polypeptides, calcium, and chloride on electron transfer in photosystem II

Electron paramagnetic resonance (EPR) measurements were performed on photosystem II (PSII) membranes that were treated with 2 M NaCl to release the 17- and 23-kilodalton (kDa) polypeptides. By using 75 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to limit the photosystem II samples to one stable charge separation in the temperature range of 77-273 K, we have quantitated the EPR signals of the several electron donors and acceptors of photosystem II. It was found that removal of the 17- and 23-kDa polypeptides caused low potential cytochrome b559 to become fully oxidized during the course of dark adaptation. Following illumination at 77-130 K, one chlorophyll molecule per reaction center was oxidized. Between 130 and 200 K, both a chlorophyll molecule and the S1 state were photooxidized and, together, accounted for one oxidation per reaction center. Above 200 K, the chlorophyll radical was unstable. Oxidation of the S1 state gave rise to the S2-state multiline EPR signal, which arises from the Mn site of the O2-evolving center. The yield of the S2-state multiline EPR signal in NaCl-washed PSII membranes was as high as 93% of the control, untreated PSII membranes, provided that both Ca2+ and Cl- were bound. Furthermore, the 55Mn nuclear hyperfine structure of the S2-state multiline EPR signal was unaltered upon depletion of the 17- and 23-kDa polypeptides. In NaCl-washed PSII samples where Ca2+ and/or Cl- were removed, however, the intensity of the S2-state multiline EPR signal decreased in parallel with the fraction of PSII lacking bound Ca2+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of O2 evolution by a wheat photosystem II reaction center complex isolated by a simplified method: disjunction of secondary acceptor quinone and enhanced Ca2+ demand

An O2-evolving photosystem II (PSII) reaction center complex was prepared from wheat by a simple method consisting of octylglucoside solubilization of Triton PSII particles followed by one-step sucrose density gradient centrifugation. The complex contained six species of proteins including the 33-kDa extrinsic protein with the same relative abundance as in the original PSII particles, one cytochrome b559, 4 Mn, and about 40 chlorophyll (Chl) per O2-evolving unit, and evolved O2 at a high rate of 1400-1700 mumol O2/mg Chl/h. O2 evolution by the complex was dependent on acceptor species, showing a hierarchy, ferricyanide greater than dichlorobenzoquinone greater than phenylbenzoquinone greater than dimethylbenzoquinone greater than duroquinone, and insensitive to DCMU, indicative of disjunction of the secondary quinone acceptor of PSII from the electron transport pathway. O2 evolution also showed a marked dependence on Cl- and Ca2+: about 10-fold acceleration by Cl- and an additional 2- to 3-fold by Ca2+. Comparison of the dissociation constants for Cl- and Ca2+ between the complex and NaCl-washed PSII particles revealed that octylglucoside treatment gives rise to a new Ca2+-sensitive site by removal of some unknown factor(s) other than the extrinsic 22- and 16-kDa proteins, while it preserves the Cl(-)-sensitive site as native as in NaCl-washed PSII particles. Analysis of the relationship between Cl- demand and Ca2+ demand revealed that Ca2+ absence noncompetitively inhibits the Cl(-)-supported O2 evolution, indicative of the independence of the binding site of these two factors.     

An enzyme kinetics study of the pH dependence of chloride activation of oxygen evolution in photosystem II

Oxygen evolution by photosystem II (PSII) involves activation by Cl^? ion, which is regulated by extrinsic subunits PsbQ and PsbP. In this study, the kinetics of chloride activation of oxygen evolution was studied in preparations of PSII depleted of the PsbQ and PsbP subunits (NaCl-washed and Na₂SO₄/pH 7.5-treated) over a pH range from 5.3 to 8.0. At low pH, activation by chloride was followed by inhibition at chloride concentrations >100 mM, whereas at high pH activation continued as the chloride concentration increased above 100 mM. Both activation and inhibition were more pronounced at lower pH, indicating that Cl^? binding depended on protonation events in each case. The simplest kinetic model that could account for the complete data set included binding of Cl^? at two sites, one for activation and one for inhibition, and four protonation steps. The intrinsic (pH-independent) dissociation constant for Cl^? activation, K _S, was found to be 0.9 ± 0.2 mM for both preparations, and three of the four pK _as were determined, with the fourth falling below the pH range studied. The intrinsic inhibition constant, K _I, was found to be 64 ± 2 and 103 ± 7 mM for the NaCl-washed and Na₂SO₄/pH7.5-treated preparations, respectively, and is considered in terms of the conditions likely to be present in the thylakoid lumen. This enzyme kinetics analysis provides a more complete characterization of chloride and pH dependence of O₂ evolution activity than has been previously presented.     

The PsbQ protein stabilizes the functional binding of the PsbP protein to photosystem II in higher plants

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and optimize the oxygen evolution reaction by regulating the binding properties of the essential cofactors Ca(2+) and Cl(-). PsbP induces conformational changes around the catalytic Mn cluster required for Ca(2+) and Cl(-) retention, and the N-terminal region of PsbP is essential for this reaction. It was reported that PsbQ partially restores the functional defect of N-terminal truncated PsbP [Ifuku and Sato (2002) Plant Cell Physiol. 43, 1244-1249]; however, the mechanism of this restoration is yet to be clarified. In this study, we demonstrate that PsbQ is able to restore the functional binding of mutated PsbPs. In the presence of PsbQ, ?15-PsbP, a truncated PsbP lacking 15 N-terminal residues, was able to specifically bind to NaCl-washed spinach PSII membranes and significantly restore the oxygen evolving activity. Furthermore, PsbQ was also able to compensate for the impaired ion-retention of H144A-PsbP, in which a conserved histidine at position 144 in the C-terminal domain was substituted with an alanine. Fourier transform infrared (FTIR) difference spectroscopy showed that PsbQ restored the ability of ?15- and H144A-PsbP to induce proper conformational changes during S(1) to S(2) transition. These data suggest that the major function of PsbQ is to stabilize PsbP binding, thereby contributing to the maintenance of the catalytic Mn cluster of the water oxidation machinery in higher plant PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Studies on 17,24 kD Depleted Photosystem II Membranes : I. Evidences for High and Low Affinity Calcium Sites in 17,24 kD Depleted PSII Membranes from Wheat versus Spinach

Analyses were made of the effects of extraction of the 17,24 kilodalton extrinsic proteins from spinach versus wheat photosystem II (PSII) membranes on Ca abundance and O(2) evolution capacity determined in the absence and presence of either Cl(-) or Ca(2+). Extraction of these proteins from spinach PSII routinely diminished steady state O(2) evolution by about 70% when assayed in the presence of sufficient Cl(-). Additionally, O(2) evolution of 17,24 kilodalton-less spinach PSII membranes showed about 2-fold more enhancement by Ca(2+) than by Cl(-) during assay. When the same extraction and assay procedures were applied to wheat PSII membranes, we observed, in contrast to 17,24 kilodalton-less spinach PSII, only about 50% inhibition of O(2) evolution and about 2-fold greater enhancement by Cl(-) than by Ca(2+). Irrespective of differences in the magnitude of enhancement of O(2) evolution by Ca(2+)versus Cl(-) in spinach versus wheat, the K(m) values for Cl(-) (about 1.7 millimolar) and Ca(2+) (about 1.5 millimolar) were similar for both type preparations. The abundance of Ca specifically associated with fully functional PSII (about 2 and about 3 Ca/200 chlorophyll for spinach and wheat, respectively) was diminished to about 1 per 200 chlorophyll upon 17.24 kilodalton protein depletion. Further treatment of wheat 17,24 kilodalton-less PSII in darkness with 2 molar NaCl/1 millimolar ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid/20 micromolar A23187(2) made O(2) evolution highly dependent on Ca(2+) addition, much like the 17,24 kilodalton-less spinach PSII. Analyses of this Ca(2+) effect on O(2) evolution revealed both high (K(m) about 65 micromolar) and low (K(m) about 1.5 millimolar) affinity Ca(2+) sites in wheat 17,24 kilodalton-less PSII. The results suggest that during 17,24 kilodalton extraction by NaCl, spinach PSII is more susceptible than wheat PSII to loss of high affinity Ca and irreversible inhibition of O(2) evolution.     

Enhancement by chloride ions of photoactivation of oxygen evolution in manganese-depleted photosystem II membranes

The Mn cluster that catalyzes photosynthetic oxygen evolution was removed from the photosystem II (PSII) complex by treating PSII membranes with 1.0 mM NH2OH with concomitant inactivation of oxygen evolution. The cluster was reconstituted by incubating the treated membranes with 1.0 mM Mn2+, 20 mM Ca2+, 10 microM 2,6-dichlorophenolindophenol, and Cl- under illumination with continuous or flashing light to restore the oxygen-evolving capacity. This light-dependent activation (photoactivation) of oxygen evolution did not occur to a significant extent at 3 mM Cl-, but markedly accelerated at higher Cl- concentrations without showing a saturation phenomenon even at 1 M Cl-. At 10 mM Cl- only about 10% of the oxygen-evolving activity before NH2OH treatment was restored by 5-min illumination with continuous light, whereas at 600 mM Cl- about 60% of the original activity was recovered. This acceleration resulted from at least two different actions of Cl-: (1) stabilization of the intermediate state involved in the photoactivation process and (2) increase in the quantum yield of photoactivation. The stabilization of the intermediate was saturated at about 150 mM Cl-, whereas the increase in yield did not show saturation. The Cl(-)-induced increase in quantum yield did not involve any changes in the affinity of either Mn2+ binding or Ca2+ binding for photoactivation, but was rather ascribed to a protective effect of Cl- against inhibition of photoactivation by high concentrations of Mn2+. We also found that removal of the extrinsic 33-kDa protein from the PSII complex increased the Cl- requirement for photoactivation.     

Azide as a competitor of chloride in oxygen evolution by Photosystem II.

Oxygen evolution by higher plants requires chloride, which binds to a site associated with the oxygen-evolving complex of photosystem II (PSII). In this study, the inhibitory effect of the anion azide was characterized using steady state measurements of oxygen evolution activity in PSII-enriched thylakoid membranes. N3- (7.8 mM) inhibited O2 evolution activity by 50% when a standard buffer containing chloride was used. By considering Cl- as the substrate in O2 evolution assays, we found azide to be primarily competitive with Cl- with an inhibitor dissociation constant Ki of about 0.6 mM. An uncompetitive component with a Ki ' of 11 mM was also found. Removal of the 17 and 23 kDa polypeptides resulted in a decrease in each inhibition constant. A pH dependence study of O2 evolution activity showed that the pH maximum became narrower and shifted to a higher pH in the presence of azide. Analysis of the data indicated that an acidic residue defined the low side of the pH maximum with an apparent pKa of 6.7 in the presence of azide compared with 5.5 for the control. A basic residue was also affected, exhibiting an apparent pKa of 7.1 compared with a value of 7.6 for the control. This result can be explained by a simple model in which azide binding to the chloride site moves negative charge of the anion away from the basic residue and toward the acidic residue relative to chloride. As a competitor of chloride, azide may provide an interesting probe of the oxygen-evolving complex in future studies.     

Competitive binding of acetate and chloride in photosystem II.

The binding of chloride and acetate to photosystem II (PSII) was examined to elucidate the mechanism of acetate inhibition. The mode of inhibition was studied, and individual binding sites were assigned by steady-state O2 evolution measurements in correlation with electron paramagnetic resonance (EPR) results. Two binding sites were found for acetate, one chloride-sensitive on the electron donor side and one chloride-insensitive on the electron acceptor side. The respective binding constants were as follows: KCl = 0.5 +/- 0.2 mM (chloride binding to the donor side), KI = 16 +/- 5 mM (acetate binding to the donor side), and KI' = 130 +/- 40 mM (acetate binding to the acceptor side). When acetate was bound to the acceptor side of PSII, 200 K illumination induced a narrowed form of the QA-FeII EPR signal, the yield of which was independent of the chloride concentration. When acetate was bound to the donor side, room-temperature illumination produced the S2YZ* state. EPR measurements showed that both the yield and formation rate of this state increased with acetate concentration. Increasing chloride concentrations slowed the rate of formation of the S2YZ* state, but did not affect the steady-state yield of the S2YZ* state. These findings indicate that the light-induced reactions in acetate-inhibited PSII are modulated by both donor side and acceptor side binding of acetate, while the steady-state yield of the S2YZ* state at the high PSII concentrations used for EPR measurements depends primarily on acceptor side turnover. Our data further support a close proximity of chloride to YZ*, indicating a possible role for chloride in the electron-transfer mechanism at the O2-evolving complex.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

The function of the chloride ion in photosynthetic oxygen evolution

The involvement of Cl(-) and several other monovalent anions in photosynthetic oxygen evolution was studied using photosystem II membranes depleted of Cl(-) by dialysis. The results of these studies differ significantly from results obtained using other depletion methods. Binding studies with glycerol as a cryoprotectant confirm our previous observations with sucrose of two interconvertible binding states of photosystem II with similar activities and with slow or fast exchange, respectively, of the bound ion. With glycerol, Cl(-) depletion decreased the oxygen evolution rate to 55% of that with Cl(-) present without decreasing the quantum efficiency of the reaction, supporting our previous conclusion that oxygen evolution can proceed at high rates in the absence of Cl(-). Further, after Cl(-) depletion the S(2) state multiline signal displayed the same periodic appearance with the same signal yield after consecutive laser flashes as with Cl(-) present. Br(-), I(-), and NO(3)(-), although with different capacities to reactivate oxygen evolution, also showed two binding modes. I(-) inhibited when bound in the low-affinity, fast-exchange mode but activated in the high-affinity mode. A comparison of the EPR properties of the S(2) state with these anions suggests that the nature of the ion or the binding mode only has a minor influence on the environment of the manganese. In contrast, F(-) completely inhibited oxygen evolution by preventing the S(2) to S(3) transition and shifted the equilibrium between the g = 4.1 and multiline S(2) forms toward the former, which suggests a considerable perturbation of the manganese cluster. To explain these and earlier observations, we propose that the role of chloride in the water-splitting mechanism is to participate together with charged amino acid side chains in a proton-relay network, which facilitates proton transfer from the manganese cluster to the medium. The structural requirements likely to be involved may explain the sensitivity of oxygen evolution to Cl(-) depletion or other perturbations.     

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.     

Carbonic anhydrase inhibitors. Inhibition of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila with anions

Anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), in addition to sulfonamides, which possess clinical applications. The first inhibition study of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila (Cam) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, and sulfate, etc., are also provided for comparison. The best Zn-Cam anion inhibitors were hydrogen sulfide and cyanate, with inhibition constants in the range of 50-90 microM, whereas thiocyanate, azide, carbonate, nitrite, and bisulfite were weaker inhibitors (K(I)s in the range of 5.8-11.7 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably up to concentrations of 200 mM, whereas the substrate bicarbonate behaves as a weak inhibitor (K(I)s of 42 mM). The best Co-Cam inhibitor was carbonate, with an inhibition constant of 9 microM, followed by nitrate and bicarbonate (K(I)s in the range of 90-100 microM). The metal poisons were much more ineffective inhibitors of this enzyme, with cyanide possessing an inhibition constant of 51.5mM, whereas cyanate, thiocyanate, azide, iodide, and hydrogen sulfide showed K(I)s in the range of 2.0-6.1mM. As for Zn-Cam, fluoride, chloride, and sulfate are not inhibitors of Co-Cam. These major differences between the two gamma-CAs investigated here can be explained only in part by the different geometries of the metal ions present within their active sites.     

pH optimum of the photosystem II H₂O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O₂ evolution activity

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH(-) inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O(2) evolution activity as well as the Cl(-) affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH(-). Through facilitation of efficient retention of Cl(-) in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl(-) to resist OH(-)-induced release from its site in the OEC. Preventing inhibition by OH(-) allows for normal (short) lifetimes of the S(2) and S(3) states in darkness [Roose, J. L., et al. (2011) Biochemistry 50, 5988] and for maximal steady-state activity by PSII. The data presented here indicate that activation of H(2)O oxidation occurs with a pK(a) of ～6.5, which could be a function of deprotonation of one or more amino acid residues that reside near the OEC active site on the D1 and CP43 intrinsic subunits of the PSII reaction center.     

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.     

Chloride cofactor in the photosynthetic oxygen-evolving complex studied by fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy, using midfrequency S2/S1 FTIR difference spectra, has been applied to studies of chloride cofactor in the photosynthetic oxygen-evolving complex (OEC) to determine the effects of Cl(-) depletion and monovalent anion substitution. Cl(-) depletion resulted in the disappearance of a large part of the amide I and II vibrational modes, and induced characteristic modification in the features of the stretching modes of the carboxylate ligands of the Mn cluster. The normal spectral features were largely restored by replenishment of Cl(-) except for some changes in amide bands. The overall features of Br(-) -, I(-) -, or NO3(-) -substituted spectra were similar to those of the Cl(-) -reconstituted spectrum, consistent with their ability to support oxygen evolution. In contrast, the spectrum was significantly altered by the replacement of Cl(-) with F- or CH3COO(-), which resulted in marked suppression and distortion of both the carboxylate and amide bands. The activity of oxygen evolution restored by NO3(-) was as high as that by Cl(-) when measured under limited light conditions, indicating that the NO3(-) -substituted OEC is fully active in oxygen evolution, although with a slow turnover rate. The double-difference spectrum between the 14NO3(-) -substituted and 15NO3- -substituted S2/S1 difference spectrum showed isotopic bands for asymmetric NO stretching mode in the region of 1400-1300 cm(-1) due to NO3(-) bound to the Cl(-) site. This demonstrated structural coupling between the Cl(-) site and the Mn cluster. A proposed model for the isotopic bands suggested that Cl(-) as well as NO3(-) is not directly associated with the Mn cluster and exists in a more symmetric configuration and weaker binding state in the S2 state than in the S1 state. These results also suggest that Cl(-) is required for changes in the structure of the specific carboxylate ligand of the Mn cluster as well as the peptide backbone of protein matrixes upon the transition from S1 to S2.     

Over-reduced states of the Mn-cluster in cucumber leaves induced by dark-chilling treatment

Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0 degrees C in the dark. The activity is restored by moderate illumination at room temperature. We examined the changes in the redox state of the Mn-cluster in cucumber leaves in the processes of dark-chilling inhibition and subsequent light-induced reactivation by means of thermoluminescence (TL). A TL B-band arising from S(2)Q(B)(-) charge recombination in PSII was observed upon single-flash illumination of untreated leaves, whereas four flashes were required to yield the B-band after dark-chilling treatment for 24 h. This three-step delay indicates that over-reduced states of the Mn-cluster such as the S(-2) state were formed during the treatment. Fitting analysis of the flash-number dependence of the TL intensities showed that the Mn-cluster was more reduced with a longer period of the treatment and that S(-3) was the lowest S-state detectable in the dark-chilled leaves. Measurements of the Mn content by atomic absorption spectroscopy showed that Mn atoms were gradually released from PSII during the dark-chilling treatment but re-bound to PSII by illumination at 30 degrees C. Thus, dark-chilling inhibition of oxygen evolution can be ascribed to the disintegration of the Mn-cluster due to its over-reduction. The observation of the S(-3) state in the present in vivo system strongly suggests that S(-3), which has been observed only by addition of exogenous reductants into in vitro preparations, is indeed a redox intermediate of the Mn-cluster in the processes of its disintegration and photoactivation.     

Azide as a probe of proton transfer reactions in photosynthetic oxygen evolution

In oxygenic photosynthesis, photosystem II (PSII) is the multisubunit membrane protein responsible for the oxidation of water to O₂ and the reduction of plastoquinone to plastoquinol. One electron charge separation in the PSII reaction center is coupled to sequential oxidation reactions at the oxygen-evolving complex (OEC), which is composed of four manganese ions and one calcium ion. The sequentially oxidized forms of the OEC are referred to as the S_n states. S₁ is the dark-adapted state of the OEC. Flash-induced oxygen production oscillates with period four and occurs during the S₃ to S₀ transition. Chloride plays an important, but poorly understood role in photosynthetic water oxidation. Chloride removal is known to block manganese oxidation during the S₂ to S₃ transition. In this work, we have used azide as a probe of proton transfer reactions in PSII. PSII was sulfate-treated to deplete chloride and then treated with azide. Steady state oxygen evolution measurements demonstrate that azide inhibits oxygen evolution in a chloride-dependent manner and that azide is a mixed or noncompetitive inhibitor. This result is consistent with two azide binding sites, one at which azide competes with chloride and one at which azide and chloride do not compete. At pH 7.5, the K_i for the competing site was estimated as 1 mM, and the K_i′ for the uncompetitive site was estimated as 8 mM. Vibrational spectroscopy was then used to monitor perturbations in the frequency and amplitude of the azide antisymmetric stretching band. These changes were induced by laser-induced charge separation in the PSII reaction center. The results suggest that azide is involved in proton transfer reactions, which occur before manganese oxidation, on the donor side of chloride-depleted PSII.