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EPR measurements on the effects of bicarbonate and triazine resistance on the acceptor side of Photosystem II
Abstract
CO2 depletion leads to an approximately 10-fold increase in the light-induced EPR signal at g = 1.82, attributed to the QA− · Fe2+ complex, in Photosystem II-enriched thylakoid membrane fragments. Upon reconstitution with HCO3−the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2-depleted samples. It is concluded that HCO2− strongly influences the localization and conformation of the QA− · Fe+ complex. The QA− · Fe2+ and split pheophytirr− EPR signals from triazine-resistant Brassica napus were virtually identical to those from triazine-susceptible samples, indicating that the change in the 32-kDa azidoatrazine-binding protein does not lead to a confonnational change of the Qa− · Fe2+ complex.
... In vitro HCO 3 − at the nonheme iron can be replaced by small carboxylic acids like glycolate, glyoxylate, and formate (Wydrzynski and Govindjee, 1975;Vermaas and Rutherford, 1984;Petrouleas et al., 1994;Hienerwadel and Berthomieu, 1995). In vivo, acetate affects the energetics of PSII in Chlamydomonas reinhardtii when grown under mixotrophic conditions in acetatecontaining medium (Roach et al., 2013). ...
... It is well known that small carboxylate anions can replace bicarbonate as ligand to the nonheme iron in PSII in vitro: electron transfer from Q A to Q B is slowed down in bicarbonate-depleted PSII or in PSII in which bicarbonate was replaced with formate, acetate, glyoxylate, or glycolate (Vermaas and Rutherford, 1984;Petrouleas et al., 1994;Berthomieu and Hienerwadel, 2001). Acetate or glycolate shifts the midpoint potential of the redox couple Q A /Q A − to a more positive potential also in vivo. ...
Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO2 conditions.
... The first indication that non-heme iron (NHI) may be involved in bicarbonate action was reported by Vermaas and Rutherford (1984). They showed that the addition of formate (HCO 2 -) to thylakoids increased the amplitude of the electron paramagnetic resonance (EPR) signal (g = 1.82) of Q A -Fe 2+ by ten-fold. ...
... 28,60 The electron paramagnetic resonance (EPR) experiments suggested that electron transfer was perturbed upon the replacement of bicarbonate with formate. 61 However, the H QA−QB calculated by replacing bicarbonate with formate is similar to that in the wild-type PSII (Table 1). This is in line with the EPR experiment, that showed that the main effects of formate appeared on the Q B H 2 exchange 29 (see below for further discussion). ...
In photosynthetic reaction centers from purple bacteria (PbRCs) and photosystem II (PSII), the photoinduced charge separation is terminated by an electron transfer between the primary (QA) and secondary (QB) quinones. Here, we investigate the electron transfer route, calculating the superexchange coupling (HQA-QB) for electron transfer from QA to QB in the protein environment. HQA-QB is significantly larger in PbRC than in PSII. In superexchange electron tunneling, the electron transfer via unoccupied molecular orbitals of the nonheme Fe complex (QA → Fe → QB) is pronounced in PbRC, whereas the electron transfer via occupied molecular orbitals (Fe → QB followed by QA → Fe) is pronounced in PSII. The significantly large HQA-QB is caused by a water molecule that donates the H-bond to the ligand Glu-M234 in PbRC. The corresponding water molecule is absent in PSII due to the existence of D1-Tyr246. HQA-QB increases in response to the Ser-L223···QB H-bond formation caused by an extension of the H-bond network, which facilitates charge delocalization over the QB site. This explains the observed discrepancy in the QA-to-QB electron transfer between PbRC and PSII, despite their structural similarity.
... Brinkert et al. [44] found that the removal of bicarbonate shifts the midpoint redox potential of the couple Q A /Q A −• from ∼−145 mV to −70 mV. Based on EPR measurements, Vermaas and Rutherford [45] concluded that the bicarbonate influences the conformation of the Q A -• Fe 2+ complex. Shevela et al. [46] found that when spinach thylakoids are depleted of carbon dioxide and bicarbonate, the miss probability in the Kok cycle is higher than under ambient conditions and that the addition of 5 mM bicarbonate to thylakoids depleted of inorganic carbon largely restores the original miss parameter. ...
This review deals with the production of oxygen by photo-oxidation of water, which is a topic fitting a journal devoted to oxygen. Most of the present biosphere, including mankind, depends on oxygen. Elucidating the mechanism is of importance for solving the present energy crisis. Photosynthesis evolved in bacteria, first in a form that did not produce oxygen. The oxygen-producing version arose with the advent of cyanobacteria about three billion years ago. The production of oxygen by photo-oxidation of water requires the co-operative action of four photons. These are harvested from daylight by chlorophyll and other pigments (e.g., phycobiliproteins) and are channeled to photosystem II and photosystem I. The oxygen-evolving complex resides in photosystem II, surrounded by protein subunits, and contains one ion of calcium, four ions of manganese, and a number of oxygen atoms. For each quantum of energy it receives from absorbed light, it proceeds one step through a cycle of states known as the Kok–Joliot cycle. For each turn of the cycle, one molecule of oxygen (O2) is produced.
... 59 The g = 1.82 EPR signal also undergoes a bicarbonatereversible 10-fold increase when formate is bound in isolated thylakoid fragments enriched in PS II. 60 Hence, it is possible that disruption of the quinone-iron environment in the D2-Y244 mutants, and possibly the loss of bicarbonate binding, prevented Q A − recombination with Y D + leading to the observed absence of the C band in Figure 5B. ...
Two plastoquinone electron acceptors, QA and QB, are present in Photosystem II (PS II) with their binding sites formed by the D2 and D1 proteins, respectively. A hexacoordinate non-heme iron is bound between QA and QB by D2 and D1, each providing two histidine ligands, and a bicarbonate that is stabilized via hydrogen bonds with D2-Tyr244 and D1-Tyr246. Both tyrosines and bicarbonate are conserved in oxygenic photosynthetic organisms but absent from the corresponding quinone-iron electron acceptor complex of anoxygenic photosynthetic bacteria. We investigated the role of D2-Tyr244 by introducing mutations in the cyanobacterium Synechocystis sp. PCC 6803. Alanine, histidine, and phenylalanine substitutions were introduced creating the Y244A, Y244H, and Y244F mutants. Electron transfer between QA and QB was impaired, the back-reaction with the S2 state of the oxygen-evolving complex was modified, and PS II assembly was disrupted, with the Y244A strain being more affected than the Y244F and Y244H mutants. The strains were also highly susceptible to photodamage in the presence of PS II-specific electron acceptors. Thermoluminescence and chlorophyll a fluorescence decay measurements indicated that the redox potential of the QA/QA- couple became more positive in the Y244F and Y244H mutants, consistent with bicarbonate binding being impacted. The replacement of Tyr244 by alanine also led to an insertion of two amino acid repeats from Gln239 to Ala249 within the DE loop of D2, resulting in an inactive PS II complex that lacked PS II-specific variable fluorescence. The 66 bp insertion giving rise to the inserted amino acids therefore created an obligate photoheterotrophic mutant.
... (Bi)carbonate has been consistently found coordinated to the non-heme iron atom located between the first (Q A ) and second (Q B ) plastoquinone electron carriers (Ferreira et al. 2004, Shen 2015. The non-heme iron ligating site of (bi)carbonate was first qualitatively identified by Govindjee and coworkers and has been extensively demonstrated and further localized by his group and others (Wydrzynski and Govindjee 1975, Vermaas and Rutherford 1984, Diner et al. 1991. The dissociation constant of bicarbonate from the non-heme iron site was recently established to be pH-dependent (van Rensen et al. 1999, Koroidov et al. 2014, Brinkert et al. 2016. ...
Arthrospira maxima is unique among cyanobacteria, growing at alkaline pH (<11) in concentrated (bi)carbonate (1.2 M saturated) and lacking carbonic anhydrases. We investigated dissolved inorganic carbon (DIC) roles within PSII of A. maxima cells oximetrically and fluorometrically, monitoring the light reactions on the donor and acceptor sides of PSII. We developed new methods for removing DIC based on a (bi)carbonate chelator and magnesium for (bi)carbonate ionpairing. We established relative affinities of three sites: the water-oxidizing complex (WOC), non-heme iron/QA–, and solvent-accessible arginines throughout PSII. Full reversibility is achieved but (bi)carbonate uptake requires light. DIC depletion at the non-heme iron site and solvent-accessible arginines greatly reduces the yield of O2 due to O2 uptake, but accelerates the PSII–WOC cycle, specifically the S2→S3 and S3→S0 transitions. DIC removal from the WOC site abolishes water oxidation and appears to influence free energy stabilization of the WOC from a site between CP43-R357 and Ca²⁺.
... В. Вер ма ас і А. Ре зер форд (Vermaas, Rutherford, 1984) по ка за ли: з до да ва ням фор міа ту до ти ла ко їдів ам п лі ту да ЕПР сиг на лу g = 1,82, що на ле жить до за лі зо-се мі хі но но во го ком плек су Q A -Fe 2+ , збіль-шу єть ся вде ся те ро. Ефект за мі щен ня бі кар бо на ту на фор мі ат де таль но оха рак те ри зо ва но за до по могою ЕПР-спек трів ком плек су Q A -Fe-Q B з ви да ленням і до да ван ням бі кар бо на ту (Bowden et al., 1991;Hermes et al., 2011). ...
... The Hill reaction is significantly and reversibly inhibited in chloroplasts of higher plants depleted of bicarbonate (HCO3) in the presence of formate (see reviews by Vermaas and Govindjee 1981, Blubaugh and Govindjee 1988a). A recent hypothesis (see Blubaugh and Govindjee 1988a) is that there are two major sites of 'bicarbonate effect' on the electron acceptor side of photosystem II (PS II): (1) as a ligand to Fe z+ in QA-Fe-QB complex that keeps the D 1 and D2 proteins in their proper functional conformation (see supportive data and arugments in Vermaas and Rutherford 1984, Michel and Deisenhofer 1988); (2) as a participant in the protonation of Q~. ...
In this communication, evidence is presented from the kinetics of QA (-) decay (where QA is the first plastoquinone electron acceptor of photosystem II) and oxygen evolution for the requirement of bicarbonate in the electron transport in a cyanobacteriumSynechocystis (Pasteur Culture Collection 6803). A large slowing down of QA (-) oxidation, measured from the variable chlorophylla fluorescence after saturating actinic flashes, was observed in the thylakoids ofSynechocystis 6803 depleted of bicarbonate in the presence of 25 mM formate. Qualitatively similar results were obtained with DCMU-treated thylakoids. This shows that bicarbonate depletion inhibits electron transport on the acceptor side of photosystem II between QA and the plastoquinone (PQ) pool in cyanobacteria. Addition of 2.5 mM HCO3 (-) fully reversed the inhibition of electron flow caused by bicarbonate depletion. Two exponential phases of QA (-) decay, a fast one and a slow one, were observed with halftimes of approx. 400 μs (fast) and 26 ms (slow) at pH 6.5. At pH 7.5, these phases were approx. 330 μs (fast) and 21 ms (slow), respectively. The amplitude, but not the halftime, of the fast component decreased by about 70% (pH 6.5) or 50% (pH 7.5); this was accompanied by a concomittant increase in the slow phase. Twenty mM bicarbonate stimulated, by a factor of 4, the Hill reaction in bicarbonate-depletedSynechocystis cells. This effect is independent of CO2 fixation as it was observed even in the presence of an inhibitor DBMIB.
... The two most likely candidates for the high-affmity sites are: a species liganded to Fe 2+, as previously proposed by Michel and Deisenhofer [19], and the other complexed with an arginine residue, as su88ested by Shipman [20]. The ligand bound to Fe 2+ would account for several observations: the large increase in the fight-induced EPR signal at g= 1.82, attributed to the Q~-Fe 2+ complex, upon HCO~-depletion [21]; the inability of high-potential quinones to oxidize the Fe 2+ in the presence, but not in the absence, of formate [22]; and the apparent lack of a homologous sequence in PS II which would correlate with a ...
In HCO3−-depleted thylakoids in which the basal activity was less than 7% of the fully restored activity after readdition of HCO3−, the restored activity at a half-saturating HCO3− concentration was non-linear with the chlorophyll concentration. This indicates that there was still some endogenous HCO3− remaining in these thylakoids, even though they appeared to be well depleted of HCO3−. A kinetic analysis of the activity curve for these thylakoids, as a function of HCO3− concentration, indicates that there are at least two high-affinity sites of HCO3− in Photosystem II (PS II), apparently with cooperative binding. An additional low-affinity site has been shown to exist earlier (Stemler, A. (1977) Biochim. Biophys. Acta 460, 511–522; Blubaugh, D.J. and Govindjee (1984) Z. Naturforsch. 39c, 378–381). Illumination was shown to convert non-exchanging membrane HCO3− to an exchanging one. It is suggested that HCO3− is an essential activator for PS II electron transport, and that a complete removal of HCO3− would result in zero activity.
... The non-heme iron at the acceptor side of PSII is coordinated by four histidine residues and an exchangeable bidentate ligand to bicarbonate [15]. Low temperature electron paramagnetic resonance (EPR) measurements have shown that small carboxylate anions influence the environment of the non-heme iron [16,17]. Carboxylate anions do not only affect the acceptor side of PSII, but also the donor side. ...
Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (TAP). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, TAP-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less (1)O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of (1)O2 originating from charge recombination events in PSII, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA(-)) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB(-)) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to (1)O2 production. Acetate treatment of PSII-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on PSII independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated PSII particles was detected by low temperature EPR spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting PSII charge recombination events and photoinhibition.
... • In 1984, Vermaas and Rutherford [143] were among the first ones to focus on the relationship of bicarbonate to the Q A -NHI-Q B niche of PSII. They discovered that removal of HCO 3 − /CO 2 , in PSII membrane fragments from Brassica napus, led to a very large increase in the EPR signal at g = 1.82 that is due to the Q A − Fe 2 + complex, and, that this effect was fully reversible when bicarbonate was added back. ...
Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.
The effect of bicarbonate (HCO3−) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3− enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the QA quinone leads to the release of HCO3− from its binding site on the non-heme iron and (ii) the Em potential of the QA/QA•− couple increases when HCO3− dissociates. This suggested a protective/regulatory role of HCO3− as it is known that increasing the Em of QA decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with 13C isotope labeling of HCO3− allows donor-and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.
IntroductionNMR Spectra of QuinonesIR Spectra of QuinonesUV/VIS Spectra of QuinonesEsr/Endor Spectra of Quinone Ion RadicalsElectrochemical MethodsChromatography of QuinonesChemical Methods
Conclusions
Acknowledgements
The main electron transfer events in PS2 are:
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The reaction center of Photosystem II contains an iron atom which is likely to be located between the acceptor-side quinones QA and QB, in analogy with the structure in photosynthetic purple bacteria. The iron, which is probably not directly involved in the electron transfer between the quinones, can be oxidized by strong oxidants, such as ferricyanide and high-potential semiquinones in plants (but not in purple bacteria) and may then be observed by EPR (1,2). This offers an additional possibility to probe the local environment of the primary iron-quinone acceptor other than that provided by the reduced complex, QA-Fe, which is EPR detectable in plants as well as in bacteria. Measurements, utilizing the sensitivity of EPR to changes in the ligand geometry around the Fe(III) ion, have detected distortions in the protein environment when quinone acceptor analogs or acceptor-side inhibitors bind (2,3). In this communication we report on EPR studies of the acceptor side in the cyanobacterium Anacystis nidulans which allow comparisons with the quinone-iron complex in plants.
Oxygen evolving photosystem two (PS2) particles isolated using Triton X-100 consist of a number of polypeptides. These include the polypeptides termed D1 and D2 and the two apoproteins of cytochrome b559.The proteins D1 and D2 which bind the primary electron transport components show considerable sequence homology with the purple bacterial reaction centre proteins L and M. Computer models have indicated that D1 and D2 each have five transmembrane helices, and that the binding regions for electron acceptors Qa and Qb reside in the loops between helices IV and V on the stromal side of the thylakoid membrane [1].
The herbicide binding domain of Photosystem 2 (PS2) has been localised to a region of the D1 polypeptide connecting the putative 4th and 5th membrane spanning alpha helices based on photoaffinity labelling studies, the locations of point mutations affecting herbicide binding, and homology with the purple bacterial reaction centre whose structure has been resolved. Although some commercial PS2 herbicides bind to the bacterial reaction centre, many other classes of PS2 herbicides, such as the phenylureas and the phenols, do not, indicating significant differences in architecture between PS2 and the bacterial reaction centre in the herbicide binding domain. Our studies are aimed at elucidating the tertiary structure of the herbicide binding domain of PS2 particularly with respect to phenylurea binding.
Electron transfer through the plastoquinone acceptors of photosystem II (PS II) is inhibited by a number of monovalent anions. These include formate, nitrite and acetate [1–3]. However, the HCO-3 anion possesses the unique ability to reverse this inhibition. We have suggested that the ability of HCO-3 to facilitate electron flow may involve participation in the protonation reactions of the two-electron gate [4]. To test this hypothesis we have studied the kinetics of QA reoxidation in control, anion inhibited/HCO-3-depleted and HCO-3-restored samples as a function of pH and flash frequency. Below, we present some preliminary findings from this work.
Bicarbonate (or CO2) was shown by Warburg and Krippahl [1] to stimulate electron transport during the Hill reaction. This phenomenon has been referred to as the bicarbonate (HCO3−) effect. The electron transport chain can be dissected into a number of clearly defined partial reactions through the addition of specific inhibitors and electron donors and acceptors. By applying this approach (see e.g., [2, 3]) the HCO3− effect has been shown to be associated with the acceptor side of Photosystem II (PS II):
$${{H}_{2}}O\to OEC\to Z\to P680\to Pheo\to {{Q}_{A}}\to {{Q}_{B}}\to PQ$$ (1)
Photosystem II (PS II) is a pigment-protein complex in thylakoid membranes from all oxygenic photosynthetic organisms (cyanobacteria and photosynthetic eukaryotes). It catalyzes the light-induced reduction of plastoquinone by water through a number of redox reactions. The electron transport chain in PS II is composed of various protein-bound components, which are held in close proximity and suitable orientation with respect to each other by the protein environment, so that a rapid and efficient electron transport is feasible. The PS II complex consists of at least five integral membrane proteins in the thylakoid (together forming the PS II “core complex”), in addition to several peripheral proteins. Many of these polypeptides interact with one or more components of the electron transport chain or with light-harvesting pigments, such that these protein ligands can fulfill a specific function in PS II.
Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a non-heme Fe2+ located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA•- and QB•- are magnetically couple to the Fe2+ giving weak and complex EPR signals. The distorted octahedral Fe2+ has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB•- Fe2+ EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA•- Fe2+ in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA•- Fe2+ and QB•- Fe2+ EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD•). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB•-Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB•-/QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA•-Fe2+ state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII.On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.
It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQA complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.
Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.
The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQA complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the QB site.
We report the detection of a “split” electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S1 state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa•- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the “S1 split signal”, which we provisionally assign to S1X•, where X could be YZ• or Car•+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40−50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).
Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones Q(A) and Q(B) in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to these of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents, Q-band EPR yielded the principal values of the g-tensor for PQ-9(.-) in 2-propanol and Q(A)(.-) in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9(.-) in vitro and on QA(.-) in the iron-depleted PS II samples, For the former a complete set of isotropic H-1 hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. H-1-ENDOR spectra of Q(A)(.-) in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of Q(A) and specific protein residues, The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.
The nonheme iron of the photosystem II reaction center was converted to its low-spin state (S = 0) by treatment with CN-. This allowed the study of the plastoquinone, Q(A)(-) anion radical by electron spin-echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of the ESEEM data of Q(A)(-) in N-14- and N-15-labeled PSII demonstrates the existence of a protein nitrogen nucleus coupled to the Q(A)(-). The N-14 coupling is characterized by a quadrupolar coupling constant e(2)qQ/4h = 0.82 MHz, an asymmetry parameter eta = 0.45, and hyperfine coupling constant A similar to 2.1 MHz. The N-15 hyperfine coupling is characterized by T = 0.41 MHz and alpha(iso) similar to 3.3 MHz. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acids thought to be close to the Q(A)(-) in PSII. Based on a comparison of the N-14 ESEEM with N-14-NQR and N-14-ESEEM data from the literature, the most likely candidate is the amide nitrogen of the peptide backbone of Ala261 of the polypeptide D2, although the indole nitrogen of Trp254 and the imino nitrogen of His215 of D2 also remain candidates.
Site directed mutagenesis studies of reaction centers (RCs) have so far focused on the L- and M-subunits, because they bind all the cofactors involved in the light-induced charge separation processes. However, the H-subunit gene, puh4, of the RC has also been isolated and sequenced from Rb. sphaemides, Rb. Capsularus, and Rps. Viridis, and site-directed mutagenesis studies can be expected in the near future. This chapter addresses significant advances made through the mutagenesis of the L- and M-subunits in understanding the roles of key residues in specific functions of the RC. The effects of the mutations have been dramatic and unexpected including the heterodimer mutants substitution of BphA by Bchl and, by opposite extremes of functional disturbance, the mutation of Asp1213 and of GIuM234. Part of the unexpectedness of these and many other mutations is the capacity of the RC to accommodate radical changes to assemble and, to a large extent, to function.
We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO2−-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO3− removal are reversed by the addition of 5 mM HCO3−. The initial first-order component for the oxidation of QA− (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by QB (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO3−-depleted membranes. After a single actinic flash, the half-time of QA− decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO3−-depleted membranes behave as control membranes with respect to electron flow from QA to QB or to QB−. The time (t50) at which the [QA−] is 50% of the maximum [QA−] during the back reaction between QA− and the S2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO3−-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of QA− with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO3−-depleted membranes. The apparent equilibrium for the sharing of an electron between QA and QB is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes (Kapp ≈ 16) as compared to the restored or control membranes (Kapp ≈ 62); there was no difference in Kapp at pH 7.75. Estimates of the operating redox potential for the QB/QB− couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO3− to act as a proton donor to the protein dissociable group believed to participate in the protonation of QB−. Finally, the possibility of HCO3− being a ligand to Fe2+ in the QA-Fe-QB complex of the PS II reaction center is also discussed.
In this paper four major points with respect to HCO3−-reversible inhibition in spinach thylakoid membranes, depleted of HCO3− in the presence of inhibitory anions, are established. (1) The oxidation of QA− (QA is the primary quinone acceptor of Photosystem II) by QB (QB is the secondary quinone acceptor of Photosystem II) or QB−, following one or two actinic flashes, respectively, exhibits a smaller t50 time at which [QA−] is 50% of maximum [QA−]) after a flash at pH 7.5 than at pH 6.5 (2) The characteristic oscillations, due to differential rates of QA− oxidation by QB or QB−, observed in the fluorescence flash pattern, generated by assaying the chlorophyll a fluorescence at specific times after an actinic flash and plotting these data as a function of flash number, are lost (i.e., the turnover of two electron gate is hampered). (3) At 1 Hz, the slowest oxidation of QA−, as indicated by f50 values, depends on both the pH and flash number: at pH 6.5, t50 for QA− decay reaches a maximum value after only three flashes (one turnover of the two-electron gate), whereas at pH 7.5, the t50 is increased further until after five flashes (two turnovers of the two-electron gate). (4) The t50 values of QA− oxidation also depend on the actinic flash frequency: at 5 Hz, [QA−] reaches its maximum after flash 5 even at pH 6.5. The increase observed in the t50 values of QA− oxidation in treated membranes is accompanied by the presence of slow components of QA− oxidation in the 0.1–10 s range which can achieve an amplitude of more than 70%. These components are suggested to be related to protonation steps involved in the quinone acceptor complex of Photosystem II and support the conclusion that the rate-limiting step in electron transfer in HCO3−-depleted thylakoids may be the protonation of QB− and possibly QB2−. A working hypothesis is presented that explains the flash, frequency and pH dependence of QA− decay observed in this paper.
Photoinhibition of isolated thylakoids results in inactivation of Photosystem II electron transport and proteolysis of the D1 reaction center protein. At low, non-freezing temperatures the mechanism of inactivation for Photosystem II electron transport can be experimentally studied without interference of secondary damaging effects, since the degradation of the D1-protein does not occur. Here we have applied electron paramagnetic resonance (EPR) spectroscopy to characterize the sequential events leading to inhibition of PS II electron transport and triggering of the D1-protein for degradation at 2 C. Two principle kinetics of inactivation and damage were observed: (i) inactivation with a half-time of 1 to 1.5 h in case of steady-state electron transport through Photosystem II, the induction of the S2-state multiline EPR signal, the EPR signal from QA-Fe2− and lowering of the Fv/Fm fluorescence ratio. This is explained by over-reduction of the first quinone acceptor (QA) leading to impairment of its function. (ii) Inhibition with a half-time of 3–4 h in case of EPR Signal IIslow; inhibition of the primary charge separation reaction and release of manganese from its site in the oxygen evolving system. We were also able, for the first time, to follow the kinetics for the triggering of the D1-protein for degradation. This triggering followed the slower kinetic phase and is likely to be the result of conformational changes in the protein induced by the highly reactive singlet oxygen. Additionally, a dark-stable cationic radical with g = 2.0031 and 10–11 G wide was progressively induced during the inhibition and was tentatively attributed to a carotenoid cation.
Electron paramagnetic resonance (EPR) spectroscopy has been applied in an investigation on the mechanism for photoinhibition of the electron transport in Photosystem II. The experiments were performed in vitro in thylakoid membranes and preparations of Photosystem-II-enriched membranes. Photoinhibition resulted in inhibition of the oxygen evolution and EPR measurements of the S2 state multiline EPR signal show that its induction by illumination at 198 K was decreased with the same kinetics as the oxygen evolution. Further EPR measurements show that the reduction of QA was inhibited with the same kinetics as the oxygen evolution. The amount of photoreducible pheophytin was estimated from photoaccumulation experiments under reducing conditions and the results show that the primary charge separation reaction was inhibited much slower than the oxygen evolution or the reduction of QA. These results indicate that photoinhibition inhibits the electron transfer between pheophytin and QA probably by impairment of the function of QA. In the inhibited centers the primary charge separation reaction is still operational. It is suggested that the event leading to photoinhibition of the electron transport is the double reduction of QA which then leaves its site. Photoinhibition also results in rapid oxidation of cytochrome b-559 and a change of cytochrome b-559 from its high potential form to its low potential form. The reaction is quantitative and proceeds with the same kinetics as the inhibition of oxygen evolution. The potential shift of cytochrome b-559 suggests that photoinhibition induces early conformational changes in Photosystem II.
Electron transfer in irreversibly acetate-inhibited PS II preparations from Synechococcus is investigated with EPR. A 1.4 mT wide EPR line with a decay rate consistent with the P+680 Q−A back reaction is tentatively assigned to be a broadened P+680 spectrum. With reversible inhibition of water cleavage by the ADRY reagent FCCP added with acetate electron transfer is studied as well. Under these conditions the spectrum of the immediate donor to P+680 is the same as the spectrum of Z+ in systems with irreversibly inhibited water cleavage (signal II fast) and the same as that of D+ (signal II slow). The consequences are discussed.
In the companion paper (Petrouleas, V., Deligiannalcis, Y. and Diner, B.A. (1994) Biochim. Biophys. Acta 1188, 260–270) we examined the effects of a number of carboxylate anions on the EPR signals associated with the iron quinone complex, the ligand field parameters of the Fe3+, and the redox properties of the iron. In this paper, we show that three representative anions, glycolate, glyoxylate, and oxalate, compete with NO, formate and bicarbonate for binding to the iron. Furthermore, the bound anions affect diversely the electron transfer rate. Glycolate has an extreme effect, similar to what is observed with high levels of formate, and is characterised by a dissociation constant, Kd, ∼ 0.5–0.7 mM. Oxalate gives a marked slowing of the rate of Q−A oxidation on all flashes but preserves a marked oscillation of the rate of period two. Glyoxylate appears to have an intermediate effect. These results offer new information on the stereochemistry of the binding of dissociable ligands to the non-heme iron of PS II and a tool for probing the redox chemistry of the iron and the electron transfer properties of the iron-quinone complex.
We have examined the effects of a number of carboxylate anions on the iron-quinone complex of Photosystem II (PS II). Typical effects are the following. In the state Q−AFe2+ oxalate enhances significantly the g = 1.84 EPR resonance while, for example, glycolate and glyoxylate suppress it. The anions have variable effects on the iron midpoint potential. Formate and oxalate raise significantly the Em of the iron. Glycolate lowers the Em significantly and the Em shows a weak pH dependence. In the presence of glycolate the native plastosemiquinone () can oxidise the iron. Glyoxylate also lowers the Em, but the Em shows a greater pH dependence than with glycolate but still weaker than the −60 mV/pH unit of the untreated iron. The Fe3+ EPR spectra are characterised by small but distinct shifts, while in addition an unusual resonance at close to g = 4.3 is observed. These as well as the temperature dependence of the spectra are analysed by a spin-Hamiltonian model. Comparison with competition studies in the companion paper indicates that the anions bind as iron ligands displacing bicarbonate.
We have investigated the EPR characteristics of native QB and QB analogues in higher plant PS II. We show that, as in cyanobacteria, an interaction between QA and QB iron-semiquinones (QA−-Fe2+-QB−) is observed which gives an EPR signal near g=1.6. Bicarbonate binding close to the non-haem iron is required to observe this interaction. The EPR signal of QB semiquinone is weak and difficult to distinguish from that of QA. The appearance of the g=1.6 signal from QA−-Fe2+-QB− after 77 K illumination is a better marker for the presence of QB semiquinone. The yield of QB semiquinone in plant PS II is lower than found in cyanobacteria. The brominated quinones DBMIB, TBTQ and bromanil were used as QB analogues to increase the yield of QA−-Fe2+-QB−. These analogues act by forming a stable semiquinone at the QB site and not by covalent binding.
We have studied the EPR signal in PS II from Phormidium laminosum with a g-value of 1.66, which we assign to an interaction between the semiquinones of Qa and Qb and the non-heme iron. 77 K illumination of samples from dark-poised redox titrations show the rise of the signal has a midpoint potential (Em) of about +60 mV, and it is lost with an Em of about −10 mV. Under the same conditions, the rise of the g = 1.9 signal from Q·−a-Fe2+ in the dark was found to be about +10-mV. The g = 1.66 signal can also be formed with a high yield by first illuminating dark-adapted PS II particles at 293 K, followed by a short period of darkness at 273 K and subsequent illumination at 77 K. We have measured the effect on signal yield of varying the period of darkness following 293 K illumination. Over 60% of the maximum signal size is seen after 1 min darkness, and increases further over 2 h. In these samples a signal attributed to Q·−b-Fe2+ is seen prior to 77 K illumination. Confirmation of the presence of Q·t-b was obtained by reductant-linked oxidation of the non-haem iron using phenyl-para-benzoquinone (PPBQ). Samples treated with the Qb-analogue tribromotoluquinone (TBTQ) give a modified EPR signal. We propose (i) that Qb is preserved in PS II preparations from P. laminosum; (ii) that Qb-semiquinone can be readily formed and trapped by freezing; and (iii) the g = 1.66 signal arises from a coupling between the primary and secondary plastosemiquinones and the non-haem iron.
Mild trypsinization of PS II particles at pH 6.0, which almost completely blocks reoxidation of the primary plastoquinone acceptor, QA−·Fe2+, 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 QA−·Fe2+. 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 QA−·Fe2+ microenvironment is briefly discussed in relation to the functional and structural organization of the PS II acceptor side.Photosystem IISemiquinone-iron complexTrypsinHerbicidePhotosythesis
Redox titrations of QA, the first quinone electron acceptor, have been performed on Photosystem II (PS II) membranes which were either active or inactive in terms of oxygen evolution. The redox state of QA was monitored by measuring the chlorophyll fluorescence yield. When titrations were done at room temperature in the absence of mediators, an Em value of approx. −80 mV was obtained for active centres and approx. +65 mV for inactive centres. These values confirm earlier reports (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) in which measurements were made under comparable conditions. In addition, we found that these Em values were independent of pH from pH 5.5 to pH 7.5, the range of pH over which the O2-evolving enzyme is stable. To understand better the scattered values for the Em of QA which exist in the literature and to assess the validity of the present values, experiments were performed under a range of different titration conditions. Two main experimental factors were found to have a strong influence on the measured Em of QA. First, the presence of redox mediators at low ambient potentials led to an irreversible shift from the low-potential (active) form to the high-potential (inactive) form. This is attributed to the reduction of the Mn cluster which is thought to remain out of equilibrium when titrations are done without mediators. Secondly, upon freezing of samples poised at low potential a change in the redox state of QA occurred, as measured by EPR and fluorescence at low temperature. Freezing and thawing of active PS II at potentials where QA is chemically reduced results in an irreversible change in the Em of QA from the low-potential to the high-potential form. This is accompanied by inhibition of oxygen evolution. It is suggested that this effect might also be related to the reduction of the Mn cluster which is, in this case, induced by freeze-thawing in the presence of chemically reduced QA−. Based on these observations, it is suggested that most titrations of QA in active PS II that have been reported previously suffer from one or both of these unexpected technical difficulties. Thus, the Em values obtained at room temperature and without mediators are probably those which should be taken into account in understanding the energetics of PS II.
The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl- site(s) in PSII, and some even support activity. NO2- has been reported to be an activator in Cl--depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO2- with PSII. NO2- is shown to be inhibitory to PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O2-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O2 yield measurements. Enzyme kinetics analysis shows multiple sites of NO2- inhibition in PSII with significant inhibition of oxygen evolution at concentrations of NO2- below 5 mM. By EPR spectroscopy, the yield of the S2 state remains unchanged up to a concentration of 15 mM NO2-. However, the S2 state g = 4.1 signal is favored over the g = 2 multiline signal with increasing NO2- concentration. This could indicate competition of NO2- for the Cl- site at higher concentrations of NO2-. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO2-. The g = 1.9 Fe(II)-QA-• signal is replaced by a broad g = 1.6 signal in the presence of NO2-. Additionally, a g = 1.8 Fe(II)-Q-• signal is present in the dark, indicating the formation of a NO2--bound Fe(II)-QB-• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO2- on the activity of PSII is largely due to the donor side chemistry of NO2-. UV-visible spectroscopy and flash-induced polarographic O2 yield measurements indicate that NO2- is oxidized by the oxygen-evolving complex (OEC) in the higher S states, contributing to the donor-side inhibition by NO2-.
Oxidation of the reduced primary electron acceptor, QA−, of Photosystem II (PS II) in formate-treated spinach thylakoids, was inhibited more after the second than after the first actinic flash. This indicates a slowing of electron flow on the acceptor side of PS II from QA− to QB−, the semiquinone form of the secondary plastoquinone acceptor, formed by electron transfer after the first flash. A hypothesis of electron transfer on the acceptor side of PS II is proposed to accommodate the bicarbonate-reversible formate/formic acid inhibition of electron transfer after single turnover flashes. We suggest that the large inhibition in QA− oxidation after the second flash reflects a blockage of the proton uptake that stabilizes QB−. Kinetics of onset of inhibition following formate addition were followed by measuring the chlorophyll a fluorescence yield, reflecting the concentration of QA−, 1 ms after the second actinic flash as a function of time after the addition of formate. The apparent rate constants for binding and unbinding, and the dissociation constant of formate were determined in the pH range from 5.5 to 7.5. The rate of onset of inhibition following formate addition, reflecting formate or formic acid binding, was highly dependent on the medium pH. Measurements on the initial binding rate, when one of the two (HCO2−/HCOOH) equilibrium species was kept constant and the other varied, suggested that formic acid is the binding species. This conclusion was consistent with the observed pH dependence of formate binding.
The effects of NO and fluoride on the iron-quinone complex of the acceptor side of Photosystem II (PSII) are examined by X- and Q-band EPR spectroscopy. It is found that the EPR signal of the iron-nitrosyl complex changes upon addition of F-. The change is determined to be due to a superhyperfine interaction between the electronic spin (S = 3/2) and the fluorine nuclear spin (I = 1/2), indicating that both F- and NO are bound to the same nonheme iron. To the best of our knowledge, this is the first report of simultaneous binding of both F- and NO to the same iron of a mononuclear nonheme protein. On the basis of an analysis of the hyperfine interaction, a cis F−Fe−NO coordination is indicated. Upon illumination of (NO, Cl-)- or (NO, F-)-treated PSII membranes at 200 K, new X- and Q-band EPR signals are observed in B1||B and B1B modes. Quantitative simulations of these signals provide an unambiguous assignment to the iron-quinone complex, QA-{FeNO},7 of the acceptor site of PSII. The exchange interaction between the iron-nitrosyl complex (S = 3/2) and the semiquinone QA- radical (S = 1/2) is determined to be J = +0.5 cm-1 (F-) and +1.3 cm-1 (Cl-) for Hex = JS1S2. A distribution in the exchange coupling is required to satisfactorily simulate the EPR spectra. This distribution is correlated to small structural variations of the iron-quinone acceptor side of PSII.
The iron quinone-complex of the reaction centers of photosystem II and the purple non-sulphur photosynthetic bacteria contains two quinones, QA and QB connected in series with respect to electron transfer, and separated by a non-heme iron coordinated by amino acid residues. It is the site of inhibition of many of the common photosynthetic herbicides, which act by displacing QB from the center. The complex is responsible for reducing QB to QBH2 in two successive one-electron photo acts. OBH2 dissociates from the center, to be replaced by a new QB molecule and reduces the following membrane-bound electron-transfer complex (cytochrome b6/for b/c1). The energetic, kinetics and mechanism of complex function are reviewed here. Recent crystallographic, spectroscopic and molecular biological evidence has produced a considerable quantity of structural information on this complex. These data have given a less formal and more molecular view of how the complex functions. They have also revealed fundamental differences between the photo system II and bacterial complexes, particularly with respect to the coordination of the iron and its chemistry. The comparative anatomy of the complexes is reviewed and its implications for function discussed.
Q400, a high-potential electron acceptor associated with Photosystem II (PS II) of oxygenic photosynthesis, originally described by Ikegami and Katoh [3], has recently been identified by Petrouleas and Diner [8] as the non-heme iron of the iron-quinone complex of the PS II reaction center. This acceptor, which can function as the Fe(Ill)/Fe(II) redox couple with an Em.7, of 400 mV, demonstrates a pH-dependence of −60 mV/pH unit, indicative of a protonation reaction coupled to Fe(III) reduction.In this review, we describe the chemical and physical properties of the acceptor which led to its identification. Through a combination of optical, EPR and Mössbauer spectroscopy, we also show how the iron, unlike its bacterial reaction center homologue, is capable of redox chemistry involving the neighboring quinones, and how it serves as a sensitive spectroscopic probe, not only of its immediate coordination sphere, but of the sites at which quinones and inhibitors bind to the reaction center. A theoretical description of the Fe(III) EPR spectrum which accounts for the positions, amplitudes and energetics of the observed resonances is also presented.
The EPR spectrum at both X- and S-band (3.94 GHz) of the oxidized acceptor-side iron in photosystem II from spinach shows two absorption-type peaks at g = 8.0 and 5.6. The intensities of these peaks have been measured at X-band in the temperature range 2–10 K. All results can be fully described assuming that the EPR spectrum arises from high-spin Fe(III) with D = 1.0±0.3 cm−1 and E/D = 0.10 ±0.01. Quantifications show that the spectrum in our case represents 0.4–0.5 Fe(III) per reaction center. The EPR parameters are consistent with the iron having bicarbonate and/or tyrosine as ligands in addition to four imidazoles.
Earlier studies have demonstrated that NO binds to the non-heme iron of the PS II ferroquinone complex in competition with the physiological ligand CO2/HCO−3 (Petrouleas, V. and Diner, B.A. (1990) Biochim. Biophys. Acta 1015, 131–140; Diner, B.A. and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141–149). We examine in this paper the effect of cyanide, also a potential iron chelator. Competition experiments involving CN− and NO show that 50 mM CN− at pH 6.5 eliminates the EPR signal at g = 4 arising from the Fe2+-NO adduct. Illumination of CN−-treated PS II preparations under conditions which induce single charge separation produces a new EPR signal at g = 1.98. The temperature and power dependence indicate that this signal is directly or indirectly associated with a transition metal species. The signal is produced with undiminished intensity upon illumination in the presence of hydroxylamine as an exogenous electron donor and of DCMU, indicating that it originates from an acceptor side species prior to the DCMU block. Upon successive one-electron charge separations the g = 1.98 signal oscillates with period of two. These results strongly suggest that the g = 1.98 signal originates from the state Q−AFe2+. An approximate titration of the g = 1.98 signal development as a function of the total cyanide concentration at pH 6.5 indicates a kd of 50–80 mM, significantly higher than the kd for cyanide-NO competition, estimated to be in the range of 10 mM. It is likely that, while displacement of NO requires the binding of one cyanide molecule, development of the modified Q−AFe2+ signal at g = 1.98 requires the binding of more than one cyanide molecules. The kinetics of the fluorescence relaxation following saturating flashes show only subtle differences over the concentration range at which cyanide displaces NO and probably bicarbonate as well. Treatment with higher concentrations of cyanide at pH 6.5 causes an inversion in the phase of the oscillation pattern that characterizes the decay of the fluorescence yield. The latter effect becomes more pronounced at higher pH levels. The absence of a slowing of the fluorescence relaxation in the presence of cyanide may indicate that CN− can fulfill the same role as bicarbonate, perhaps in promoting proton transfer coupled to interquinone electron transfer. Only the relative rates of the two interquinone electron transfer reactions are reversed.
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
The effect of specific proteolytic enzymes on variable fluorescence, p-benzoquinone-mediated oxygen evolution, PS II herbicide (atrazine and bromoxynil) binding, and protein degradation has been analyzed in isolated class II pea chloroplasts.
It was found that:
1. Trypsin and a lysine-specific protease effectively reduce the maximum chlorophyll-a fluorescence yield, whereas the initial fluorescence remains almost constant. At the same number of enzymatic activity units both proteases have practically the same effect.
2. Trypsin and a lysine-specific protease inhibit the p-benzoquinone-mediated flash-induced oxygen evolution with trypsin being markedly more effective at the same number of activity units of both enzymes. Unstacked thylakoids exhibit a higher sensitivity to proteolytic degradation by both enzymes.
3. Trypsin and a lysine-specific protease reduce the binding capacity of [ ¹⁴ C]atrazine, but enhance that of [ ¹⁴ C]bromoxynil (at long incubation times trypsin treatment also impairs bromoxynil binding). At the same specific activity a markedly longer treatment is required for the lysine-specific protease in order to achieve the same degree of modification as with trypsin.
4. Trypsin was found to attack the rapidly-turned-over 32 kDa-protein severely, whereas the lysine-specific protease does not modify this polypeptide. On the other hand, the lysine-specific protease attacks the light harvesting complex II.
5. Under our experimental conditions an arginine-specific protease did not affect chlorophyll-a fluorescence yield, p-benzoquinone-mediated oxygen evolution, herbicide binding and the poly- peptide pattern.
Based on these results a mechanism is proposed in which an as yet unidentified polypeptide with exposable lysine residues, as well as the lysine-free “QB-protein” regulate the electron transfer from Q ⁻ A to Q B and are involved in herbicide binding.
Bicarbonate depletion of chloroplast thylakoids reduces the affinity of the herbicide, ioxynil, to its binding site in Photosystem (PS) II. This herbicide is found to be a relatively more efficient inhibitor of the Hill reaction when HCO−3 is added to CO2-depleted thylakoids in subsaturating rather than in saturating concentrations. The reason for this dependence of the inhibitor efficiency on the HCO−3 concentration is that the inactive HCO−3-deficient PS II reaction chains bind less ioxynil than the active PS II electron-transport chains that have bound HCO−3, and, thus, after addition of a certain amount of ioxynil the concentration of the free herbicide increases when the HCO−3 concentration decreases. Therefore, the inhibition of electron transport by ioxynil increases at decreasing HCO−3 levels. Measurements on the effects of modification of lysine and arginine residues on the rate of electron transport are also presented: the rate of modification is faster in the presence than in the absence of HCO−3. Therefore, we suggest that surface-exposed lysine or arginine residues are not involved in binding of HCO−3 (or CO2 or CO2−3) to its binding protein, but that HCO−3 influences the conformation of its binding environment such that the affinity for certain herbicides and the accessibility for amino acid modifiers are changed.
We investigated the effect of HCO−3 addition to CO2-depleted thylakoids by means of fluorescence techniques. (1) In the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), the net reduction of the primary quinone-type electron acceptor (Q) of Photosystem (PS) II is about 2-times faster in the absence of HCO−3 than in its presence, whether normal, heat-treated or NH2OH-treated samples are used. This effect of HCO−3 is, therefore, not on the O2-evolving apparatus. It is, however, interpreted to be due to an influence of HCO−3 on the kinetics of the reduction of Q, perhaps combined with an effect on the back reaction of Q− with P-680+, the oxidized form of the PS II reaction center chlorophyll a. (2) Fluorescence experiments in the absence of diuron indicate that the absence of HCO−3 results in a complete block at the quinone level; the area over the fluorescence induction curve in the absence of HCO−3 was found to be 2.2-times higher in the absence than in the presence of diuron, pointing to a complete block of BH2 oxidation in the absence of HCO−3. (3) No change in the midpoint potential of Q is observed when HCO−3 is added to CO2-depleted membranes. HCO−3 not only has a large (on/off) effect on the reoxidation of BH2, but also a smaller effect between P-680 and Q. We propose that HCO−3 binding to its specific site in the thylakoid membrane results in a conformational change, allowing normal electron transport between the two photosystems.
— Using high-intensity actinic light, the chlorophyll a fluorescence transient from HCO-3-depleted chloroplasts shows a rapid initial rise (O → I) followed by a slow phase (I → P). In the presence of HCO-3, the O → I rise is delayed but the I → P phase is much more rapid. Using low-intensity actinic light, the chlorophyll a fluorescence transient from 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU)-treated chloroplasts is delayed in the presence of HCO-3. Bicarbonate increases the amount of delayed light emission from chloroplasts given 10 s illumination with weak blue light (0·4 W/m2). DCMU greatly increases the amount of delayed light seen in the presence of HCO-3 under these conditions but decreases the amount seen in the absence of HCO-3. It is suggested that HCO-3 may somehow form or stabilize, in the dark, a number of reaction centers corresponding to the S1 state in the model of B. Forbush, B. Kok and M. McGloin (Photochem. Photobiol. 14, 307–321, 1971).
Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (Em ⋍ −50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (Em ⋍ −275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the Em-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
Photosystem 2 preparations with very high rates of oxygen evolution from the thermophilic cyanobacterium Phormidium laminosum have been studied by EPR spectrometry. In the presence of DCMU the g = 1.82 signal of the iron—quinone electron acceptor (Q) can be observed. It is proposed that DCMU is necessary to disrupt a magnetic interaction, between the semiquinone forms of Q and the secondary acceptor B, which otherwise prevents detection of the Q−Fe signal. A doublet EPR signal arising from magnetic interaction between Q−Fe and the reduced intermediary electron acceptor pheophytin (I−), and a spin-polarized triplet signal assumed to arise from the back reaction between I− and P680+ can also be seen. Preliminary redox titrations of Q reduction have been carried out, indicating Em ⋍ 0 mV.
Bicarbonate (or carbon dioxide) is required for electron transport in isolated broken pea chloroplasts. The site of action of the bicarbonate ion is between the primary electron acceptor of Photosystem 2, Q, and the plastoquinone pool. After trypsin treatment the Hill reaction with ferricyanide does not require bicarbonate. Photosystem 2 inhibiting herbicides act also at this site. Therefore, a possible interaction of bicarbonate and these herbicides in their effect on photosynthetic electron transport was studied.
The reciprocal of the Hill reaction rate in CO2-depleted chloroplasts was plotted against the reciprocal of added bicarbonate concentration in the absence and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-methoxy-4,6-bis (ethylamino)-1,3,5-triazine (simeton) or 4,6-dinitro-o-cresol (DNOC). From these Lineweaver-Burk plots we concluded that DCMU and simeton inhibit both bicarbonate binding and Vmax. There is a purely competitive inhibition of bicarbonate binding by DNOC. We suggest that DNOC may exert its inhibition of electron transport by removing bicarbonate from its binding site.
In isolated thylakoid membranes of Synechococcus leopoliensis we did not find a bicarbonate effect nor inhibition by DNOC after Q, indicating that in the thylakoids of this blue-green alga the binding site for bicarbonate and DNOC between Q and plastoquinone is absent.
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 ratio increases to about 0.2. This is explained by a relatively fast donation () 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 () 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.
Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe2+ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe2+ has low symmetry, with zero-field splitting parameters of |D1|=7.0±1.3 cm−1 and |E1|=1.7±0.5 cm−1 and for the redox state Q1−Fe2+Q2−. The rhombic character of the ligand field is increased in the redox state Q1Fe2+Q−2, where . This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe2+. There exists an electron-spin exchange interaction between Fe2+ and Q−1 and Q−2, having magnitudes |J1|=0.12±0.03 cm−1 and , respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe2+ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe2+ and Q−1 and Q−2. Selective line-broadening of some of the EPR transitions, involving Q− coupling to the magnetic sublevels of the Fe2+ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 Ȧ between Fe2+ and Q−1, thus placing Q1 outside the immediate coordination shell of Fe2+.
The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1.1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes.2.2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200–350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200–400 μs. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1.3.3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q−B → QB−, and following the second flash, Q−B− → QB2−. Benzoquinone at low concentrations oxidises a residual fraction of B− which is usually reduced in the dark before the flash sequence.4.4. A faster component in the decay () following the first flash titrated in over the range Eh > 350 mV. The binary oscillation was still apparent but delayed by one flash.5.5. We discuss the relative redox potentials of the couples and , and the role of the component which titrates in at Eh > 350 mV.
In thylakoid membranes incubated in the dark with ferricyanide, an auxiliary acceptor (Q400) associated with Photosystem II becomes oxidized. It has been reported that, based on oxygen flash-yield data, electron flow to Q400 did not occur in ‘bicarbonate-depleted’ (formate-pretreated) samples. Contrary to this earlier report, we find, based on oxygen flash-yield data and chlorophyll a fluorescence-transient measurements, that Q400 is active as an electron acceptor in formate-pretreated samples. It is concluded that the effect of formate pretreatment is on the flow of electrons between Q, B and the plastoquinone pool and not the flow to Q400. We also believe that another auxiliary acceptor of Photosystem II exists under conditions of formate pretreatment and pH larger than 7.0. This belief is based on increased double advancement in the oxygen flash-yield pattern and increased area above the chlorophyll a fluorescence-rise curve. The double advancement in the oxygen pattern shows a second-order dependence on flash intensity. These effects are eliminated by bicarbonate addition or shifts to lower values of pH such as 6.8. This new acceptor is believed to be different from Q400.
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1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kappa b) of [14C]atrazine to thylakoid membranes indicating a decreased affinity of atrazine to membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.
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