Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Properties of inactive Photosystem II centers

A fraction (usually in the range of 10–25%) of PS II centers is unable to transfer electrons from the primary quinone acceptor Q_A to the secondary acceptor Q_B. These centers are inactive with respect to O₂ evolution since their reopening after photochemical charge separation to the S₂O_A ^- state involves predominantly a back reaction to S₁Q_A in the few seconds time range (slower phases are also occurring). Several properties of these centers are analyzed by fluorescence and absorption change experiments. The initial rise phase F_o-F_pl of fluorescence induction under weak illumination reflects both the closure of inactive centers and the modulation of the fluorescence yield by the S-states of the oxygen-evolving system: We estimate typical relative amplitudes of these contributions as, respectively, 65 and 35% of the F_o-F_pl amplitude. The half-rise time of this phase is significantly shorter than for the fluorescence induction in the presence of DCMU (in which all centers are involved). This finding is shown to be consistent with inactive centers sharing the same light-harvesting antenna as normal centers, a view which is also supported by comparing the dependence of the fluorescence yield on the amount of closed active or inactive centers estimated through absorption changes. It is argued that the exponential kinetics of the F_o-F_pl phase does not indicate absence of excitation energy transfer between the antennas of inactive and active centers. We show that the acceptor dichlorobenzoquinone does not restore electron transfer in inactive centers, in disagreement with previous suggestions. We confirm, however, the enhancement of steady-state electron flow caused by this quinone and suggest that it acts by relieving a blocking step involved in the reoxidation of a fraction of the plastoquinone pool. Part of the discrepancies between the present results and those from previous literature may arise from the confusion of inactive centers characterized on a single turnover basis and PS II centers that become blocked under steady-state conditions because of deficient reoxidation of their secondary acceptors.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - PS photosystem     

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: Cytochromec oxidoreductase?

The Q cycle mechanism of thebc ₁ complex requires two quinone reaction centers, the hydroquinone oxidation (Q_P) and the quinone reduction (Q_N) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of Q_P site inhibitors to thebc ₁ complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutantbc ₁ complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochromeb and parts of the iron-sulfur protein. At least two different binding sites for Q_P site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochromeb, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the Q_P pocket is proposed.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Thermoluminescence and Delayed Luminescence Investigation of the Green Alga,Chlamydobotrys stellata

Thermoluminescence and delayed luminescence investigations of the autotrophically and photoheterotrophically cultivated green alga, Chlamydobotrys stellata, demonstrated that both the thermoluminescence and delayed luminescence yields are much lower in the photoheterotophic algae than in the autotrophic ones due to an efficient luminescence quenching of unknown mechanism. The relative contributions of the so called Q (S₂Q^?_A charge recombination) and B (S₂Q^?_B and S₃Q^?_B charge recombinations) thermoluminescence bands to the glow curve as well as the Q_A(S₂Q^?_B charge recombination) and Q_B (S₂Q^?_B and S₃Q^?_B charge recombinations) delayed luminescence components to the delayed luminescence decay of autotrophically and photoheterotrophically cultivated Chl. stellata were compared using a computer assisted curve resolution method. It was found that, while in the autotrophic cells the area of the B band was considerably larger than of the Q band, in photoheterotrophic cells the Q band was more effectively charged than the B band. In the delayed luminescence decay curves measured in the seconds to minutes time region the amplitude of the Q_A component relative to that of the Q_B component was larger in the photoheterotrophic cells than in the autotrophic ones. These observations demonstrate that, after light-induced charge separation in the photosystem II reaction centers of autotrophic cells, electrons are “quasipermanently” stored mainly in the secondary quinone acceptor pool, Q_B but in the nonquenched photosystem II reaction centers of photoheterotrophic cells the main reservoir of electrons is the primary quinone acceptor, Q_A. This behaviour indicates an inhibition of electron transport in the photoheterotrophic alga at the level of the secondary quinone acceptor, Q_B.     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, Q_A, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320–330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - F_o initial fluorescence level using dark-adapted thylakoids - Inactive reaction centers reaction centers inactive in plastoquinone reduction - PS II Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II Department of Plant Biology, University of IllinoisDepartment of Physiology & Biophysics, University of Illinois     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

Kinetics of the oxidation—reduction reactions of the photosystem II quinone acceptor complex,and the pathway for deactivation

When the photosystem II quinone acceptor complex has been singly reduced to the state Q_AQ^?_B, there is a 22 s half-time back-reaction of Q^?_B with an oxidized photosystem II donor (S₂), directly measured here for the first time. From the back-reaction kinetics with and without inhibitors, kinetic and equilibrium parameters have been estimated. We suggest that the state Q_AQ^?_B of the complex is formed by a second-order reaction of vacant reaction centers in the state Q^?_A with plastoquinone from the pool, and discuss the physico-chemical parameters involved.     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

Influence of magnetic fields on the P-870 triplet state in Rps. sphaeroides reaction centers

Magnetic fields influence two properties of the P-870 triplet state observed in Rps. sphaeroides reaction centers: the yield of formation and the kinetics of decay. These effects have been studied in reaction centers which were prepared in three different states: state Q_A ^–, state Q_A ^2– and state (– Q_A) (Q_A depleted). The triplet yields decrease with increasing magnetic fields, with B1/2's of about 140, 41 and 57 Gauss, respectively. The half-time of ³P-870 decay is not influenced by the field in state Q_A ^–; it increases at increasing fields, in state Q_A ^2– and state (– Q_A), with the same B1/2 as the triplet yield. These results are discussed in the framework of current theories of the radical-pair dynamics and of the mechanism of triplet decay.Abbreviations I primary electron acceptor - LDAO lauryldimethylamine oxide - P-870 primary electron donor - Q_A first quinone acceptor - SDS sodium dodecylsulfate - YAG Yttrium Aluminum Garnet     

Modification of quinone electrochemistry by the proteins in the biological electron transfer chains: examples from photosynthetic reaction centers

Quinones such as ubiquinone are the lipid soluble electron and proton carriers in the membranes of mitochondria, chloroplasts and oxygenic bacteria. Quinones undergo controlled redox reactions bound to specific sites in integral membrane proteins such as the cytochrome bc₁ oxidoreductase. The quinone reactions in bacterial photosynthesis are amongst the best characterized, presenting a model to understand how proteins modulate cofactor chemistry. The free energy of ubiquinone redox reactions in aqueous solution and in the Q_A and Q_B sites of the bacterial photosynthetic reaction centers (RCs) are compared. In the primary Q_A site ubiquinone is reduced only to the anionic semiquinone (Q^•−) while in the secondary Q_B site the product is the doubly reduced, doubly protonated quinol (QH₂). The ways in which the protein modifies the relative energy of each reduced and protonated intermediate are described. For example, the protein stabilizes Q^•− while destabilizing Q⁼ relative to aqueous solution through electrostatic interactions. In addition, kinetic and thermodynamic mechanisms for stabilizing the intermediate semiquinones are compared. Evidence for the protein sequestering anionic compounds by slowing both on and off rates as well as by binding the anion more tightly is reviewed.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Characterization of the alterations of the chlorophyll a fluorescence induction curve after addition of Photosystem II inhibiting herbicides

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo′), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo′ and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S₀ state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, Q_B. The addition of a herbicide causes a transfer of the electron on Q_B to the primary quinone acceptor, Q_A, and displacement of Q_B by the herbicide; the reduced Q_A leads to a higher Fo′. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q_A, non-heme Fe²⁺ ion and neighboring residues_. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q_A state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q_A-His^M219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P⁺/Q_A⁻ recombination rate temperature dependence.     

Characterization of a semi-stable,charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q_A to Q_B electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870⁺ with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870⁺. In reaction centers depleted of Q_B, where forward electron transfer from Q_A is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870⁺ could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of the Primary Electron Transfer Rate in Photosynthetic Reaction Centers by Reduction of a Secondary Acceptor

Photosynthetic application of picosecond spectroscopic techniques to bacterial reaction centers has led to a much greater understanding of the chemical nature of the initial steps of photosynthesis. Within 10 ps after excitation, a charge transfer complex is formed between the primary donor, a “special pair” of bacteriochlorophyll molecules, and a transient acceptor involving bacteriopheophytin. This complex subsequently decays in about 120 ps by donating the electron to a metastable acceptor, a tightly bound quinone.

Recent experiments with conventional optical and ESR techniques have shown that when reaction centers are illuminated by a series of single turnover flashes in the presence of excess electron donors and acceptors, a stable, anionic ubisemiquinone is formed on odd flashes and destroyed on even flashes, suggesting that the acceptor region contains a second quinone that acts as a two-electron gate between the reaction center and subsequent electron transport events involving the quinone pool.

Utilizing standard picosecond techniques, we have examined the decay of the charge transfer complex in reaction centers in the presence of the stable semiquinone, formed by flash illumination with a dye laser 10 s before excitation by a picosecond pulse. In this state the decay rate for the charge transfer complex is considerably slower than when no electron is present in the quinone acceptor region. This indicates fairly strong coupling between constituents of the reaction center-quinone acceptor complex and may provide a probe into the relative positions of the various components.

     

The primary structure of D1 near the QB pocket influences oxygen evolution

Photosystem II (PS II) is the site of oxygen evolution. Activation of dark adapted samples by a train of saturating flashes produces oxygen with a yield per flash which oscillates with a periodicity of four. Damping of the oxygen oscillations is accounted for by misses and double hits. The mechanisms hidden behind these parameters are not yet fully understood. The components which participate in charge transfer and storage in PS II are believed to be anchored to the heterodimer formed by the D1 and D2 proteins. The secondary plastoquinone acceptor Q_B binds on D1 in a loop connecting the fourth and fifth helices (the Q_B pocket). Several D1 mutants, mutated in the Q_B binding region, have been studied over the past ten years.In the present report, our results on nine D1 mutants of Synechocystis PCC 6714 and 6803 are analyzed. When oxygen evolution is modified, it can be due to a change in the electron transfer kinetics at the level of the acceptor side of PS II and also in some specific mutants to a long ranging effect on the donor side of PS II. The different properties of the mutants enable us to propose a classification in three categories. Our results can fit in a model in which misses are substantially determined by the fraction of centers which have Q_A ^- before each flash due to the reversibility of the electron transfer reactions. This idea is not new but was more thoroughly studied in a recent paper by Shinkarev and Wraight (1993). However, we will show in the discussion that some doubts remain as to the true origin of misses and double hits.Abbreviations BQ p-benzoquinone - Chl chlorophyll - D1 and D2 proteins of the core of PS II - DCMU 3-(3,4-dichlorophenyl)-1,1 dimethyl urea - OEC oxygen evolving complex - P₆₈₀ chlorophyll center of PS II acting as the primary donor - PS II Photosystem II - Q_A and Q_B primary and secondary quinone electron acceptor - TL thermoluminescence     

Carboxylate shifts steer interquinone electron transfer in photosynthesis

Understanding the mechanisms of electron transfer (ET) in photosynthetic reaction centers (RCs) may inspire novel catalysts for sunlight-driven fuel production. The electron exit pathway of type II RCs comprises two quinone molecules working in series and in between a non-heme iron atom with a carboxyl ligand (bicarbonate in photosystem II (PSII), glutamate in bacterial RCs). For decades, the functional role of the iron has remained enigmatic. We tracked the iron site using microsecond-resolution x-ray absorption spectroscopy after laser-flash excitation of PSII. After formation of the reduced primary quinone, Q_A⁻, the x-ray spectral changes revealed a transition (t_½ ≈ 150 μs) from a bidentate to a monodentate coordination of the bicarbonate at the Fe(II) (carboxylate shift), which reverted concomitantly with the slower ET to the secondary quinone Q_B. A redox change of the iron during the ET was excluded. Density-functional theory calculations corroborated the carboxylate shift both in PSII and bacterial RCs and disclosed underlying changes in electronic configuration. We propose that the iron-carboxyl complex facilitates the first interquinone ET by optimizing charge distribution and hydrogen bonding within the Q_AFeQ_B triad for high yield Q_B reduction. Formation of a specific priming intermediate by nuclear rearrangements, setting the stage for subsequent ET, may be a common motif in reactions of biological redox cofactors.