The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II

The PsbL protein is a 4.5 kDa subunit at the monomer–monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~ 15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ?(N6–N8), ?(P11–V12) and ?(E13–N15), have been created. The ?(N6–N8) and ?(P11–V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ?(E13–N15) cells had the most severe phenotype. The Δ(E13–N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13–N15) strain indicate that the binding of plastoquinone to the Q_B-binding site had been altered such that the affinity of Q_B is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ?(E13–N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from Q_A through indirect interactions with the D–E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Binding affinity of bicarbonate and formate in herbicide-resistant D1 mutants of Synechococcus sp. PCC 7942

We examined the effects of mutations at amino acid residues S264 and F255 in the D1 protein on the binding affinity of the stimulatory anion bicarbonate and inhibitory anion formate in Photosystem II (PS II) in Synechococcus sp. PCC 7942. Measurements on the rates of oxygen evolution in the wild type and mutant cells in the presence of different concentrations of formate with a fixed bicarbonate concentration and vice versa, analyzed in terms of an equilibrium activator-inhibitor model, led to the conclusion that the equilibrium dissociation constant for bicarbonate is increased in the mutants, while that of the formate remains unchanged (11±0.5 mM). The hierarchy of the equilibrium dissociation constant for bicarbonate (highest to lowest, ±2 M) was: D1-F255L/S264A (46 M)>D1-F255Y/ S264A (31 M)D1-S264A (34 M)D1-F255Y (33 M)>wild type (25 M). The data suggest the importance of D1-S264 and D1-F255 in the bicarbonate binding niche. A possible involvement of bicarbonate and these two residues in the protonation of Q_B ^-, the reduced secondary plastoquinone of PS II, in the D1 protein is discussed.Abbreviations Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - MES 2-[N-morpholino]ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - Q_A bound plastoquinone, a one-electron acceptor in Photosystem II - Q_B another bound plastoquinone, a two-electron acceptor in Photosystem II This paper is dedicated to the memory of my dear friend Robin Hill-Govindjee.     

Functional complementation of the Arabidopsis thaliana psbo1 mutant phenotype with an N-terminally His6-tagged PsbO-1 protein in photosystem II

The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine₆-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of Q_B and the delayed exchange of Q_BH₂ with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His₆-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS II_α/PS II_β reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His₆-tagged PsbO-1 protein restored efficient electron transfer to Q_B, while in the presence of DCMU, charge recombination between Q_A⁻ and the S₂ state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His₆-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined.     

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Evidence from thermoluminescence for bicarbonate action on the recombination reactions involving the secondary quinone electron acceptor of Photosystem II

In this paper, we present the first measurements on thermoluminescence from isolated thylakoids to probe the recombination reactions of S₂ (or possibly S₃) with Q^?_B or Q^?_A, after bicarbonate depletion and its readdition. The effects of bicarbonate depletion on the S₂Q^?_B (or S₃O^?_B) thermoluminescence band was (1) a 6–10°C shift to a higher temperature; (2) a reduction in its intensity upon prolonged depletion; and (3) elimination after the first few flashes of the characteristic period four oscillations in its intensity as a function of the flash number. On the other hand, addition of diuron (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea), which blocks electron flow from Q^?_A to Q_B, produced the same thermoluminescence band, at about + 20°C, assigned to S₂Q^?_A recombination, in both depleted and reconstituted samples. These results suggest (1) the initial effect of bicarbonate depletion is to increase the activation energy for S₂(S₃)Q^?_B recombination; (2) with further depletion, the incidence of this recombination decreases and the cycling of the S₂Q^?_B and S₃Q^?_B recombination is inhibited through effects at the Q_B apoprotein; and (3) the depletion effects are fully reversible. It is suggested that a conformational change of the PS II complex in the region of the Q_B apoprotein is responsible for these effects.     

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.     

Nuclear mutations affecting plastoquinone accumulation in maize

We have isolated and characterized two nuclear mutations which affect plastoquinone accumulation in maize. The mutations, hcf103 and hcf114, modify the same genetic locus. Plants homozygous for either mutant allele exhibit reduced PS II electron transport activity, reduced variable chlorophyll fluorescence and reduced delayed fluorescence yield. In these ways, hcf103 and hcf114 resemble previously described PS II mutants which lack stably assembled PS II reaction center complexes. However, unlike most previously described PS II mutants, hcf103 and hcf114 possess stable membrane-associated PS II complexes. Plastoquinone (PQ-9), which performs a variety of redox functions essential to normal non-cyclic electron transport, is severely depleted in the mutants. The lack of PS II electron transport activity is attributed to the absence of PQ-9. This is the first report of mutants deficient in PQ which do not also suffer serious pleiotropic defects.Abbreviations PS II Photosystem II - PQ plastoquinone - Q_A and Q_B primary and secondary stable electron acceptors of PS II - HPLC high pressure liquid chromatography - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

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     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

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Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.     

Bicarbonate effects in leaf discs from spinach

In this paper, we show the unique role of bicarbonate ion in stimulating the electron transfer of photosystem II (PS II) in formate-treated leaf discs from spinach. This is referred to as the bicarbonate effect and is independent of the role of CO₂ in CO₂ fixation. It is shown to have two sites of action:

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

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     

Oxygen-evolving system and secondary quinonic acceptors are highly reduced in dark adapted Euglena cells: A thermoluminescence study

Characteristics of thermoluminescence glow curves were compared in three types of Euglena cells: (i) strictly autotrophic, Cramer and Myers cells; (ii) photoheterotrophic cells sampled from an exponentially growing culture containing lactate as substrate repressing the photosynthetic activity; (iii) semiautotrophic cells, sampled when the lactate being totally exhausted, the photosynthesis was enhanced.In autotrophic and semiautotrophic cells, composite curves were observed after series of two or more actinic flashes fired at –10°C, which can be deconvoluted into a large band peaking in the range 12–22°C and a smaller one near 40°C, This second band presents the characteristics of a typical B band (due to S_2/3Q_B ^- recombination), whereas the first one resembled the band, shifted by -15–20°C, which is observed in herbicide resistant plants. The amplitude of this major band, which was in all cases very low after one flash, exhibited oscillations of period four but rapidly damping, with maxima after two and six flashes. In contrast, photoheterotrophic Euglena displayed single, non-oscillating curves with maxima in the range 5–10°C.In autotrophic and semiautotrophic cells, oxidizing pretreatments by either a preillumination with one or more (up to twenty-five) flashes, or a far-red preillumination in the presence of methylviologen, followed by a short dark period, induced thermoluminescence bands almost single and shifted by +3–5°C, or +12°C, respectively. In autotrophic cells, far-red light plus methyl viologen treatment induced a band peaking at 31°C, as in isolated thylakoids from Euglena or higher plants, while it had barely any effect in photoheterotrophic cells.Due to metabolic activities in dark-adapted cells, a reduction of redox groups at the donor and acceptor sides of PS II dark-adapted cells is supposed to occur. Two different explanations can be proposed to explain such a shift in the position of the main band in dark-adapted autotrophic control. The first explanation would be that in these reducing conditions a decreasing value of the equilibrium constant for the reaction: S_nQ_A ^-Q_BS_nQ_AQ_B ^-, would determine the shift of the main TL band towards low temperatures, as observed in herbicide resistant material. The second explanation would be that the main band would correspond to peak III already observed in vivo and assigned to S_2/3Q_B ^2- recombinations.Abbreviations CM Cramer and Myers - D₁ a 32 kDa protein component of the PS II reaction center, psbA.gene product - D₂ a 34 kDa protein component of the PS II reaction center, psbD gene product - FR lar-red illumination - Lexpo and Lstat cells from lactate culture samples at exponential and stationary phase of growth - MV methylviologen - pBQ parabenzoquinone - PQ plastoquinone - PS II photosystem II - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - TL thermoluminescence     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

The lumenal loop connecting transmembrane helices I and II of the D1 polypeptide is important for assembly of the photosystem two complex

Current structural models indicate that the D1 and D2 polypeptides of the Photosystem two reaction center complex (PS II RC) each span the thylakoid membrane five times. In order to assess the importance of the lumenal extrinsic loop that connects transmembrane helices I and II of D1 we have constructed five deletion mutants and two double mutants in the cyanobaterium Synechocystic sp. PCC 6803. Four of the deletion mutants (59–65, 69–74, 79–86 and 109–110) are obligate photoheterotrophs unable to accumulate D1 in the membrane as assayed by immunoblotting experiments or pulse-labelling experiments using [³⁵S]-methionine. In contrast deletion mutant 100 which lacks A100 behaved very similarly to the WT control strain in terms of photoautotrophic growth rate, saturated rates of oxygen evolution, flash-induced oxygen evolution, fluorescence induction and decay, and thermoluminescence. 100 is the first example of an internal deletion on the lumenal side of the D1 polypeptide that is benign to photosystem two function. Double mutant D103G/E104A also behaves similarly to the WT control strain leading to the conclusion that residues D103 and E104 are unlikely to be involved in ligating the metal ions Mn or Ca²⁺, which are needed for photosynthetic oxygen evolution. Double mutant, G109A/G110A, was constructed to assess the significance of this GlyGly motif which is also conserved in the L subunit of purple bacterial reaction centres. The G109A/G110A mutant is able to evolve oxygen at approximately 50–70% of WT rates but is unable to grow phatoautotrophically apparently because of an enhanced sensitivity to photoinactivation than the WT control strain. A photoautotropic revertant was isolated from this strain and shown to result from a mutation that restored the WT codon at position 109. Pulse-chase experiments in cells using [³⁵S]-methionine showed that resistance to photoinhibition in the revertant correlated with an enhanced rate of incorporation of D1 into the membrane compared to mutant G109A/G110A. The sensitivity to photoinhibition shown by the G109A/G110A mutant is therefore consistent with a perturbation to the D1 repair cycle possibly at the level of D1 synthesis or incorporation of D1 into the PS II complex.Abbreviations DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes- 4-morpholineethanesulfonic acid - PCR- polymerase chain reaction - PS II- Photosystem II - TL- thermoluminescence - PQ- plastoquinone - PS II^–- absence of PS II activity - PS^–- incapable of photoautotrophic growth - Q_A- primary plastoquinone electron acceptor - Q_B- secondary plastoquinone electron acceptor - SDS- sodium dodecyl sulphate     

The molecular mechanism of the bicarbonate effect at the plastoquinone reductase site of photosynthesis

It has been known for some time that bicarbonate reverses the inhibition, by formate under HCO₃ ^--depletion conditions, of electron transport in thylakoid membranes. It has been shown that the major effect is on the electron acceptor side of photosystem II, at the site of plastoquinone reduction. After presenting a historical introduction, and a minireview of the bicarbonate effect, we present a hypothesis on how HCO₃ ^- functions in vivo as (a) a proton donor to the plastoquinone reductase site in the D1-D2 protein; and (b) a ligand to Fe²⁺ in the Q_A-Fe-Q_B complex that keeps the D1-D2 proteins in their proper functional conformation. They key points of the hypothesis are: (1) HCO₃ ^- forms a salt bridge between Fe²⁺ and the D2 protein. The carboxyl group of HCO₃ ^- is a bidentate ligand to Fe²⁺, while the hydroxyl group H-bonds to a protein residue. (2) A second HCO₃ ^- is involved in protonating a histidine near the Q_B site to stabilize the negative charge on Q_B. HCO₃ ^- provides a rapidly available source of H⁺ for this purpose. (3) After donation of a H⁺, CO₃ ^2- is replaced by another HCO₃ ^-. The high pKa of CO₃ ^2- ensures rapid reprotonation from the bulk phase. (4) An intramembrane pool of HCO₃ ^- is in equilibrium with a large number of low affinity sites. This pool is a H⁺ buffering domain functionally connecting the external bulk phase with the quinones. The low affinity sites buffer the intrathylakoid [HCO₃ ^-] against fluctuations in the intracellular CO₂. (5) Low pH and high ionic strength are suggested to disrupt the HCO₃ ^- salt bridge between Fe²⁺ and D₂. The resulting conformational change exposes the intramembrane HCO₃ ^- pool and low affinity sites to the bulk phase.Two contrasting hypotheses for the action of formate are: (a) it functions to remove bicarbonate, and the low electron transport left in such samples is due to the left-over (or endogenous) bicarbonate in the system; or (b) bicarbonate is less of an inhibitor and so appears to relieve the inhibition by formate. Hypothesis (a) implies that HCO₃ ^- is an essential requirement for electron transport through the plastoquinones (bound plastoquinones Q_A and Q_B and the plastoquinone pool) of photosystem II. Hypothesis (b) implies that HCO₃ ^- does not play any significant role in vivo. Our conclusion is that hypothesis (a) is correct and HCO₃ ^- is an essential requirement for electron transport on the electron acceptor side of PS II. This is based on several observations: (i) since HCO₃ ^-, not CO₂, is the active species involved (Blubaugh and Govindjee 1986), the calculated concentration of this species (220 M at pH 8, pH of the stroma) is much higher than the calculated dissociation constant (Kd) of 35–60 M; thus, the likelihood of bound HCO₃ ^- in ambient air is high; (ii) studies on HCO₃ ^- effect in thylakoid samples with different chlorophyll concentrations suggest that the left-over (or endogenous) electron flow in bicarbonate-depleted chloroplasts is due to left-over (or endogenous) HCO₃ ^- remaining bound to the system (Blubaugh 1987).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (common name: diuron) - PSII photosystem II - Q_A first plastoquinone electron acceptor of PSII - Q_B second plastoquinone acceptor of PS II