Herbicide effect on the hydrogen-bonding interaction of the primary quinone electron acceptor QA in photosystem II as studied by Fourier transform infrared spectroscopy

The redox potential of Q(A) in photosystem II (PSII) is known to be lower by approximately 100 mV in the presence of phenolic herbicides compared with the presence of DCMU-type herbicides. In this study, the structural basis underlying the herbicide effects on the Q(A) redox potential was studied using Fourier transform infrared (FTIR) spectroscopy. Light-induced Q(A)(-)/Q(A) FTIR difference spectra of Mn-depleted PSII membranes in the presence of DCMU, atrazine, terbutryn, and bromacil showed a strong CO stretching peak of Q(A)(-) at 1,479 cm(-1), while binding of phenolic herbicides, bromoxynil and ioxynil, induced a small but clear downshift by approximately 1 cm(-1). The CO peak positions and the small frequency difference were reproduced in the S(2)Q(A)(-)/S(1)Q(A) spectra of oxygen-evolving PSII membranes with DCMU and bromoxynil. The relationship of the CO frequency with herbicide species correlated well with that of the peak temperatures of thermoluminescence due to S(2)Q(A)(-) recombination. Density functional theory calculations of model hydrogen-bonded complexes of plastoquinone radical anion showed that the small shift of the CO frequency is consistent with a change in the hydrogen-bond structure most likely as a change in its strength. The Q(A)(-)/Q(A) spectra in the presence of bromoxynil, and ioxynil, which bear a nitrile group in the phenolic ring, also showed CN stretching bands around 2,210 cm(-1). Comparison with the CN frequencies of bromoxynil in solutions suggested that the phenolic herbicides take a phenotate anion form in the Q(B) pocket. It was proposed that interaction of the phenolic C-O(-) with D1-His215 changes the strength of the hydrogen bond between the CO of Q(A) with D2-His214 via the iron-histidine bridge, causing the decrease in the Q(A) redox potential.     

Herbicide effect on the photodamage process of photosystem II: fourier transform infrared study

The photodamage process of photosystem II by strong illumination was investigated by examining the herbicide effects on the photoinactivation of redox cofactors. O(2)-evolving photosystem II membranes from spinach in the absence of herbicide and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and bromoxynil were subjected to strong white-light illumination at 298K, and the illumination-time dependence of the activities of Q(A), the Mn cluster, and P680 were monitored using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The decrease in the Q(A) activity was suppressed and accelerated by DCMU and bromoxynil, respectively, in comparison with the sample without herbicide. The intensity change in the S(2)/S(1) FTIR signal of the Mn cluster exhibited a time course virtually identical to that in the Q(A) signal in all the three samples, suggesting that the loss of the S(1)→S(2) transition was ascribed to the Q(A) inactivation and hence the Mn cluster was inactivated not faster than Q(A). The decrease in the P680 signal was always slower than that of Q(A) keeping the tendency of the herbicide effect. Degradation of the D1 protein occurred after the P680 inactivation. These observations are consistent with the acceptor-side mechanism, in which double reduction of Q(A) triggers the formation of (1)O(2)* to promote further damage to other cofactors and the D1 protein, rather than the recently proposed mechanism that inactivation of the Mn cluster initiates the photodamage. Thus, the results of the present study support the view that the acceptor-side mechanism dominantly occurs in the photodamage to PSII by strong white-light illumination.     

Probing the quinone binding site of photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of herbicides

The main cofactors involved in Photosystem II (PSII) oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is predominantly encoded by either the psbA(1) or the psbA(3) gene, the expression of which depends on the environmental conditions. In this work, the Q(B) site properties in PsbA1-PSII and PsbA3-PSII were probed through the binding properties of DCMU, a urea-type herbicide, and bromoxynil, a phenolic-type herbicide. This was done by using helium temperature EPR spectroscopy and by monitoring the time-resolved changes of the redox state of Q(A) by absorption spectroscopy in PSII purified from a His(6)-tagged WT strain expressing PsbA1 or from a His(6)-tagged strain in which both the psbA(1) and psbA(2) genes have been deleted and which therefore only express PsbA3. It is shown that, in both PsbA1-PSII and PsbA3-PSII, bromoxynil does not bind to PSII when Q(B) is in its semiquinone state which indicates a much lower affinity for PSII when Q(A) is in its semiquinone state than when it is in its oxidized state. This is consistent with the midpoint potential of Q(A)(-)/Q(A) being more negative in the presence of bromoxynil than in its absence [Krieger-Liszkay and Rutherford, Biochemistry 37 (1998) 17339-17344]. The addition in the dark of DCMU, but not that of bromoxynil, to PSII with a secondary electron acceptor in the Q(B)(-) state induces the oxidation of the non-heme iron in a fraction of PsbA3-PSII but not in PsbA1-PSII. These results are explained as follows: i) bromoxynil has a lower affinity for PSII with the non-heme iron oxidized than DCMU therefore, ii) the midpoint potential of the Fe(II)/Fe(III) couple is lower with DCMU bound than with bromoxynil bound in PsbA3-PSII; and iii) the midpoint potential of the Fe(II)/Fe(III) couple is higher in PsbA1-PSII than in PsbA3-PSII. The observation of DCMU-induced oxidation of the non-heme iron leads us to propose that Q(2), an electron acceptor identified by Joliot and Joliot [FEBS Lett. 134 (1981) 155-158], is the non-heme iron.     

UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Direct mediatorless electron transport between the monolayer of photosystem II and poly(mercapto-p-benzoquinone) modified gold electrode--new design of biosensor for herbicide detection

Photosystem II (PSII) modified gold electrodes have been prepared providing mediatorless electron transport on the basis of electrodeposited conductive layer poly-mercapto-p-benzoquinone (polySBQ). Such electrodes are suitable in construction of biosensors for PSII inhibiting herbicides. PolySBQ layer was synthesized on (i) screen-printed gold electrodes and (ii) gold microelctodes in an array on silicon substrate, by electrochemical-oxidation of sulpho-p-benzoquinone (SBQ) at +650 mV versus Ag/AgCl. The basic properties of polySBQ layer were characterized using linear sweep voltammetry and atomic force microscopy (AFM). The typical redox response for quinones was observed. The optimal length of the polymer providing direct electron transfer (DET) was found to be very close to 30 nm. PSII particles isolated from the thermophilic cyanobacteria Synechococcus bigranulatus were physically adsorbed on the polySBQ covered gold electrodes. The generation of photocurrent was observed at E=+250 mV (versus Ag/AgCl) without addition of any mediator. The basic properties of DET were studied. We concluded that: (i) PSII active in DET is immobilized in form of monolayer; (ii) the charge transport from PSII to gold working electrode (AuWE) is fast and dominated by the rate of the enzymatic reaction; (iii) polySBQ layer drains electrons from the Q(A) pocket of the photosystem since the electrode activity is inhibited by specific inhibitor, i.e. diuron (DCMU); (iv) the stability of the photosystem immobilized on gold electrodes by using polySBQ is comparable to the stability of PSII in solution under the same experimental conditions; (v) the inhibition of the photosystem by herbicide DCMU follows the sigmoid dependence; (vi) I(50) as well as limit of detection (LOD) show an improved sensitivity compared to other published biosensing systems using PSII as bioactive part.     

Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) > 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.     

Identification of the proton pathway in bacterial reaction centers: decrease of proton transfer rate by mutation of surface histidines at H126 and H128 and chemical rescue by imidazole identifies the initial proton donors.

The pathway for proton transfer to Q(B) was studied in the reaction center (RC) from Rhodobacter sphaeroides. The binding of Zn(2+) or Cd(2+) to the RC surface at His-H126, His-H128, and Asp-H124 inhibits the rate of proton transfer to Q(B), suggesting that the His may be important for proton transfer [Paddock, M. L., Graige, M. S., Feher, G. and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. To assess directly the role of the histidines, mutant RCs were constructed in which either one or both His were replaced with Ala. In the single His mutant RCs, no significant effects were observed. In contrast, in the double mutant RC at pH 8.5, the observed rates of proton uptake associated with both the first and the second proton-coupled electron-transfer reactions k(AB)(()(1)()) [Q(A)(-)(*)Q(B)-Glu(-) + H(+) --> Q(A)(-)(*)Q(B)-GluH --> Q(A)Q(B)(-)(*)-GluH] and k(AB)(()(2)()) [Q(A)(-)(*)Q(B)(-)(*) + H(+) --> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)], were found to be slowed by factors of approximately 10 and approximately 4, respectively. Evidence that the observed changes in the double mutant RC are due to a reduction in the proton-transfer rate constants are provided by the observations: (i) k(AB)(1) at pH approximately pK(a) of GluH became biphasic, indicating that proton transfer is slower than electron transfer and (ii) k(AB)(2) became independent of the driving force for electron transfer, indicating that proton transfer is the rate-limiting step. These changes were overcome by the addition of exogenous imidazole which acts as a proton donor in place of the imidazole groups of His that were removed in the double mutant RC. Thus, we conclude that His-H126 and His-H128 facilitate proton transfer into the RC, acting as RC-bound proton donors at the entrance of the proton-transfer pathways.     

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus

Binding of herbicides to photosystem II inhibits the electron transfer from Q(A) to Q(B) due to competition of herbicides with plastoquinone bound at the Q(B) site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q(B) sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.     

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].     

Kinetics and pathways of charge recombination in photosystem II

The mechanism of charge recombination of the S(2)Q(A)(-) state in photosystem II was investigated by modifying the free energy gap between the quinone acceptor Q(A) and the primary pheophytin acceptor Ph. This was done either by changing the midpoint potential of Ph (using mutants of the cyanobacterium Synechocystis with a modified hydrogen bond to this cofactor), or that of Q(A) (using different inhibitors of the Q(B) pocket). The results show that the recombination rate is dependent on the free energy gap between Ph and Q(A), which confirms that the indirect recombination pathway involving formation of Ph(-) has a significant contribution. In the mutant with the largest free energy gap, direct electron transfer from Q(A)(-) to P(+) predominates. The temperature dependence of the recombination rate was investigated, showing a lower activation enthalpy in this mutant compared with the WT. The data allow the determination of the rate of the direct route and of its relative weight in the various strains. The set of currently accepted values for the midpoint potentials of the Q(A)/Q(A)(-), Ph/Ph(-), and P(+)/P* couples is not consistent with the relatively rapid rate of the indirect recombination pathway found here, nor with the 3% yield of delayed fluorescence as previously estimated by de Grooth and van Gorkom (1981, Biochim. Biophys. Acta 635, 445-456). It is argued that a likely explanation is that the midpoint potentials of the two latter couples are more positive than believed due to electrostatic interactions. If such is the case, the estimation of the midpoint potential of the P(+)/P and S(2)/S(1) couples must also be revised upward, with values of 1260 and 1020 mV, respectively.     

Electrostatic role of the non-heme iron complex in bacterial photosynthetic reaction center

To elucidate the role of the non-heme iron complex (Fe-complex) in the electron transfer (ET) events of bacterial photosynthetic reaction centers (bRC), we calculated redox potentials of primary/secondary quinones Q(A/B) (E(m)(Q(A/B))) in the Fe-depleted bRC. Removing the Fe-complex, the calculated E(m)(Q(A/B)) are downshifted by approximately 220 mV/ approximately 80 mV explaining both the 15-fold decrease in ET rate from bacteriopheophytin (H(A)(-)) to Q(A) and triplet state occurrence in Fe-depleted bRC. The larger downshift in E(m)(Q(A)) relative to E(m)(Q(B)) increases the driving-energy for ET from Q(A) to Q(B) by 140 meV, in agreement with approximately 100 meV increase derived from kinetic studies.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q(B) site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q(B) sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q(A) to Q(B) electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q(B) site inhibitors, DCMU and atrazine. In the sensitive centers, the S(2)Q(A)(-) state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S(2)Q(A)(-) state than the S(1)Q(A) state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S(2)-multiline electron paramagnetic resonance (EPR) signal and the 'split' EPR signal, originating from the S(2)Y(Z) state showed no significant changes upon binding of phenolic inhibitors at the Q(B) site. We thus propose a working model where Q(A) redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q(B) niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q(B) site.     

Replacement of photosynthetic electron transport inhibitors by silicomolybdate

KCN-treated spinach chloroplasts, their photosystem I being ineffective, exhibit a single reaction site for silicomolybdate. Using this heteropolyanion as electron acceptor, photosynthetic oxygen evolution is partially inhibited by ureas, triazines, or phenylpyridazinone herbicides, their inhibitory effect depending on the concentration of silicomolybate. Labelled atrazine attached to isolated chloroplast material is competitively replaced by silicomolybdate in the same manner as e.g. ureas complete with a triazine herbicide. – It is concluded (1) that silicomolybdate is bound and reduced at the herbicide-binding protein, and (2) that the inhibition of silicomolybdate reduction by herbicides such as DCMU is due to loss of reaction sites for silicomolybdate.     

Characterization of photosystem ii electron acceptors in Phormidium laminosum

Chlorophyll a fluorescence has been used to monitor the redox state of the primary electron acceptor of photosystem II (PS II) in the blue-green alga Phormidium laminosum during equilibrium titrations. The shape of induction curves measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) have been analyzed. The induction curves were very similar in unfractionated thylakoid membranes and PS II particles. In both, the fast (alpha) phase was sigmoidal, and was followed by a slow (beta) exponential tail. Thus, the structural organization and complexity of the particles (J. M. Bowes and P. Horton, Biochim, Biophys. Acta 680, 127-133 (1982), as indicated by the occurrence of energy transfer between alpha centers and presence of beta centers, must preexist in the membranes. Redox titration of the initial level of fluorescence indicated the presence of a single quencher QH in the unfractionated thylakoids, midpoint potential: Em7.0 approximately -35 mV (n = 1). Thus, the occurrence of a single acceptor is characteristic for P. laminosum and the absence of a low potential acceptor in PS II particles (J.M. Bowes, P. Horton, and D.S. Bendall, FEBS Lett. 135, 261-264 (1981] was not the result of its removal during their preparation. The midpoint potential of Q varied by -60 mV/pH unit in PS II particles and membrane fragments, with a pK at pH greater than 8.5 (particles) and at pH 7.5 (fragments). In PS II particles, DCMU raised the pK by approximately 0.5 pH units. It is argued that the pH dependence of Q is conferred by protonation of a protein which accompanies its reduction rather than protonation of the semiquinone Q X itself.     

How does the QB site influence propagate to the QA site in photosystem II?

The redox potential of the primary quinone Q(A) [E(m)(Q(A))] in photosystem II (PSII) is lowered by replacement of the native plastoquinone (PQ) with bromoxynil (BR) at the secondary quinone Q(B) binding site. Using the BR-bound PSII structure presented in the previous Fourier transform infrared and docking calculation studies, we calculated E(m)(Q(A)) considering both the protein environment in atomic detail and the protonation pattern of the titratable residues. The calculated E(m)(Q(A)) shift in response to the replacement of PQ with deprotonated BR at the Q(B) binding site [ΔE(m)(Q(A))(PQ→BR)] was -55 mV when the three regions, Q(A), the non-heme iron complex, and Q(B) (Q(B) = PQ or BR), were treated as a conjugated supramolecule (Q(A)-Fe-Q(B)). The negative charge of BR apparently contributes to the downshift in ΔE(m)(Q(A))(PQ→BR). This downshift, however, is mostly offset by the influence of the residues near Q(B). The charge delocalization over the Q(A)-Fe-Q(B) complex and the resulting H-bond strength change between Q(A) and D2-His214 are crucial factors that yield a ΔE(m)(Q(A))(PQ→BR) of -55 mV by (i) altering the electrostatic influence of the H-bond donor D2-His214 on E(m)(Q(A)) and (ii) suppressing the proton uptake events of the titratable residues that could otherwise upshift ΔE(m)(Q(A))(PQ→BR) during replacement of PQ with BR at the Q(B) site.     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.     

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.