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.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B(A)), bacteriopheopytin (H(A)), the primary quinone (Q(A)) to the secondary quinone (Q(B)). Although the non-heme iron complex (Fe complex) is located between Q(A) and Q(B), it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q(A) and Q(B) in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q(A/B) (E(m)(Q(A/B))) and the Fe complex (E(m)(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E(m)(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E(m)(Fe) downshift by 230 mV upon formation of Q(A)(-) (but not Q(B)(-)) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Function of redox-active tyrosine in photosystem II

Water oxidation at photosystem II Mn-cluster is mediated by the redox-active tyrosine Y(Z). We calculated the redox potential (E(m)) of Y(Z) and its symmetrical counterpart Y(D), by solving the linearized Poisson-Boltzmann equation. The calculated E(m)(Y( )/Y(-)) were +926 mV/+694 mV for Y(Z)/Y(D) with the Mn-cluster in S2 state. Together with the asymmetric position of the Mn-cluster relative to Y(Z/D), differences in H-bond network between Y(Z) (Y(Z)/D1-His(190)/D1-Asn(298)) and Y(D) (Y(D)/D2-His(189)/D2-Arg(294)/CP47-Glu(364)) are crucial for E(m)(Y(Z/D)). When D1-His(190) is protonated, corresponding to a thermally activated state, the calculated E(m)(Y(Z)) was +1216 mV, which is as high as the E(m) for P(D1/D2). We observed deprotonation at CP43-Arg(357) upon S-state transition, which may suggest its involvement in the proton exit pathway. E(m)(Y(D)) was affected by formation of P(D2)(+) (but not P(D1)(+)) and sensitive to the protonation state of D2-Arg(180). This points to an electrostatic link between Y(D) and P(D2).     

Influence of the PsbA1/PsbA3, Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone Q(A) in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry

Ca(2+) and Cl(-) ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr(2+) and Br(-), respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn?Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330-13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn(4)Ca cluster in the S? state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone electron acceptor Q(A), E(m)(Q(A)/Q(A)(-)), were investigated. Since the previous studies on the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E(m)(Q(A)/Q(A)(-)). Here we show that i) the E(m)(Q(A)/Q(A)(-)) was up-shifted by ca. +38mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca(2+)/Sr(2+) exchange up-shifted the E(m)(Q(A)/Q(A)(-)) by ca. +27mV, whereas the Cl(-)/Br(-) exchange hardly influenced E(m)(Q(A)/Q(A)(-)). On the basis of the results of E(m)(Q(A)/Q(A)(-)) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.     

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.     

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.     

Energetics of quinone-dependent electron and proton transfers in Rhodobacter sphaeroides photosynthetic reaction centers

Proteins bind redox cofactors, modifying their electrochemistry and affinity by specific interactions of the binding site with each cofactor redox state. Photosynthetic reaction centers from Rhodobacter sphaeroides have three ubiquinone-binding sites, Q(A), and proximal and distal Q(B) sites. Ubiquinones, which can be doubly reduced and bind 2 protons, have 9 redox states. However, only Q and Q(-) are seen in the Q(A) site and Q, Q(-), and QH(2) in the proximal Q(B) site. The distal Q(B) function is uncertain. Multiple conformation continuum electrostatics (MCCE) was used to compare the ubiquinone electrochemical midpoints (E(m)) and pK(a) values at these three sites. At pH 7, the Q(A)/Q(A)(-) E(m) is -40 mV and proximal Q(B)/Q(B)(-) -10 mV in agreement with the experimental values (assuming a solution ubiquinone E(m) of -145 mV). Q(B) reduction requires changes in nearby residue protonation and SerL223 reorientation. The distal Q(B)/Q(B)(-) E(m) is a much more unfavorable -260 mV. Q(A) and proximal Q(B) sites generally stabilize species with a -1 charge, while the distal Q(B) site prefers binding neutral species. In each site, the dianion is destabilized because favorable interactions with the residues and backbone increase with charge (q), while the unfavorable loss of solvation energy increases with q(2). Therefore, proton binding before a second reduction, forming QH and then QH(-), is always preferred to forming the dianion (Q(-)(2)). The final product QH(2) is higher in energy at the proximal Q(B) site than in solution; therefore, it binds poorly, favoring release. In contrast, QH(2) binds more tightly than Q at the distal Q(B) site.     

Redox potential of quinones in photosynthetic reaction centers from Rhodobacter sphaeroides: dependence on protonation of Glu-L212 and Asp-L213

The absolute values of the one-electron redox potentials of the two quinones (Q(A) and Q(B)) in bacterial photosynthetic reaction centers from Rhodobacter sphaeroides were calculated by evaluating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation at pH 7.0. The redox potential for Q(A) was calculated to be between -173 and -160 mV, which is close to the lowest measured values that are assumed to refer to nonequilibrated protonation patterns in the redox state Q(A)(-). The redox potential of quinone Q(B) is found to be about 160-220 mV larger for the light-exposed than for the dark-adapted structure. These values of the redox potentials are obtained if Asp-L213 is nearly protonated (probability 0.75-1.0) before and after electron transfer from Q(A) to Q(B), while Glu-L212 is partially protonated (probability 0.6) in the initial state Q(A)(-)Q(B)(0) and fully protonated in the final state Q(A)(0)Q(B)(-). Conversely, if the charge state of the quinones is varied from Q(A)(-)Q(B)(0) to Q(A)(0)Q(B)(-) corresponding to the electron transfer from Q(A) to Q(B), Asp-L213 remains protonated, while Glu-L212 changes its protonation state from 0.15 H(+) to fully protonated. In agreement with results from FTIR spectra, there is proton uptake at Glu-L212 going along with the electron transfer, whereas Asp-L213 does not change its protonation state. However, in our simulations Asp-L213 is considered to be protonated rather than ionized as deduced from FTIR spectra. The calculated redox potential of Q(A) shows little dependence on the charge state of Asp-L213, which is due to a strong coupling with the protonation state of Asp-M17 but increases by 50 mV if Glu-L212 changes from the ionized to the protonated charge state. Both are in agreement with fluorescence measurements observing the decay of SP(+)Q(A)(-) in a wide pH regime. The computed difference in redox potential of Q(B) in the light-exposed and dark-adapted structure was traced back to the hydrogen bond of Q(B) with His-L190 that is lost in the dark-adapted structure and the charge of the non-heme iron atom, which is closer to Q(B) in the light-exposed than in the dark-adapted structure.     

Probing subtle coordination changes in the iron-quinone complex of photosystem II during charge separation,by the use of NO

The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.     

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

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.     

Short hydrogen bond between redox-active tyrosine Y(Z) and D1-His190 in the photosystem II crystal structure

The crystal structure of photosystem II (PSII) analyzed at a resolution of 1.9 ? revealed a remarkably short H-bond between redox-active tyrosine Y(Z) and D1-His190 (2.46 ? donor-acceptor distance). Using large-scale quantum mechanical/molecular mechanical (QM/MM) calculations with the explicit PSII protein environment, we were able to reproduce this remarkably short H-bond in the original geometry of the crystal structure in the neutral [Y(Z)O···H···N(ε)-His-N(δ)H···O═Asn] state, but not in the oxidized states, indicating that the neutral state was the one observed in the crystal structure. In addition to the appropriate redox/protonation state of Y(Z) and D1-His190, we found that the presence of a cluster of water molecules played a key role in shortening the distance between Y(Z) and D1-His190. The orientations of the water molecules in the cluster were energetically stabilized by the highly polarized PSII protein environment, where the Ca ion of the oxygen-evolving complex (OEC) and the OEC ligand D1-Glu189 were also involved.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.     

Assignment of the Qy absorbance bands of photosystem II chromophores by low-temperature optical spectroscopy of wild-type and mutant reaction centers

Photosystem II (PSII) contains a collection of pheophytins (Pheo) and chlorophylls (Chl) that have unique absorbance spectra depending on their electronic structure and the surrounding protein environment. Despite numerous efforts to identify the spectra of each cofactor, differing assignments of the chromophore absorbance bands and electrochromic effects have led to conflicting models of pigment organization and chromophore interactions in PSII. We have utilized low-temperature measurements on well-defined redox states, together with the use of site-directed mutants, to make spectral assignments of several reaction center (RC) chromophores. Cryogenic (77 K) optical spectroscopy has been used to trap the bound redox-active quinone, Q(A), in the reduced form and measure the effect of the redox state of Q(A) on PSII chromophores without interference from other redox-active cofactors. The Q(A)(-) minus Q(A) difference spectrum contains a number of features that represent the perturbation of Pheo and Chl absorbance bands upon Q(A) reduction. Using site-directed mutants in which the axial ligand of the D1-side monomeric core Chl, P(A), is changed (D1-H198Q) or the hydrogen-bonding environment of the D1-side Pheo is modified (D1-Q130E), we have assigned the Q(y)() absorbance bands of four chromophores shifted by Q(A) reduction including both RC Pheos, the D1-side monomeric accessory Chl (B(A)), and one other Chl in PSII. The absorbance maximum of B(A) was identified at 683.5 nm from least-squares fits of the D1-H198Q minus wild type (WT) Q(A)(-) minus Q(A) double-difference spectrum; this assignment provides new evidence of a secondary effect of site-directed mutation on a RC chromophore. The other chromophores were assigned from simultaneous fits of the WT and D1-Q130E spectra in which the parameters of only the D1-side Pheo were allowed to vary. The D1-side and D2-side Pheos were found to have lambda(max) values at 685.6 and 669.3 nm, respectively, and another Chl influenced by Q(A)(-) was identified at 678.8 nm. These assignments are in good agreement with previous spectral analyses of intact PSII preparations and reveal that the number of chromophores affected by Q(A) reduction has been underestimated previously. In addition, the assignments are generally consistent with chromophore positions that are similar in the PSII RC and the bacterial photosynthetic RC.     

No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.     

Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp. PCC6803

Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex. The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550). Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium. Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant. The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins. S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays. Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible.     

Molecular interference of Cd(2+) with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.     

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.     

Changes in the redox potential of primary and secondary electron-accepting quinones in photosystem II confer increased resistance to photoinhibition in low-temperature-acclimated Arabidopsis

Exposure of control (non-hardened) Arabidopsis leaves for 2 h at high irradiance at 5 degrees C resulted in a 55% decrease in photosystem II (PSII) photochemical efficiency as indicated by F(v)/F(m). In contrast, cold-acclimated leaves exposed to the same conditions showed only a 22% decrease in F(v)/F(m). Thermoluminescence was used to assess the possible role(s) of PSII recombination events in this differential resistance to photoinhibition. Thermoluminescence measurements of PSII revealed that S(2)Q(A)(-) recombination was shifted to higher temperatures, whereas the characteristic temperature of the S(2)Q(B)(-) recombination was shifted to lower temperatures in cold-acclimated plants. These shifts in recombination temperatures indicate higher activation energy for the S(2)Q(A)(-) redox pair and lower activation energy for the S(2)Q(B)(-) redox pair. This results in an increase in the free-energy gap between P680(+)Q(A)(-) and P680(+)Pheo(-) and a narrowing of the free energy gap between primary and secondary electron-accepting quinones in PSII electron acceptors. We propose that these effects result in an increased population of reduced primary electron-accepting quinone in PSII, facilitating non-radiative P680(+)Q(A)(-) radical pair recombination. Enhanced reaction center quenching was confirmed using in vivo chlorophyll fluorescence-quenching analysis. The enhanced dissipation of excess light energy within the reaction center of PSII, in part, accounts for the observed increase in resistance to high-light stress in cold-acclimated Arabidopsis plants.     

The mechanism of UV-A radiation-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence

The UV-A (320-400 nm) component of sunlight is a significant damaging factor of plant photosynthesis, which targets the photosystem II complex. Here we performed a detailed characterization of UV-A-induced damage in photosystem II membrane particles using EPR spectroscopy and chlorophyll fluorescence measurements. UV-A irradiation results in the rapid inhibition of oxygen evolution accompanied by the loss of the multiline EPR signal from the S(2) state of the water-oxidizing complex. Gradual decrease of EPR signals arising from the Q(A)(-)Fe(2+) acceptor complex, Tyr-D degrees, and the ferricyanide-induced oxidation of the non-heme Fe(2+) to Fe(3+) is also observed, but at a significantly slower rate than the inhibition of oxygen evolution and of the multiline signal. The amplitude of Signal II(fast), arising from Tyr-Z degrees in the absence of fast electron donation from the Mn cluster, was gradually increased during the course of UV-A treatment. However, the amount of functional Tyr-Z decreased to a similar extent as Tyr-D as shown by the loss of amplitude of Signal II(fast) that could be measured in the UV-A-treated particles after Tris washing. UV-A irradiation also affects the relaxation of flash-induced variable chlorophyll fluorescence. The amplitudes of the fast (600 micros) and slow (2 s) decaying components, assigned to reoxidation of Q(A)(-) by Q(B) and by recombination of (Q(A)Q(B))(-) with donor side components, respectively, decrease in favor of the 15-20 ms component, which reflects PQ binding to the Q(B) site. In the presence of DCMU, the fluorescence relaxation is dominated by a 1 s component due to recombination of Q(A)(-) with the S(2) state. After UV-A radiation, this is partially replaced by a much faster component (30-70 ms) arising from recombination of Q(A)(-) with a stabilized intermediate PSII donor, most likely Tyr-Z degrees. It is concluded that the primary damage site of UV-A irradiation is the catalytic manganese cluster of the water-oxidizing complex, where electron transfer to Tyr-Z degrees and P(680)(+) becomes inhibited. Modification and/or inactivation of the redox-active tyrosines and the Q(A)Fe(2+) acceptor complex are subsequent events. This damaging mechanism is very similar to that induced by the shorter wavelength UV-B (280-320) radiation, but different from that induced by the longer wavelength photosynthetically active light (400-700 nm).