An EPR study of the pH dependence of formate effects on Photosystem II.

Effects of formate on rates of O(2) evolution and electron paramagnetic resonance (EPR) signals were observed in the oxygen evolving PS II membranes as a function of pH. In formate treated PS II membranes, decrease in pH value resulted in the inhibition of the O(2) evolving activity, a decrease in the intensity of S(2) state multiline signal but an increase in the intensity of the Q(A)(-)Fe(2+) EPR signal. Time-resolved EPR study of the Y(Z)(*) decay kinetics showed that the light-induced intensity of Y(Z)(*) EPR signal was proportional to the formate concentration. The change in the pH affected both the light-induced intensities and the decay rates of Y(Z)(*), which was found to be faster at lower pH. At 253 K, t(1/e) value of Y(Z)(*) decay kinetics was found to be 8-10 s at pH 6.0 and 18-21 s at pH 5.0. The results presented here indicate that the extent of inhibition at the donor and the acceptor side of PS II due to formate is pH dependent, being more effective at lower pH.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Nonlineal relationship between g=2 doublet and multiline signals in Ca(2+)-depleted Photosystem II

Illuminating of the Ca(2+)-depleted PS II in the S(2) state for a short period induced the doublet signal at g=2 with concomitant diminution of the multiline signal, both in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In the absence of DCMU, the doublet signal decayed (t(1/2) approximately 7 min) during subsequent dark incubation at 273 K and the multiline signal was regenerated to the original amplitude with the same kinetics of the doublet decay. In the presence of DCMU, the doublet signal decayed much faster (t(1/2) approximately 1 min) by charge recombination with Q(A)(-), while the time course of the multiline recovery was inherently identical with that observed in the absence of DCMU. A simple theoretical consideration indicates the direct conversion from the doublet-signal state to the multiline state with no intermediate state between them. Lengthy dark storage at 77 K led to disappearance of the DCMU-affected doublet signal and a Fe(2+)/Q(A)(-) electron spin resonance (ESR) signal, but no recovery of the multiline signal. Notably, the multiline signal was restored by subsequent dark incubation at 273 K. The charge recombination between Q(A)(-) and the doublet signal species led to a thermoluminescence band at 7 degrees C in a medium at pH 5.5. The peak position shifted to 17 degrees C at pH 7.0, presumably due to a pH-dependent change in the redox property of a donor-side radical species responsible for the doublet signal. Based on these results, redox events in the Ca(2+)-depleted PS II are discussed in contradistinction with the normal processes in oxygen-evolving PS II.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca2+-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca(2+)-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y(Z)*. The ratio of the intensities between the g=2 broad signal and the Y(Z)* signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y(Z)* signal decayed together in parallel with the appearance of the S(2) state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S(1)X* state in the transition from the S(1) to the S(2) state, where X* represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y(Z)*. Possible reactions in the S state transitions in Ca(2+)-depleted PS II were discussed.     

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions.

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.     

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.     

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).     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P(680+)Q(A-) limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P(680+) by the secondary electron donor Tyrosine Z cannot occur because Y(Z) is already oxidized. The 1.4 ms recombination is seen in Y(Z)-less mutants of the cyanobacterium Synechocystis. However, the rate of P(680+)Q(A-) recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P(680+) reduction by Y(Z) were obtained, and the rate of the competing P(680+)Q(A-) recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y(Z)(ox) slightly decreased the efficiency of excitation trapping but did not seem to accelerate P(680+)Q(A-) recombination. The two P(680+)Q(A-) lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

The differences in microenvironments and functions of tyrosine radicals YZ and YD in photosystem II studied by EPR

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) were performed to investigate the difference in microenvironments and functions between tyrosine Z (Y(Z)) and tyrosine D (Y(D)). Mn-depletion or Ca(2+)-depletion causes extension of the lifetime of tyrosine radical Y(Z)(*), which can be trapped by rapid freezing after illumination at about 250 K. Above pH 6.5, Y(Z)(*) radical in Mn-depleted PS II shows similar EPR and ENDOR spectra similar to that of Y(D)(*) radical, which are ascribed to a typical neutral tyrosine radical. Below pH 6.5, Y(Z)(*) radical shows quite different EPR and ENDOR spectra. ENDOR spectra show the spin density distribution of the low-pH form of Y(Z)(*) that has been quite different from the high-pH form of Y(Z)(*). The spin density distribution of the low-pH Y(Z)(*) can be explained by a cation radical or the neutral radical induced by strong electrostatic interaction. The pH dependence of the activation energy of the recombination rate between Y(Z)(*) and Q(A)(-) shows a gap of 4.4 kJ/mol at pH 6.0-6.5. In the Ca(2+)-depleted PS II, Y(Z)(*) signal was the mixture of the cation-like and normal neutral radicals, and the pH dependence of Y(Z)(*) spectrum in Ca(2+)-depleted PS II is considerably different from the neutral radical found in Mn-depleted PS II. Based on the recent structure data of cyanobacterial PS II, the pH dependence of Y(Z)(*) could be ascribed to the modification of the local structure and hydrogen-bonding network induced by the dissociation of ASP170 near Y(Z).     

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.     

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA. Despite a significant decrease in the rate of O(2) evolution after this treatment, only small changes in the P680*+ reduction kinetics were observed. Loss of Ca(2+) did not affect P680*+ reduction associated with electron transfer from Y(Z). Since much larger changes in the P680*+ reduction kinetics of intact PS II occurred at comparable free-Ca(2+) concentrations in the presence of EGTA, we conclude that EGTA influenced the P680*+ reduction kinetics by directly interacting with PS II rather than by lowering the free Ca(2+) concentration of the surrounding media. Notwithstanding these effects, we show that useful information about Ca(2+) binding to PS II can be obtained when direct interaction of EGTA is taken into account.     

Interactions of Chloride and Formate at the Donor and the Acceptor Side of Photosystem II

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S₂ state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe₂+Q–A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The DeltaPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y(D)(ox) radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S(2)Q(A)(-) and S(2)Q(B)(-) charge recombinations were stabilized in DeltaPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y(D)(+)Q(A)(-) recombination, pointed to the donor side modifications in DeltaPsbR. EPR measurements revealed that S(1)-to-S(2)-transition and S(2)-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q(A) to Q(B) electron transfer in DeltaPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.     

pH-dependent characteristics of Y(Z) radical in Ca(2+)-depleted photosystem II studied by CW-EPR and pulsed ENDOR

The Y(Z)-tyrosine radical was trapped by freezing immediately after illumination in Ca(2+)-depleted Photosystem II (PS II) membranes and the pH-dependent characteristics of the radical were investigated using CW-EPR and pulsed ENDOR. The spectrum of the Y*(Z) radical trapped in the Y*(Z)S(1) state at pH 5.5 was cation-like as reported in Mn-depleted PS II (H. Mino et al., Spectrochim. Acta A 53 (1997) 1465-1483). By illuminating the PS II-retaining S(2) state, the Y*(Z) radical and a broad doublet signal formed in the g approximately 2 region were trapped concomitantly. The spectrum of the trapped Y*(Z) radical in the Y*(Z)S(2) state was cation-like at pH 5.5 but the pulsed ENDOR measurements reveals the involvement of the neutral Y*(Z) radical in the doublet signal. At pH 7.0, the resulting Y*(Z) signal was the mixture of the cation-like and neutral radical spectra, and considerably different from the neutral radical found in Mn-depleted PS II. pH-Dependent changes in the properties of the Y*(Z) radical are discussed in relation to the redox events occurring in Ca(2+)-depleted PS II.     

Inhibition of photosystem II of spinach by the respiration inhibitors piericidin A and thenoyltrifluoroacetone

The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm'-F)/Fm' and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between QA, primary quinone acceptor in PS II, and QB, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q(B), out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.     

Electrogenic reactions and dielectric properties of photosystem II

This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P(680) and primary quinone acceptor Q(A); (ii) P(680)(+) reduction by the redox-active tyrosine Y(Z) of polypeptide D1; (iii) oxidation of Mn cluster by Y(Z)(ox) followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor Q(B). The electrogenicity related to (i) proton-coupled electron transfer between Q(A)(-) and preoxidized non-heme iron (Fe(3+)) in native and (ii) electron transfer between protein-water boundary and Y(Z)(ox) in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and Y(Z) radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S1 and S3 states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11-15 signal, were detected in Ca2+-depleted PS II. The g=11-15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S2 in Ca2+-depleted PS II. The singlet-like signal was associated with the g=11-15 signal but not with the Y(Z) (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y(Z) radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y(Z) radical was discussed.     

The position of cytochrome b(559) relative to Q(A) in photosystem II studied by electron-electron double resonance (ELDOR)

The electron-electron double resonance (ELDOR) method was applied to measure the dipole interaction between cytochrome (Cyt) b(+)(559) and the primary acceptor quinone (Q(-)(A)), observed at g=2.0045 with the peak to peak width of about 9 G, in Photosystem II (PS II) in which the non-heme Fe(2+) was substituted by Zn(2+). The paramagnetic centers of Cyt b(+)(559)Y(D)Q(-)(A) were trapped by illumination at 273 K for 8 min, followed by dark adaptation for 3 min and freezing into 77 K. The distance between the pair Cyt b(+)(559)-Q(-)(A) was estimated from the dipole interaction constant fitted to the observed ELDOR time profile to be 40+/-1 A. In the membrane oriented PS II particles the angle between the vector from Q(A) to Cyt b(559) and the membrane normal was determined to be 80+/-5 degrees. The position of Cyt b(559) relative to Q(A) suggests that the heme plane is located on the stromal side of the thylakoid membrane. ELDOR was not observed for Cyt b(+)(559) Y(D) spin pair, suggesting the distance between them is more than 50 A.     

Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.