Effects of formate binding on the quinone-iron electron acceptor complex of photosystem II

EPR was used to study the influence of formate on the electron acceptor side of photosystem II (PSII) from Thermosynechococcus elongatus. Two new EPR signals were found and characterized. The first is assigned to the semiquinone form of Q(B) interacting magnetically with a high spin, non-heme-iron (Fe2(+), S=2) when the native bicarbonate/carbonate ligand is replaced by formate. This assignment is based on several experimental observations, the most important of which were: (i) its presence in the dark in a significant fraction of centers, and (ii) the period-of-two variations in the concentration expected for Q(B)(?-) when PSII underwent a series of single-electron turnovers. This signal is similar but not identical to the well-know formate-modified EPR signal observed for the Q(A)(?-)Fe2(+) complex (W.F.J. Vermaas and A.W. Rutherford, FEBS Lett. 175 (1984) 243-248). The formate-modified signals from Q(A)(?-)Fe2(+) and Q(B)(?-)Fe2(+) are also similar to native semiquinone-iron signals (Q(A)(?-)Fe2(+)/Q(B)(?-)Fe2(+)) seen in purple bacterial reaction centers where a glutamate provides the carboxylate ligand to the iron. The second new signal was formed when Q(A)(?-) was generated in formate-inhibited PSII when the secondary acceptor was reduced by two electrons. While the signal is reminiscent of the formate-modified semiquinone-iron signals, it is broader and its main turning point has a major sub-peak at higher field. This new signal is attributed to the Q(A)(?-)Fe2(+) with formate bound but which is perturbed when Q(B) is fully reduced, most likely as Q(B)H? (or possibly Q(B)H(?-) or Q(B)(2?-)). Flash experiments on formate-inhibited PSII monitoring these new EPR signals indicate that the outcome of charge separation on the first two flashes is not greatly modified by formate. However on the third flash and subsequent flashes, the modified Q(A)(?-)Fe2(+)Q(B)H? signal is trapped in the EPR experiment and there is a marked decrease in the quantum yield of formation of stable charge pairs. The main effect of formate then appears to be on Q(B)H? exchange and this agrees with earlier studies using different methods.     

Semiquinone-iron complex of photosystem II: EPR signals assigned to the low-field edge of the ground state doublet of QA•-Fe2+ and QB•-Fe2+

The quinone-iron complex of the electron acceptor complex of Photosystem II was studied by EPR spectroscopy in Thermosynechococcus elongatus. New g ～ 2 features belonging to the EPR signal of the semiquinone forms of the primary and secondary quinone, i.e., Q(A)(?-)Fe(2+) and Q(B)(?-)Fe(2+), respectively, are reported. In previous studies, these signals were missed because they were obscured by the EPR signal arising from the stable tyrosyl radical, TyrD(?). When the TyrD(?) signal was removed, either by chemical reduction or by the use of a mutant lacking TyrD, the new signals dominated the spectrum. For Q(A)(?-)Fe(2+), the signal was formed by illumination at 77 K or by sodium dithionite reduction in the dark. For Q(B)(?-)Fe(2+), the signal showed the characteristic period-of-two variations in its intensity when generated by a series of laser flashes. The new features showed relaxation characteristics comparable to those of the well-known features of the semiquinone-iron complexes and showed a temperature dependence consistent with an assignment to the low-field edge of the ground state doublet of the spin system. Spectral simulations are consistent with this assignment and with the current model of the spin system. The signal was also present in Q(B)(?-)Fe(2+) in plant Photosystem II, but in plants, the signal was not detected in the Q(A)(?-)Fe(2+) state.     

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.     

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.     

Electron paramagnetic resonance study of the electron transfer reactions in photosystem II membrane preparations from Arabidopsis thaliana

Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe2(+) and Phe- Q(A)- Fe2(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S?- and S?-states of CaMn?O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn?O(x)-cluster in the S?-, S?-, S?-, and the S?-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b??? were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.     

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.     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

Iron-blocking the high-affinity Mn-binding site in photosystem II facilitates identification of the type of hydrogen bond participating in proton-coupled electron transport via YZ

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y(Z)(*) at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK(app) of the hydrogen bond partner for Y(Z) (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E(a)) for Y(Z)(*) reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK(app) (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E(a) is equal to 42.2 +/- 2.9 kJ/mol in PSII(-Mn) and 46.4 +/- 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK(app), the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E(a) = 36.1 +/- 7.5 kJ/mol), which is much lower than the E(a) of the corresponding component observed for Y(Z)(*) reduction in PSII(-Mn) samples (48.1 +/- 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y(Z) and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D(2)O to insert D(+), which has an energetic barrier distinct from H(+), into the LBHB. Measurement of the pH effects on the rates of Y(Z)(*) reduction in PSII(-Mn, +Fe) samples blocked in D(2)O shows a shift of the pK(app) from 6.1 to 7.6, and an increase in the E(a) of the slow component. This approach was also used to measure the stability of the Y(Z)(*) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y(Z)(*) radical is stable below 190 K, but half of the Y(Z)(*) EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y(Z)(*) radical is unstable at a much lower temperature (77 K). However, the insertion of D(+) into the hydrogen bond between Y(Z) and base B during the blocking process increases the temperature stability of the Y(Z)(*) EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y(Z) in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y(Z) and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].     

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).     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.     

Binding of amines to the O2-evolving center of photosystem II

The binding of several primary amines to the O2-evolving center (OEC) of photosystem II (PSII) has been studied by using low-temperature electron paramagnetic resonance (EPR) spectroscopy of the S2 state. Spinach PSII membranes treated with NH4Cl at pH 7.5 produce a novel S2-state multiline EPR spectrum with a 67.5-G hyperfine line spacing when the S2 state is produced by illumination at 0 degrees C [Beck, W. F., de Paula, J. C., & Brudvig, G. W. (1986) J. Am. Chem. Soc. 108, 4018-4022]. The altered hyperfine line spacing and temperature dependence of the S2-state multiline EPR signal observed in the presence of NH4Cl are direct spectroscopic evidence for coordination of one or more NH3 molecules to the Mn site in the OEC. In contrast, the hyperfine line pattern and temperature dependence of the S2-state multiline EPR spectrum in the presence of tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol, or CH3NH2 at pH 7.5 were the same as those observed in untreated PSII membranes. We conclude that amines other than NH3 do not readily bind to the Mn site in the S2 state because of steric factors. Further, NH3 binds to an additional site on the OEC, not necessarily located on Mn, and alters the stability of the S2-state g = 4.1 EPR signal species. The effects on the intensities of the g = 4.1 and multiline EPR signals as the NH3 concentration was varied indicate that both EPR signals arise from the same paramagnetic site and that binding of NH3 to the OEC affects an equilibrium between two configurations exhibiting the different EPR signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

Acclimation to the growth temperature and thermosensitivity of photosystem II in a mesophilic cyanobacterium, Synechocystis sp. PCC6803

Differences in the temperature dependence and thermosensitivities of PSII activities in Synechocystis sp. PCC6803 grown at 25 and 35 degrees C were studied. Hill reactions in cells, thylakoid membranes and purified PSII core complexes were measured at high temperatures or at their growth temperatures after high-temperature treatments. In the presence of 2,5-dichloro-p-benzoquinone as an electron acceptor, which can accept electrons directly from Q(A), the temperature dependence of the oxygen-evolving activity was almost the same in thylakoid membranes and in the purified PSII complexes from cells grown at 25 or 35 degrees C. When duroquinone, which accepts electrons only through Q(B) plastoquinone, was used as an electron acceptor, the temperature dependence was the same for purified PSII core complexes but was different between thylakoids isolated from the cells grown at 25 and 35 degrees C. No remarkable difference was observed in protein compositions between thylakoids and between purified PSII complexes from cells grown at 25 or 35 degrees C. However, the fluidity of thylakoids, measured by electron flow to P700, was affected by the growth temperature. These results suggest that one of the major factors which cause the changes in the thermosensitivity of PSII is the change in the fluidity of thylakoid membranes. As for the acclimation of PSII in thylakoids to high temperatures, one of the main causes is the decrease in the high-temperature-induced formation of non-Q(B) PSII due to the decreased fluidity in the cells grown at 35 degrees C.     

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.     

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue

New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl- facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl- depletion or F- substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl- and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 +/- 0.005, a symmetric line shape with line width = 16 +/- 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 +/- 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (less than 1 mu B2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 A of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops.(ABSTRACT TRUNCATED AT 250 WORDS)