Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II_slow and Signal II_fast of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y_Z and Y_D of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043 ± 0.0003 and a line width of 10 ± 1 G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q_A in the absence of magnetic interaction with the non-heme Fe²⁺. We conclude that peroxynitrite impairs PSII electron transport in the Q_AFe²⁺ niche.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Influence of the redox potential of the primary quinone electron acceptor on photoinhibition in photosystem II

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.     

Crystallization and electron paramagnetic resonance characterization of the complex of photosystem I with its natural electron acceptor ferredoxin

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of photosystem II.

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.     

Absence of the psbH gene product destabilizes photosystem II complex and bicarbonate binding on its acceptor side in Synechocystis PCC 6803.

The PsbH protein, a small subunit of the photosystem II complex (PSII), was identified as a 6-kDa protein band in the PSII core and subcore (CP47-D1-D2-cyt b-559) from the wild-type strain of the cyanobacterium Synechocystis PCC 6803. The protein was missing in the D1-D2-cytochrome b-559 complex and also in all PSII complexes isolated from IC7, a mutant lacking the psbH gene. The following properties of PSII in the mutant contrasted with those in wild-type: (a) CP47 was released during nondenaturing electrophoresis of the PSII core isolated from IC7; (b) depletion of CO2 resulted in a reversible decrease of the QA- reoxidation rate in the IC7 cells; (c) light-induced decrease in PSII activity, measured as 2,5-dimethyl-benzoquinone-supported Hill reaction, was strongly dependent on the HCO3- concentration in the IC7 cells; and (d) illumination of the IC7 cells lead to an extensive oxidation, fragmentation and cross-linking of the D1 protein. We did not find any evidence for phosphorylation of the PsbH protein in the wild-type strain. The results showed that in the PSII complex of Synechocystis attachment of CP47 to the D1-D2 heterodimer appears weakened and binding of bicarbonate on the PSII acceptor side is destabilized in the absence of the PsbH protein.     

Characterization of the complex interaction between the electron acceptor silicomolybdate and Photosystem II

Silicomolybdate (SiMo) and its effects on thylakoids have been characterized to evaluate its use as a probe for Photosystem II (PS II). It can accept electrons at two places in the electron transport chain: one at PS II and the other at PS I. In the presence of 1 M 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) only the site at PS II is available. It is suggested that SiMo must disp;ace bicarbonate from its binding site to be able to function as an electron acceptor. This displacement is non-competitive. The binding of SiMo is inhibited differentially by PS II inhibitors: dinoseb>ioxynil> diuron. This difference is determined by the different positions of the inhibitors within the Q_B binding niche and their interaction with bicarbonate. The experimental results show that the SiMo-binding niche is located between the parallel helices of the D1 and D2 proteins of PS II, close to the non-heme iron. We conclude that SiMo is an electron acceptor with unique characteristics useful as a probe of the acceptor side of PS II.     

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.     

Effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the secondary electron acceptor B of photosystem II

Measurements of chlorophyll fluorescence have been used to monitor electron transport from the primary electron acceptor of photosystem II, Q, to the secondary acceptor, B, in chloroplasts in either the presence or the absence of the plastoquinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Electron transport is markedly slower from Q^? to either B or B^? in the presence of DBMIB. Binary oscillations in the rate of reoxidation of Q^? (equivalent to the reactions Q^?B → QB^? and Q^?B^? → QB^2?) after each of a series of flashes were of a phase opposite to those observed in the absence of DBMIB (J. M. Bowes, and A. R. Crofts, (1980) Biochim. Biophys. Acta590, 573–584). The results confirm that inhibition of electron transport by DBMIB in chloroplasts is not restricted to an inhibition of electron transfer from the plastoquinone pool, but that there is also a specific interaction between the reduced form of the inhibitor and the secondary electron acceptor B. Models are discussed to account for the mechanism of this interaction.     

Fourier transform infrared spectrum of the secondary quinone electron acceptor Q(B) in photosystem II

Fourier transform infrared difference spectra upon single reduction of the secondary quinone electron acceptor Q(B) in photosystem II (PSII), without a contribution from the electron donor-side signals, were obtained for the first time using Mn-depleted PSII core complexes of the thermophilic cyanobacterium Thermosynechococcus elongatus. The Q(B)(-)/Q(B) difference spectrum exhibited a strong C...O stretching band of the semiquinone anion at 1480 cm(-)(1), the frequency higher by 2 cm(-)(1) than that of the corresponding band of Q(A)(-), in agreement with the previous S(2)Q(B)(-)/S(1)Q(B) spectrum of the PSII membranes of spinach [Zhang, H., Fischer, G., and Wydrzynski, T. (1998) Biochemistry 37, 5511-5517]. Also, several peaks originating from the Fermi resonance of coupled His modes with its strongly H-bonded NH vibration were observed in the 2900-2600 cm(-)(1) region, where the peak frequencies were higher by 7-24 cm(-)(1) compared with those of the Q(A)(-)/Q(A) spectrum. These frequency differences suggest that H-bond interactions of the CO groups, especially with a His side chain, are different between Q(B)(-) and Q(A)(-). Furthermore, a prominent positive peak was observed at 1745 cm(-)(1) in the C=O stretching region of COOH or ester groups in the Q(B)(-)/Q(B) spectrum. The peak frequency was unaffected by D(2)O substitution, indicating that this peak does not arise from a COOH group but probably from the 10a-ester C=O group of the pheophytin molecule adjacent to Q(B). The absence of protonation of carboxylic amino acids upon Q(B)(-) formation in contrast to the previous observation in the purple bacterium Rhodobacter sphaeroides suggests that the protonation mechanism of Q(B) in PSII is different from that of bacterial reaction centers.     

Extraction of electron acceptor A1 from pea photosystem I

Photosystem I particles were washed twice in either 0, 50 or 100% water-saturated diethyl ether. It was found that the characteristic electron spin resonance signal associated with electron acceptor A₁ was progressively lost with increasing percentage saturation of the ether. Light-induced electron flow to the terminal iron-sulphur acceptors was inhibited although these acceptors were still present and could be chemically reduced. The kinetics of optical measurements of P700⁺ re-reduction at 820 nm following ether washing were consistent with removal of electron acceptor A₁.     

Characterization of the manganese O2-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramagnetic resonance and X-ray absorption spectroscopy

The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.     

Redox properties and regulatory mechanism of the iron-quinone electron acceptor in photosystem II as revealed by FTIR spectroelectrochemistry

Photosynthesis Research - Photosystem II (PSII) performs oxidation of water and reduction of plastoquinone through light-induced electron transfer. Electron transfer reactions at individual redox...     

Interactions of photosystem II with bicarbonate, formate and acetate

In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO₂ by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (β₁), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40–50 mM formate or acetate results in a significant increase in the miss parameter and to an ∼50% (formate) and ∼10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased β₁ parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2–10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S₀, S₂, and S₃ states were measured, and no differences were found between the two sample types. (2) The S₁, S₀, S₋₁, and S₋₂ states were probed by incubation with small concentrations of NH₂OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent S_i/S_−i state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO₂ concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).     

Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone Q(B) in the photosystem II reaction center

Phosphatidylglycerol (PG), a ubiquitous constituent of thylakoid membranes of chloroplasts and cyanobacteria, is demonstrated to be essential for the functionality of plastoquinone electron acceptor Q(B) in the photosystem II reaction center of oxygenic photosynthesis. Growth of the pgsA mutant cells of Synechocystis sp. PCC6803 that are defective in phosphatidylglycerolphosphate synthase and are incapable of synthesizing PG, in a medium without PG, resulted in a 90% decrease in PG content and a 50% loss of photosynthetic oxygen-evolving activity as reported [Hagio, M., Gombos, Z., Várkonyi, Z., Masamoto, K., Sato, N., Tsuzuki, M., and Wada, H. (2000) Plant Physiol. 124, 795-804]. We have studied each step of the electron transport in photosystem II of the pgsA mutant to clarify the functional site of PG. Accumulation of Q(A)(-) was indicated by the fast rise of chlorophyll fluorescence yield under continuous and flash illumination. Oxidation of Q(A)(-) by Q(B) plastoquinone was shown to become slow, and Q(A)(-) reoxidation required a few seconds when measured by double flash fluorescence measurements. Thermoluminescence measurements further indicated the accumulation of the S(2)Q(A)(-) state but not of the S(2)Q(B)(-) state following the PG deprivation. These results suggest that the function of Q(B) plastoquinone was inactivated by the PG deprivation. We assume that PG is an indispensable component of the photosystem II reaction center complex to maintain the structural integrity of the Q(B)-binding site. These findings provide the first clear identification of a specific functional site of PG in the photosynthetic reaction center.