Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 mol O₂/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl₂ or diphenylcarbazide, or diuron as an inhibitor of electron transfer from Q_A, the primary bound quinone acceptor, to Q_B, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to F_max, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680⁺ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H₂O₂, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O₂ with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680⁺, as well as by oxygen reduction by still unidentified components of PSII.     

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.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.     

Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states

It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH₂NH₂ at 20 °C in the CO₂/HCO₃⁻-depleted buffer the S-state populations are: 42% of S₋₃, 42% of S₋₂, 16% of S₋₁ and even formal S₋₄ state is reached, while in the presence of 2 mM NaHCO₃, the same treatment produces 30% of S₋₃, 38% of S₋₂, and 32% of S₋₁ and there is no indication of the S₋₄ state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.     

Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states

It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH(2)NH(2) at 20 degrees C in the CO(2)/HCO(3)(-)-depleted buffer the S-state populations are: 42% of S(-3), 42% of S(-2), 16% of S(-1) and even formal S(-4) state is reached, while in the presence of 2 mM NaHCO(3), the same treatment produces 30% of S(-3), 38% of S(-2), and 32% of S(-1) and there is no indication of the S(-4) state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.     

Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II

Since the end of the 1950s hydrogencarbonate ('bicarbonate') is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn(4)O(x)Ca cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (~0.3) HCO(3)(-) per PSII upon addition of formate. The same amount of HCO(3)(-) release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO(3)(-) from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO(3)(-) is corroborated by showing that a reductive destruction of the Mn(4)O(x)Ca cluster inside the MIMS cell by NH(2)OH addition does not lead to any CO(2)/HCO(3)(-) release. We note that even after an essentially complete HCO(3)(-)/CO(2) removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain > or =75% of their initial flash-induced O(2)-evolving capacity. We therefore conclude that HCO(3)(-) has only 'indirect' effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.     

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

Photosynthetica - The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt...     

Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II

Since the end of the 1950s hydrogencarbonate (‘bicarbonate’) is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn₄O_xCa cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (≈ 0.3) HCO₃⁻ per PSII upon addition of formate. The same amount of HCO₃⁻ release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO₃⁻ from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO₃⁻ is corroborated by showing that a reductive destruction of the Mn₄O_xCa cluster inside the MIMS cell by NH₂OH addition does not lead to any CO₂/HCO₃⁻ release. We note that even after an essentially complete HCO₃⁻/CO₂ removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain ≥ 75% of their initial flash-induced O₂-evolving capacity. We therefore conclude that HCO₃⁻ has only ‘indirect’ effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

Water exchange in manganese-based water-oxidizing catalysts in photosynthetic systems: From the water-oxidizing complex in photosystem II to nano-sized manganese oxides

The water-oxidizing complex (WOC), also known as the oxygen-evolving complex (OEC), of photosystem II in oxygenic photosynthetic organisms efficiently catalyzes water oxidation. It is, therefore, responsible for the presence of oxygen in the Earth's atmosphere. The WOC is a manganese–calcium (Mn₄CaO₅(H₂O)₄) cluster housed in a protein complex. In this review, we focus on water exchange chemistry of metal hydrates and discuss the mechanisms and factors affecting this chemical process. Further, water exchange rates for both the biological cofactor and synthetic manganese water splitting are discussed. The importance of fully unveiling the water exchange mechanism to understand the chemistry of water oxidation is also emphasized here. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.     

Interaction of Zn2+ with the donor side of Photosystem II

The inhibitory effect of Zn²⁺ on photosynthetic electron transport was investigated in native and CaCl₂-treated (depleted in extrinsic polypeptides) Photosystem II (PS II) submembrane preparations. Inhibition of 2,6-dichlorophenolindophenol photoreduction by Zn²⁺ was much stronger in protein-depleted preparations in comparison to the native form. It was found that Ca²⁺ significantly reduced the inhibition in the native PS II preparations, as did Mn²⁺ in a combination with H₂O₂ in the protein-depleted counterparts. No other tested monovalent or divalent cations could replace Ca²⁺ or Mn²⁺ in the respective experiments. Diphenylcarbazide could partially relieve (40–45%) the inhibition in both types of preparations. The above indicates the presence of an active Zn²⁺ inhibitory site on the donor side of PS II. However, neither Ca²⁺ nor Mn²⁺ could completely prevent inhibition by high concentrations of Zn²⁺ (>1 mM). We propose that elevated levels of Zn²⁺ strongly perturb the conformation of the PS II core complex and might also affect the acceptor side of the photosystem.Abbreviations PMSF phenylmethanesulfonyl fluoride - MES 2-(N-morpholino)ethane sulphonic acid - Chl chlorophyll - PS II Photosystem II - DCIP 2,6-dichlorophenolindophenol - DPC sym-diphenylcabazide - DCBQ 2,5-dichlorobenzoquinone     

Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.     

Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

Reconstitution of water-oxidizing complex in manganese-depleted photosystem 2 preparations with synthetic manganese complexes

Four synthetic manganese complexes in which Mn atoms have different coordination environments and valence states were used to reconstitute water-oxidizing complex (WOC) in Mn-depleted photosystem 2 preparations. Three Mn-complexes restored a significant rate of electron transfer and oxygen evolution except one complex in which Mn atom ligated to the O-atoms within the ligands by covalent linkage. The effect of coordination environment of the Mn-atom within the Mn-complexes on their efficiencies in reconstituting the electron transport and oxygen evolution was analysed.     

Structure of an active water molecule in the water-oxidizing complex of photosystem II as studied by FTIR spectroscopy

The vibrations of a water molecule in the water-oxidizing complex (WOC) of photosystem II were detected for the first time using Fourier transform infrared (FTIR) spectroscopy. In a flash-induced FTIR difference spectrum upon the S(1)-to-S(2) transition, a pair of positive and negative bands was observed at 3618 and 3585 cm(-1), respectively, and both bands exhibited downshifts by 12 cm(-1) upon replacement of H(2)(16)O by H(2)(18)O. Upon D(2)O substitution, the bands largely shifted down to 2681 and 2652 cm(-1). These observations indicate that the bands at 3618 and 3585 cm(-1) arise from the O-H stretching vibrations of a water molecule, probably substrate water, coupled to the Mn cluster in the S(2) and S(1) states, respectively. The band frequencies indicate that the O-H group forms a weak H-bond and this H-bonding becomes weaker upon S(2) formation. Intramolecular coupling with the other O-H vibration of this water molecule was studied by a decoupling experiment using a H(2)O/D(2)O (1:1) mixture. The downshifts by decoupling were estimated to be 4 and 12 cm(-1) for the 3618 (S(2)) and 3585 cm(-1) (S(1)) bands, both of which were much smaller than 52 cm(-1) of water in vapor, indicating that the observed water has a considerably asymmetric structure; i.e., one of the O-H groups is weakly and the other is strongly H-bonded. The smaller coupling in the S(2) than the S(1) state means that this H-bonding asymmetry becomes more prominent upon S(2) formation. Such a structural change may facilitate the proton release reaction that takes place in the later step by lowering the potential barrier. The present study showed that FTIR detection of the O-H vibrations is a useful and promising method to directly monitor the chemical reactions of substrate water and clarify the molecular mechanism of photosynthetic water oxidation.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.