Hydrogen Peroxide and Superoxide Anion Radical Photoproduction in PSII Preparations at Various Modifications of the Water-Oxidizing Complex

The photoproduction of superoxide anion radical (O2−•) and hydrogen peroxide (H2O2) in photosystem II (PSII) preparations depending on the damage to the water-oxidizing complex (WOC) was investigated. The light-induced formation of O2−• and H2O2 in the PSII preparations rose with the increased destruction of the WOC. The photoproduction of superoxide both in the PSII preparations holding intact WOC and the samples with damage to the WOC was approximately two times higher than H2O2. The rise of O2−• and H2O2 photoproduction in the PSII preparations in the course of the disassembly of the WOC correlated with the increase in the fraction of the low-potential (LP) Cyt b559. The restoration of electron flow in the Mn-depleted PSII preparations by exogenous electron donors (diphenylcarbazide, Mn2+) suppressed the light-induced formation of O2−• and H2O2. The decrease of O2−• and H2O2 photoproduction upon the restoration of electron transport in the Mn-depleted PSII preparations could be due to the re-conversion of the LP Cyt b559 into higher potential forms. It is supposed that the conversion of the high potential Cyt b559 into its LP form upon damage to the WOC leads to the increase of photoproduction of O2−• and H2O2 in PSII.


Introduction
Photosystem II (PSII) is a pigment-protein complex built into the thylakoid membrane. The main function of PSII is the light-induced oxidation of water to molecular oxygen with a transfer of electrons to the pool of plastoquinones. Recent crystallographic investigations of cyanobacterial PSII showed that a minimal structure capable of photosynthetic water oxidation and oxygen evolution (the so-called core complex of PSII) contains at least 20 protein subunits, 35 chlorophyll (Chl) molecules, 12 molecules of carotenoids, and at least 14-20 integral lipid molecules per monomer [1][2][3]. The light-induced charge separation with the formation of an oxidized primary electron donor, P 680 +• (the strongest biological oxidant, with a redox potential of 1.1-1.27 V [4,5]), occurs in the photochemical reaction centre (RC) consisting of main proteins, D1 (PsbA) and D2 (PsbD), and cytochrome b 559 (Cyt b 559 ). P 680 +• oxidizes TyrZ (tyrosine residue of D1 protein) with the formation of TyrZ • , which in turn takes an electron from the Mn 4 CaO 5 cluster, the inorganic core of the water-oxidizing complex (WOC). The sequential absorption of photons and charge separation in the RC result in the formation of intermediate states (S 0 -S 4 ) of the WOC, and the transition from S 4 to S 0 is accompanied by the oxygen release. An integral part of the reaction centre is Cyt b 559 , which participates in redox reactions and, in comparison with other redox components of the RC, is not located inside the D1/D2 heterodimer. Cyt b 559 can be found in at least four different redox forms: the Cyt b 559 high-potential (HP) form (E = from + 350 mV to + 450 mV), in intermediate-potential (IP) form (E = from + 125 to +240 mV), enzyme-catalyzed dismutation. Using a luminol-peroxidase method for the detection of H 2 O 2 , it was shown that the light-induced yield of the H 2 O 2 in isolated oxygen-evolving PSII membrane fragments was slight (about 0.01 H 2 O 2 molecules per RC and saturating flash) [16,17]. Possible donors of electrons to O 2 can be the reduced forms of the primary electron acceptor pheophytin (Pheo − ) [18], the primary (Q A − ) and secondary (Q B − ) quinone electron acceptors [19], plastosemiquinone (PQH • ) (where O 2 −• is produced via the proportion between plastoquinone (PQ) and plastoquinol (PQH 2 )) [20,21], and LP cytochrome Cyt b 559 [22,23]. For a detailed description of O 2 -• and H 2 O 2 photoproduction in PSII, see also [24,25]. It was shown that the treatments leading to the perturbation of the PSII donor side increased H 2 O 2 photoproduction [16,17,26,27]. It was assumed that the increase of H 2 O 2 photoproduction in the PSII after a partial injury of the WOC could be associated with the replacement of the four-electron (with the release of O 2 ) by the two-electron (with the production of H 2 O 2 ) oxidation of water [16,27]. However, using isotope-labelled water in combination with a detection system for H 2 O 2 showed that the oxygen in H 2 O 2 formed during the illumination of NaCI-wash PSII membranes did not originate from water [26]. Thus

Functional Activity in PSII Preparations at Various Modifications of the WOC
The investigation of H 2 O 2 and O 2 −• photoproduction in PSII was carried out on the PSII membranes and the PSII core complexes with different degrees of damage to the WOC: untreated, and NaCl-, CaCl 2 -, and NH 2 OH-treated PSII. The step-by-step disassembly of the WOC led to the suppression of PSII activity (oxygen-evolving activity and photoinduced ∆F). The yield of photoinduced ∆F was decreased by 20% and 30% after NaCl and CaCl 2 treatments of PSII membranes, respectively (Figure 1(I)A, curves 2 and 3). The complete removal of Mn ions from the WOC by NH 2 OH treatment led to a 5-fold decrease in the ∆F (Figure 1(I)A, curve 4) due to the loss of electron donation from the Mn-containing WOC to the PS II reaction centre (RC), which is in accordance with previous publications [28]. The photosynthetic oxygen evolution was more sensitive to the treatments in comparison with the ∆F (Figure 1(II) A). The rate of photosynthetic oxygen evolution in the untreated PSII membranes was about 600 µmol O 2 (mg Chl h) −1 . The treatment of PSII membranes with NaCl and CaCl 2 resulted in a decrease in the rate of photosynthetic O 2 evolution by 30% and 90%, respectively. The Mn removal from the WOC completely inhibited the oxygen-evolving activity of PSII and resulted in O 2 photoconsumption which, as was shown earlier, was associated with both the photoformation of organic hydroperoxides on the donor side via a radical chain mechanism and with the photoproduction of H 2 O 2 on the acceptor side of PSII [29][30][31].
of the oxygen-evolving activity in the core complexes can be associated with the partial removal of PsbP and PsbQ proteins during ion exchange chromatography, since the concentration of MgSO4 used to elute the PSII cores was about 100 mM. It was shown that the release of PsbP and PsbQ proteins from the WOC suppressed PSII oxygen-evolving activity and the addition of CaCl2 reconstituted high rates of oxygen evolution in the PS II preparations deprived of these proteins [32]. Due to this reason, only NH2OH treatment was performed to modify the WOC in the PSII core complexes. In comparison with PSII membranes (where the release of Mn from the WOC resulted in a drastic decrease in the ΔF), the yield of F in the Mn-depleted PSII core complexes was about two times less than in the untreated ones ( Figure 1(I)В). A similar yield of F was also observed in Mndepleted PSII core complexes which were obtained by isolation from Mn-depleted PSII membranes. This may be due to the removal of a quinone from the QB site, since the QB quinone can release from its binding site during the isolation of PSII core complexes [33]. Even though the yield of F in the Mn-depleted PSII core complexes was sufficiently high, the ability of the samples to perform photosynthetic oxygen evolution was completely lost (Figure 1(II)B, curve 4).  (1) and after modification of the water-oxidizing complex caused by treatments with NaCl (2), CaCl2 (3), and NH2OH (4). The measurements of ΔF were done in a medium containing 50 mM MES-NaOH (pH 6.5), 35 mM NaCl and 0.4 M sucrose at a Chl concentration of 10 µg/ml. , switching of the measuring light; ↑ and ↓, actinic light on and off, respectively. (II) Kinetics of oxygen evolution in PSII membranes (A) and PSII core complexes (B) before (1) and after modification of the water-oxidizing complex caused by treatments with NaCl (2), CaCl2 (3), and NH2OH (4). The measurements were made in the medium containing 50 mM MES-NaOH (pH 6.5), 35 mM NaCl, 0.4 M sucrose at a Chl concentration of 10 µg/ml for the PSII membranes and at 5 µg/ml for the PSII core complexes in the presence of 1 mM K3[Fe(CN)6] and 100 µM DCBQ. (1)-oxygen evolution in the PSII core complexes was done in the presence of 5 mM CaCl2.↑ and ↓light ( > 650 nm, 1500 µmol photon s −1 m −2 ) on and off, respectively.

The Ratio in Redox Forms of Cyt b559 in PSII Preparations at Various Modifications of the WOC
In addition to the suppression of the PSII functional activity, the destruction of the WOC changed the ratio in redox forms of Cyt b559 in the PSII membranes ( Table 1). The contents of HP, IP, and LP Cyt b559 in the untreated PSII membranes were 57%, 9%, and 34%, respectively. The treatment and PSII core complexes (B) before (1) and after modification of the water-oxidizing complex caused by treatments with NaCl (2), CaCl 2 (3), and NH 2 OH (4). The measurements of ∆F were done in a medium containing 50 mM MES-NaOH (pH 6.5), 35 mM NaCl and 0.4 M sucrose at a Chl concentration of 10 µg/mL. ∆, switching of the measuring light; ↑ and ↓, actinic light on and off, respectively. (II) Kinetics of oxygen evolution in PSII membranes (A) and PSII core complexes (B) before (1) and after modification of the water-oxidizing complex caused by treatments with NaCl (2), CaCl 2 (3), and NH 2 OH (4). The measurements were made in the medium containing 50 mM MES-NaOH (pH 6.5), 35 mM NaCl, 0.4 M sucrose at a Chl concentration of 10 µg/ml for the PSII membranes and at 5 µg/ml for the PSII core complexes in the presence of 1 mM K 3 [Fe(CN) 6 ] and 100 µM DCBQ. (1 )-oxygen evolution in the PSII core complexes was done in the presence of 5 mM CaCl 2 .↑ and ↓ -light (λ > 650 nm, 1500 µmol photon s −1 m −2 ) on and off, respectively.
The PSII core complexes showed maximal oxygen-evolving activity (about 1300 µmol O 2 (mg Chl h) −1 ) only in the presence of exogenous Ca 2+ (Figure 1(II)B, curves 1 and 1'). The CaCl 2 dependence of the oxygen-evolving activity in the core complexes can be associated with the partial removal of PsbP and PsbQ proteins during ion exchange chromatography, since the concentration of MgSO 4 used to elute the PSII cores was about 100 mM. It was shown that the release of PsbP and PsbQ proteins from the WOC suppressed PSII oxygen-evolving activity and the addition of CaCl 2 reconstituted high rates of oxygen evolution in the PS II preparations deprived of these proteins [32]. Due to this reason, only NH 2 OH treatment was performed to modify the WOC in the PSII core complexes. In comparison with PSII membranes (where the release of Mn from the WOC resulted in a drastic decrease in the ∆F), the yield of ∆F in the Mn-depleted PSII core complexes was about two times less than in the untreated ones ( Figure 1(I)B). A similar yield of ∆F was also observed in Mn-depleted PSII core complexes which were obtained by isolation from Mn-depleted PSII membranes. This may be due to the removal of a quinone from the Q B site, since the Q B quinone can release from its binding site during the isolation of PSII core complexes [33]. Even though the yield of ∆F in the Mn-depleted PSII core complexes was sufficiently high, the ability of the samples to perform photosynthetic oxygen evolution was completely lost (Figure 1(II)B, curve 4).

The Ratio in Redox Forms of Cyt b 559 in PSII Preparations at Various Modifications of the WOC
In addition to the suppression of the PSII functional activity, the destruction of the WOC changed the ratio in redox forms of Cyt b 559 in the PSII membranes ( Table 1). The contents of HP, IP, and LP Cyt b 559 in the untreated PSII membranes were 57%, 9%, and 34%, respectively. The treatment of PSII membranes with 1 M NaCl caused a slight decrease in the content of HP Cyt b 559 and an increase of its IP form without changing the content of LP Cyt b 559 . A much stronger disturbance of the WOC induced by the treatment of PSII membranes with 1 M CaCl 2 was accompanied by a significant decrease in the proportion of HP Cyt b 559 and increase of IP and LP Cyt b 559 ; thus, the ratio of the redox form of Cyt b 559 in the samples was about 20% of the HP form, 35% of the IP form, and 45% of the LP form. In the Mn-depleted PSII membranes, most of Cyt b 559 was in the LP (52%) and the IP (31%) forms, and only 17% was in the HP form. The similar interrelationship between the state of the WOC and the ratio in the redox forms of Cyt b 559 in PSII preparations was shown previously [9,10]. In contrast to PSII membranes, untreated PSII core complexes contained about 12 % of HP Cyt b 559 , and this percentage did not change after the removal of Mn from the WOC. However, the untreated and Mn-depleted PSII core complexes considerably differed in the content of IP and LP Cyt b 559 : For the untreated samples, the contents of the IP and LP forms were 45% and 43%, respectively, while Mn-depleted samples contained 21% of the IP form and 67 % of the LP form (Table 1).  Figure 2A illustrates the dependence of H 2 O 2 photoproduction in the PSII membranes, varying in the degree of damage to the WOC, on the duration of illumination. The photoproduction of H 2 O 2 by PSII membranes increased with the increasing destruction of the WOC. If, before treatments, the PSII membranes produced about 0.014 µmol H 2 O 2 per mg Chl for 30 s of illumination (λ > 600 nm, 1500 µmol photon s −1 m −2 ), then after NaCl, CaCl 2 , and NH 2 OH treatments, the yield of H 2 O 2 was 0.014, 0.018, and 0.045 µmol H 2 O 2 per mg Chl, respectively. It appears from this that the Mn-depleted PSII membranes, in which the electron supply from water to the reaction centre was inhibited, produced three times more H 2 O 2 than other samples. However, the capability of Mn-depleted PSII membranes to the light-induced production of H 2 O 2 decreased during illumination. As a consequence, the amount of H 2 O 2 produced by the Mn-depleted PSII membranes with 3 min of lighting was close to that generated by untreated samples. Ono and Inoue [34] showed that a gradual release of Mn from the WOC in the CaCl 2 -washed PSII membranes took place, and the Mn abundance in the samples decreased to about one half of the initial level after incubation in CaCl 2 -free medium at 0 • C under darkness for 7 h. In our case, the incubation time of the CaCl 2 -treated PSII membranes at 0 • C did not exceed 30 minutes, since a small aliquot of the samples was thawed for each series of measurements. In this regard, the number of reaction centres containing two manganese ions should be small based on the total number of reaction centres. Nevertheless, the CaCl 2 -treated samples containing about two Mn ions per RC were specially prepared. The rates of H 2 O 2 and O 2 −• photoproduction in these samples were two times higher than those of the CaCl 2 -treated PSII membranes containing four Mn ions per RC (data not presented). photoproduction in Mn-depleted PSII core complexes calculated for 30 s after the start of continuous illumination (λ > 600 nm, 1500 µmol photon s −1 m −2 ) was four times higher than the untreated samples (16 µmol and 4 µmol H2O2 per mg Chl h, respectively). However, the suppression of H2O2 production in Mn-depleted PSII core complexes during illumination or at increasing light intensity occurred slower than in the Mn-depleted PSII membranes.   Figure 2B shows the dependence of the rate of H 2 O 2 photoproduction by the PSII membranes on light intensity. The rate of H 2 O 2 production was calculated by monitoring the concentration of H 2 O 2 formed upon 1 min illumination of the samples. The rate of H 2 O 2 photoproduction in untreated PSII membranes at 250 µmol photons m −2 s −1 was equal to 0.5 µmol H 2 O 2 (mg Chl h) −1 , and it increased two times after CaCl 2 treatment of the PSII membranes and five times after Mn removal. The difference in the rate of H 2 O 2 photoproduction between the untreated and Mn-depleted PSII membranes gradually decreased with increasing light intensity, to the extent that at the photosynthetic photon flux density (PPFD) of 3000 µmol of photons m −2 s −1 , the rate of H 2 O 2 photoproduction by Mn-depleted PSII membranes was only two times higher than in untreated ones (4.7 and 2.8 µmol H 2 O 2 (mg Chl h) −1 , respectively). At the same time, the difference in the rates of H 2 O 2 production between untreated and NaCl-and CaCl 2 -treated PSII membranes upon the increase of PPFD was practically unchanged. Similar to PSII membranes, the removal of Mn clusters from the PSII core complexes stimulated the photoproduction of H 2 O 2 ( Figure 3). The rate of H 2 O 2 photoproduction in Mn-depleted PSII core complexes calculated for 30 s after the start of continuous illumination (λ > 600 nm, 1500 µmol photon s −1 m −2 ) was four times higher than the untreated samples (16 µmol and 4 µmol H 2 O 2 per mg Chl h, respectively). However, the suppression of H 2 O 2 production in Mn-depleted PSII core complexes during illumination or at increasing light intensity occurred slower than in the Mn-depleted PSII membranes.
In addition to H 2 O 2 , other species of peroxides (such as organic hydroperoxides) also can be formed upon the illumination of PSII preparations, which is especially applicable to the Mn-depleted samples [29][30][31]. To obtain insight into the specificity of homovanilic acid (HVA) for other peroxide species, the reaction of the probe with two peroxides-m-chloroperbenzoic acid (MCPBA) as a model of a lipophilic hydroperoxide and tert-butyl hydroperoxide (TBHP) as a hydrophilic hydroperoxide-was examined. The addition of MCPBA or TBHP at a concentration even ten times higher than H 2 O 2 resulted in only a slight increase in the fluorescence intensity of HVA, indicating that the contribution of hydroperoxides (which could be formed on the donor side of PSII) was negligible (Supplementary Materials Figure S1).  In addition to H2O2, other species of peroxides (such as organic hydroperoxides) also can be formed upon the illumination of PSII preparations, which is especially applicable to the Mn-depleted samples [29][30][31]. To obtain insight into the specificity of homovanilic acid (HVA) for other peroxide species, the reaction of the probe with two peroxides-m-chloroperbenzoic acid (MCPBA) as a model of a lipophilic hydroperoxide and tert-butyl hydroperoxide (TBHP) as a hydrophilic hydroperoxide-was examined. The addition of MCPBA or TBHP at a concentration even ten times higher than H2O2 resulted in only a slight increase in the fluorescence intensity of HVA, indicating that the contribution of hydroperoxides (which could be formed on the donor side of PSII) was negligible (supplementary materials Figure 1S).

Photoproduction of O2 −• in PSII Preparations at Various Modification of the WOC
The main path of H2O2 production in PSII is the disproportion of superoxide anion radicals, which are from the one-electron reduction of O2 on the acceptor side of PSII. The photoproduction of O2 -• in the PSII preparations was investigated using Cyt c. To distinguish the photoreduction of Cyt c related to O2 −• from its reduction by reduced electron carriers on the acceptor side of PSII [35], the measurements were performed both in the absence and in the presence of superoxide dismutase (SOD). The photoreduction of Cyt c in untreated PSII membranes as well as in NaCl-and CaCl2treated PSII membranes in the absence of SOD occurred with equal rates ( Figure 4A-C, curve 1). The rate of Cyt c photoreduction in the Mn-depleted PSII membranes was much higher in comparison with other samples (especially during the first 10 seconds of illumination ( Figure 4D, curve 1). The SOD added to the PSII membranes suppressed the Cyt c photoreduction and degree of the suppression depending on the destruction of the WOC ( Figure 4A-D, curve 2). The inhibition of the Cyt c photoreduction with SOD was equal to 50%, 60%, and 76% in the untreated PSII, NaCl-, and CaCl2-treated PSII membranes, respectively. The addition of SOD completely suppressed Cyt c photoreduction by the Mn-depleted PSII membranes, and negative A550 was observed ( Figure 4D, curve 2) which, as was shown recently [35], is associated with photooxidation of reduced Cyt c on the donor side of PSII. Figure 4E shows the kinetics of the Cyt c photoreduction after the subtraction of the kinetics measured in the presence of SOD, which demonstrates O2 −• -dependent Cyt c reduction.  Figure 4D, curve 1). The SOD added to the PSII membranes suppressed the Cyt c photoreduction and degree of the suppression depending on the destruction of the WOC ( Figure 4A-D, curve 2). The inhibition of the Cyt c photoreduction with SOD was equal to 50%, 60%, and 76% in the untreated PSII, NaCl-, and CaCl 2 -treated PSII membranes, respectively. The addition of SOD completely suppressed Cyt c photoreduction by the Mn-depleted PSII membranes, and negative ∆A 550 was observed ( Figure 4D, curve 2) which, as was shown recently [35], is associated with photooxidation of reduced Cyt c on the donor side of PSII. Figure 4E shows the kinetics of the Cyt c photoreduction after the subtraction of the kinetics measured in the presence of SOD, which demonstrates O 2 −• -dependent Cyt c reduction. These data indicate that the increase in the damage to the WOC stimulates O 2 −• photoproduction by PSII membranes. The removal of Mn clusters from the PSII core complexes also led to a significant increase in the rate of Cyt c photoreduction. However, in contrast to PSII membranes, the addition of SOD completely suppressed the Cyt c photoreduction both in untreated and Mn-depleted PSII core complexes ( Figure 5A, curves 3 and 4), indicating that the samples were not capable of reducing Cyt c by electron carriers. Figure 5B shows the Cyt c reduction associated with the light-induced O 2 −• formation in PSII core complexes.
As can be seen from the figure, Mn removal from the WOC led to a significant (more than five times) stimulation of O 2 −• photoproduction in PSII core complexes. a significant (more than five times) stimulation of O2 −• photoproduction in PSII core complexes. The addition of 20 µM diuron led to the almost complete suppression of H2O2 and O2 −• photoproduction in all the samples. This demonstrates that H2O2 and O2 −• photoproduction is linked to electron transport in PSII.  and after modification of the water-oxidizing complex caused by treatments of NaCl (2), CaCl 2 (3) and NH 2 OH (4). The kinetics was obtained by the subtraction of the kinetics of Cyt c photoreduction measured in the presence of SOD from that measured in the absence of SOD.
concentration of 10 µg/ml. Up and down arrows indicate light on and off, respectively. (E) Kinetics of Cyt c reduction associated with the light-induced O2 −• formation in the PSII membranes before (1) and after modification of the water-oxidizing complex caused by treatments of NaCl (2), CaCl2 (3) and NH2OH (4). The kinetics was obtained by the subtraction of the kinetics of Cyt c photoreduction measured in the presence of SOD from that measured in the absence of SOD.  Table 2 shows the comparison in the rates of H2O2 and O2 −• photoproduction in PSII preparations at various modifications of the WOC. As illustrated above, the ability of the Mn-depleted PSII preparations to produce H2O2 and O2 −• was significantly decreased during illumination as a consequence of their sensitivity to light. Therefore, the rates were calculated for 30 s after the start of illumination ( > 600 nm, 1500 µmol photon m -2 s -1 ) of the PSII preparations. The rate of light-induced formation of O2 -• and Н2О2 in the PSII preparations rose with the increasing destruction of the WOC, and the photoproduction of O2 −• in all samples was almost two times higher than H2O2. The data suggest that all or most of the H2O2 comes from O2 −• dismutation, where two molecules of O2 −• form one peroxide molecule.

Effect of Exogenous Electron Donors on the Photoproduction of O 2 −• and H 2 O 2 in Mn-Depleted PSII Preparations
Exogenous electron donors effectively restore photoinduced ∆F as a result of an increase in electron flow to the PSII reaction centre [28]. Figure 6I shows the Cyt c reduction associated with O 2 −• photoproduction in Mn-depleted PSII membranes ( Figure 6IA) and in Mn-depleted PSII core complexes ( Figure 6IB) upon the addition of 50 µM diphenylcarbazide (DPC). The restoration of electron flow in the Mn-depleted PSII preparations by DPC resulted in a three-fold suppression of O 2 −• photoproduction in PSII membranes, which was two-fold in PSII core complexes. The effect of the exogenous electron donor, Mn 2+ , on the photoproduction of H 2 O 2 in the Mn-depleted PSII preparations was studied using an H 2 O 2 -dependent couple reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB) catalyzed by peroxidase. The use of another system for the determination of H 2 O 2 was due to the fact that the electron donors used for the restoration of electron flow in the Mn-depleted PSII preparations affected the reaction of H 2 O 2 with HVA. In addition to this, Mn 2+ was used instead of DPC since DPC also affected the determination of H 2 O 2 by this measuring system. MnCl 2 (50 µM) added to the samples before illumination diminished the photoproduction of H 2 O 2 in Mn-depleted PSII membranes and core complexes by 55% and 45%, respectively ( Figure 6II, kinetics 1 and 2). Adding 50 µM MnCl 2 to the samples after illumination had practically no effect on the light-induced yield of H 2 O 2 ( Figure 6II, kinetics 1

Discussion
The obtained results demonstrate that the step-by-step disassembly of the WOC leading to the suppression of electron transport from the WOC to RC stimulates H2O2 and O2 −• photoproduction in PSII, and, among the samples, the Mn-depleted PSII preparations (which are not capable of water

Discussion
The obtained results demonstrate that the step-by-step disassembly of the WOC leading to the suppression of electron transport from the WOC to RC  (see [24,25]) and H 2 O 2 formed on the donor side when the WOC is perturbed without the release of manganese [16,27]. In our case, the stimulation of H 2 O 2 photoproduction in the PSII preparations induced by the injury of the WOC was mainly due to the increase in the O 2 −• production on the acceptor side of PSII. This conclusion has been made based on the following observations: (1)  and TyrZ • in the absence of an Mn cluster can be chlorophylls and carotenoids (their photooxidation has been shown in several works [38][39][40][41][42]), lipids in the lipid belt around D1 and D2 (their presence in the RC has been demonstrated [2,43]), the amino acid residues involved in coordination of the Mn 4 CaO 5 cluster [3], and His located in the vicinity of TyrZ. Apparently, the changes of the acceptor side caused by the modification of the WOC facilitate the photoproduction of O 2 −• . However, it cannot be excluded that the donor side of PSII also generates H 2 O 2 , especially in the case of CaCl 2 -treated PSII membranes [27,44], but its contribution seems negligible. In order to accurately estimate the contribution of the donor side, it is necessary to separate the H 2 O 2 formed on the acceptor side from the donor side. Pool PQ, pheophytin, Q A , and Cyt b 559 are considered to be the primary sources involved in O 2

−•
and H 2 O 2 photoproduction on the acceptor side (see [24,25]). It is worthwhile to consider the role of these cofactors in the enhancement of O 2 −• and H 2 O 2 photoproduction by PSII preparations after the destruction of the WOC. The pool of PQ is shown to be involved in H 2 O 2 formation within the thylakoid membrane [20,21]. The isolation of PSII preparations results in the deprivation of the PQ pool. It was shown that the PQ content was about 2.5 PQ/RC for PSII membranes [45], while the Q B quinone could be release from its binding site during the isolation of PSII core complexes (these complexes did not emit the B-band arising from S 2 Q B charge recombination, although the vacant Q B pocket preserved a high affinity for 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)) [33]. The analysis of PQ in the PSII core complexes isolated from cyanobacterium Acaryochloris marina MBIC 11017 showed that these complexes contained about 1.4 PQ per RC [46].  According to Allakhverdiev and co-workers [49], the Em (Pheo/Pheo − ) in PSII core complexes from Synechocystis sp PCC 6803 was −525 mV for untreated and about −609 mV for Mn-depleted samples. Thus, the removal of manganese from the WOC shifts the Em (Pheo/Pheo − ) towards negative values. It seems that this shift in the redox potential of Pheo would not lead to a significant increase of O 2 −• and H 2 O 2 photoproduction when the electron transport from the WOC to the RC was inhibited. In addition, the rate of H 2 O 2 photoproduction in the Mn-depleted PSII preparations at low light intensity was five times higher than that in the samples containing "native" WOC, i.e., when the accumulation of the long-lived state of Pheo − is less favorable. By contrast, the production of H 2 O 2 in the Mn-depleted PSII preparations decreased with increasing light intensity or duration of illumination. It seems that the electron transfer directly from Pheo − to O 2 is not productive, although its reduction potential favors this reaction. Perhaps this is due to the recombination between P 680 + and Pheo − (which is The Em for Q A /Q A − in intact PSII preparations isolated from spinach has values of −84 mV [51], about −162 mV [52] or −146 mV [49]. The discrepancy in the values is attributed to the removal of bicarbonate from PSII during the measurement procedure of Em (Q A /Q A − ) [53]. Thus, the redox potential of Q A is not sufficient for the effective reduction of O 2 to O 2 −• (although it is assumed that this reaction occurs because the ratio between O 2 and O 2 −• is strongly shifted towards O 2 [24]). In  [56]. Thus, the 90% suppression of the photoproduction of O 2 −• in the membranes and core complexes of PSII by diuron may also indicate that the main part of O 2 −• is not formed on the Q A site. It is probably true that this effect of diuron can be associated with the effects on the another components of the PSII reaction centre. It was shown that DCMU influenced the functioning of the WOC, the light-induced accumulation of reduced pheophytin [57], and the redox potential of HP Cyt b 559 [58]. The involvement of Cyt b 559 in O 2 reduction is presented in several works (see [15]), and all of them confirm that only LP Cyt b 559 can be involved in the reduction of O 2 to O 2 −• . The redox potential of LP Cyt b 559 varies from −40 mV to +80 mV (see [6]

Isolation of PS II Membranes and PSII Core Complexes
Oxygen-evolving PSII membrane preparations were isolated from spinach leaves according to the procedure in [59]. The samples were suspended in a medium containing 20 mM MES-NaOH (pH 6.5), 35 mM NaCl, 0.33 M sucrose, and 10% glycerol and stored at −76 • C. The isolation of PSII core complexes was performed according to the method in [60] with some modification: Bis-Tris buffer was replaced by MES. The concentration of chlorophyll (Chl) was measured as described previously [61]. The manganese content in PSII preparations was determined with an atomic absorption spectrophotometer equipped with a Kvant2A flame atomizer (Cortec, Russia).

Preparation of PSII Membranes with a Different Degree of Disassembly of the WOC and Mn-Depleted PSII Core Complexes
To obtain PSII membrane preparations with different degrees of disassembly of the WOC, the samples were treated by 1 M NaCl [62], 1 M CaCl 2 [63], or 5 mM NH 2 OH [64]. According to the literature, the first treatment results in the depletion of two extrinsic proteins (PsbP and PsbQ) of the WOC (NaCl-treated PSII), while the incubation of the PSII preparations in the presence of 1 M CaCl 2 releases all the external proteins (PsbP, PsbQ, and PsbO) from the WOC (CaCl 2 -treated PSII). Both these treatments do not extract manganese ions from the WOC, which suggests that the Mn cluster is relatively unaffected. The NH 2 OH treatment removes PsbP, PsbQ, and PsbO proteins and Mn ions from the WOC, but some amount of PsbO protein remains (Mn-depleted PSII).
Mn-depleted PSII core complexes were obtained by two approaches: (1) PSII core complexes were incubated in the presence of 5 mM NH 2 OH for 60 min, and then the samples were transferred to a Q-Sepharose column equilibrated with medium containing 20 mM MES-NaOH (pH 6.5), 35 mM NaCl, and 0.4 M sucrose with 0.03% (w/v) n-dodecyl-β-D-maltoside (medium A). After loading the samples, the column was washed with medium A with 1 mM ethylenediaminetetraacetic acid (EDTA) and then with medium A free from EDTA. The Mn-depleted PSII core complexes were eluted from the column by 100 mM MgSO 4 being added into medium A; (2) Mn-depleted PSII core complexes were obtained from Mn-depleted PSII membranes in accordance with the procedure of isolation of PSII core complexes [60].
Atomic absorption spectroscopy measurements of the manganese content in PSII membranes showed that untreated and NaCl-treated PSII preparations had 4.2 ± 0.2 atoms of manganese per PSII reaction centre, while its content was 3.8 ± 0.1 and less than 0.1 Mn per RC in the CaCl 2 -treated and the Mn-depleted PSII membranes, respectively. The content of Mn ions in PSII core complexes was 3.9 ± 0.2 for untreated and close to 0 for the NH 2 OH-treated samples.

Measurements of Functional Activity of PSII Preparations
The functional activity of PSII preparations was estimated by photoinduced changes of chlorophyll fluorescence yield (∆F) related to the photoreduction of the primary electron donor, Q A , and oxygen evolution measurements. The kinetics of photoinduced ∆F were measured in a 10 mm cuvette at room temperature by using an XE-PAM fluorometer (Walz, Germany). The photosynthetic oxygen evolution was measured in a temperature-controlled chamber by a Clark-type oxygen electrode (Hansatech Instruments, UK) at continuous illumination (λ > 600 nm, 1500 µmol photons s −1 m −2 ). The measurements were carried out at 25 • C in the presence of artificial electron acceptors for PSII 0.1 mM 2,6-dichloro-p-benzoquinone (DCBQ) and 1 mM K 3 [Fe(CN) 6 ].

Determination of H 2 O 2 Photoproduction by PSII Preparations
The photoproduction of H 2 O 2 in PSII membranes or core complexes was studied using the fluorescent probe homovanilic acid (HVA). The method is based on the H 2 O 2 -dependent oxidation of HVA mediated by horseradish peroxidase (HRP) to a highly fluorescent dimer [65]. The PSII preparations, resuspended in medium containing 20 mM MES-NaOH (pH 6.5), 35 mM NaCl, and 0.4 M sucrose at 50 µg of Chl/ml, were illuminated or kept under darkness at 25 • C. Then, an aliquot (500 µl) of the samples was added into the same volume of the reaction medium containing 100 mM Hepes (pH 7.6), 600 µM HVA, and 2 Un/ml HRP. After 30 min incubation at 37 • C, the PSII membranes were centrifuged at 12,000 g for 2 min. The supernatant was collected, and its fluorescence spectrum (350−500 nm, λex = 312 nm) was recorded with a Cary Eclipse fluorescence spectrophotometer (Agilent, USA). To remove the PSII core complexes from the solution, they were loaded on an Amicon Ultra centrifugal filter (Ultracel 30K, Merck Millipore, Germany) and centrifuged at 5000 g for 15 min. The fraction passing through the filter (free from PSII core complexes) was collected, and the fluorescence spectrum was recorded. The difference between the fluorescence spectra of illuminated and unilluminated samples, designated as the "light minus dark" fluorescence spectrum, represented the light-induced formation of H 2 O 2 . The number of H 2 O 2 formed under the illumination of the PSII preparations was calculated from the fluorescence intensity of HVA upon the addition of 5 µM H 2 O 2 . The effect of the exogenous electron donor, Mn 2+ , on the photoproduction of H 2 O 2 in Mn-depleted PSII was examined by the method based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB) in the presence of H 2 O 2 peroxidase catalyzes, with the couple reaction between MBTH and DMAB with the formation of a deep purple compound having an absorption band between 575 and 600 nm with a peak at 590 nm [27,66]. The use of this method for detecting hydrogen peroxide was because Mn 2+ did not interfere with the determination of H 2 O 2 when using this system, while the presence of Mn 2+ affected the detection of H 2 O 2 by HVA. The measurements were performed as follows: 5 mM DMAB and 0.1 mM MBTH were added to the samples illuminated in the absence or the presence of 50µM MnCl 2 , then the change at 590 nm was recorded before and after the injection of HRP (3 Un/ml).

Determination of O 2 −• Photoproduction by PSII Preparations
The light-induced generation of O 2 −• in PSII was detected by cytochrome c (Cyt c) [67,68]. PSII membranes or core complexes were resuspended at 10 µg Chl/ml in a buffer solution containing 50 mM MES-NaOH(pH 6.5), 35 mM NaCl, 0.4 M sucrose, and 10 µM Cyt c. Kinetics of absorbance changes at 550 nm related to the reduction of Cyt c upon illumination of PSII preparations with red light (λ > 600 nm, 1500 µmol (photon) s −1 m −2 ) were measured in a 10 mm cuvette at room temperature using a spectrophotometer Agilent 8453 (USA). The rate of photoreduction of Cyt c was estimated by monitoring the concentration of reduced Cyt c. The amount of reduced Cyt c was calculated using the differential extinction coefficient between ferrocytochrome c and ferricytochrome c at 550 nm (21.1 mM −1 ).

Analysis of Redox Forms of Cyt b 559 in PSII Preparations
Redox states of Cyt b 559 in PSII preparations were determined by measuring the differential (reduced-minus-oxidized) absorption spectrum of Cyt b 559 on a Shimadzu UV-1800 (Japan) spectrophotometer. To oxidize Cyt b 559 , 50 µM potassium ferricyanide was added. The reduction of the HP, IP, and LP (LP+VLP) forms of Cyt b 559 was achieved by the stepwise addition of 5 mM hydroquinone, 5 mM sodium ascorbate, and sodium dithionite, respectively. After each addition of the redox agent, a differential absorption spectrum was recorded. The content of HP Cyt b 559 was attributable to the spectra of Cyt b 559 obtained upon the addition of hydroquinone to the samples with ferricyanide. The fraction of IP Cyt b 559 was determined as the difference between the spectra of Cyt b 559 reduced by ascorbate and the spectra of Cyt b 559 reduced by hydroquinone, for the LP form of Cyt b 559 , and from the spectra of dithionite-reduced Cyt b 559 were subtracted the ascorbate-reduced spectra of Cyt b 559 .