Effects of Novel Photosynthetic Inhibitor [CuL2]Br2 Complex on Photosystem II Activity in Spinach

The effects of the novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex) on the photosystem II (PSII) activity of PSII membranes isolated from spinach were studied. The absence of photosynthetic oxygen evolution by PSII membranes without artificial electron acceptors, but in the presence of [CuL2]Br2, has shown that it is not able to act as a PSII electron acceptor. In the presence of artificial electron acceptors, [CuL2]Br2 inhibits photosynthetic oxygen evolution. [CuL2]Br2 also suppresses the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA. The inhibition of both characteristic PSII reactions depends on [CuL2]Br2 concentration. At all studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in Fv. [CuL2]Br2 causes neither changes in the F0 level nor the retardation of the photoinduced rise in FM, which characterizes the efficiency of the electron supply from the donor-side components to QA through the PSII reaction center (RC). Artificial electron donors (sodium ascorbate, DPC, Mn2+) do not cancel the inhibitory effect of [CuL2]Br2. The dependences of the inhibitory efficiency of the studied reactions of PSII on [CuL2]Br2 complex concentration practically coincide. The inhibition constant Ki is about 16 µM, and logKi is 4.8. As [CuL2]Br2 does not change the aromatic amino acids’ intrinsic fluorescence of the PSII protein components, it can be proposed that [CuL2]Br2 has no significant effect on the native state of PSII proteins. The results obtained in the present study are compared to the literature data concerning the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.


Introduction
Weeds, especially fast-growing and continuously emerging herbicide-resistant species, are one of the main factors limiting the yield of economically important crops. Advanced technology to increase yields through the creation of genetically modified plant species involves the simultaneous application of herbicides to kill weeds. Resistance to these herbicides has been created in cultivated plants through gene manipulation [1]. However, even with such approaches, it is necessary to search for more effective inhibitors that should act at the maximal low concentrations and should be capable of selectively suppressing damage near the RC and enhancing the dissipation of the incoming excitation energy into heat [36]. The fact that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not change the Cu 2+ inhibition pattern of the room temperature fluorescence induction of chloroplasts supposes that Cu 2+ and DCMU do not compete with each other, and consequently, have different inhibitory sites within PSII [36]. However, the opposite results [37] showed that the fluorescence intensity at room temperature decreased upon the addition of cupric sulfate and was partially restored by adding DCMU, testifying to the fact that Cu 2+ and DCMU do compete with each other, and consequently, the Cu 2+ inhibitory site within PSII coincides with that for DCMU, at least partially. Cu(II) has a specific inhibitory binding site by interacting with an essential amino acid group(s) that can be protonated or deprotonated at the inhibitory binding site, and the Cu(II) inhibitory mechanism is non-competitive with respect to 2,6-dichloro-1,4-benzoquinone (DCBQ) and DCMU and competitive with respect to H + [38]. Cu influences the PSII electron transport on the acceptor side of the Pheo-Fe-Q A domain [39,40]. CuCl 2 greatly reduces the binding affinity of atrazine for PSII (K D ) but does not change the number of binding sites compared to the control [41]. Furthermore, 100 µM CuSO 4 does not affect the charge separation and the formation of (P 680 + Q A − ) but changes the properties of TyrZ and/or its immediate microenvironment, thus blocking the electron transfer to P 680 + [42]. Cu 2+ (CuSO 4 ) inhibits TyrZ oxidation by P 680 + , eliminating the light-induced EPR signal II rapidly, and the effect cannot be reversed by EDTA [43]. On PSII membranes, when using picosecond time-resolved fluorescence, it was shown that Cu(II) does not affect primary radical pair formation but strongly affected the formation of a relaxed radical pair by neutralizing the negative charge on Q A − and eliminating the repulsive interaction between Pheo − and Q A − and/or by modifying the general dielectric properties of the protein region surrounding these cofactors. Furthermore, a close attractive interaction between Pheo − , Q A − , and Cu 2+ has been supposed [44]. In a PSII membrane, Cu enhances photoinhibition [45], catalyzing the production of hydroxyl radicals [45]. Ca 2+ competitively prevents Cu 2+ inhibition of O 2 evolution activity in NaCl-washed PSII membranes [46]. As part of a Cu-based complex, Cu substitutes endogenous Ca 2+ and Mn 2+ ions from the OEC [15]. Cu ions of the compounds studied in [15] and other aqua Cu-based complexes [47] affect both the TyrZ of D1 and the TyrD of D2 proteins of the PSII reaction center. CuCl 2 oxidizes both forms (LP and HP) of cytochrome b 559 , at low (1-10 µM) and higher (10-100 µM) concentrations, respectively, as well as chlorophyll Z, and these sites are a source of copper-induced fluorescence quenching and the inhibition of oxygen evolution in PSII [48]. Comprehensive reviews of the different sites and effects of Cu 2+ aqua ions on various PSII components are detailed in several publications [49,50]. The aim of this work was to investigate the possible effects of a novel Cu(II) organometallic complex (bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex) (CuL 2 ]Br 2 ) on photosystem II. To analyze the effects of this complex on the activity of PSII membranes isolated from spinach, we studied photosynthetic oxygen evolution by PSII membranes; the photoinduced changes of the PSII chlorophyll fluorescence yield (Fv) related to the photoreduction of the primary quinone electron acceptor, Q A ; as well as intrinsic fluorescence of aromatic amino acids (components of the PSII proteins); at different [CuL 2 ]Br 2 concentrations. The results obtained in the present study are compared to the literature data on the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.

Thylakoids Isolation
Thylakoids were isolated from the leaves of the greenhouse spinach (Spinacia oleracea L.) according to a method similar to the one described previously [51], and were suspended in a medium (A) containing 20 mM Mes-NaOH (pH 6.5), 0.33 M of sucrose, 15 mM of NaCl, 10 mM of MgCl 2 , and 10% glycerol and kept at −80 • C.

PSII Membranes Isolation
The oxygen-evolving PSII-enriched thylakoid membrane fragments (PSII membranes) were isolated from the leaves of the greenhouse spinach (Spinacia oleracea L.), as described earlier [52], with a little modification as in [53]. A similar PSII membrane ratio of RC (determined from pheophytin photoreduction)-to-chlorophyll molecules is about 1/200-220 was used [54]. The membranes were capable of evolving photosynthetic oxygen with rates of 400-500 µmol O 2 (mg Chl) −1 h −1 under saturating light in the presence of 0.1 mM 2,5dichloro-p-benzoquinone and 1 mM K 3 Fe(CN) 6 as electron acceptors. The PSII membranes were suspended in a medium (A) and were fast frozen for storage at −80 • C. The total chlorophyll concentration of the PSII membranes was determined using 96% (v/v) ethanol, as described earlier [55].

Photosynthetic Oxygen Evolution Measurements
The rate of steady-state light-induced oxygen evolution using the PSII membranes was measured polarographically in Medium A under continuous saturating red light (λ > 650 nm, 1200 µmol photon s −1 m −2 ) at 25 • C using a Clark-type Hansatech oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK) connected to a water-temperaturecontrolled thermostat in the presence of a combination of two artificial electron acceptors: 100 µM of a strongly lipophilic dichloro-p-benzoquinone (DCBQ) dissolved in dimethyl sulfoxide (DMSO) and 1 mM potassium ferricyanide (FeCN) as additional electron acceptors to keep DCBQ in an oxidized state [56]. The PSII membrane concentration was equivalent to 20 µg mL -1 of chlorophyll. The rates were estimated from the slope of the kinetics during the first 30 s after turning on the actinic light. Previously [57], according to the data on the dependence of the photoinduced oxygen evolution rate by PSII particles upon DCBQ concentration, it was shown that the highest oxygen yields were recorded in the presence of 25-200 µM DCBQ.

Photoinduced Changes of the PSII Chlorophyll Fluorescence Yield Measurements
The photochemical activity of the PSII was also analyzed by measuring the kinetics of the light-induced changes of the PSII chlorophyll fluorescence yield (∆F), related to the photoreduction of the PSII primary electron acceptor, plastoquinone Q A , with a pulseamplitude modulation (PAM) fluorometer (XE-PAM, Heinz Walz, Germany) and accompanying software Power Graph Professional 3.3 in Medium A in a 10-mm cuvette at room temperature. The characteristic values F V , F 0 , F M , and the maximum quantum photochemical yield of PSII (ratio F V /F M ), as well as the ratio F V /F 0 , were determined. Here, F 0 is the level of fluorescence before actinic light is applied, measured under weak measuring light illumination, and F M is the maximum fluorescence level, F V = F M − F 0 . To record the F 0 level of fluorescence, the preliminary dark-adapted sample was illuminated using weak probe pulses of measuring light (λ = 490 nm; 4 µmol m -2 s -1 ; Xenon-Measuring Flash Lamp, 64 Hz, BG39, Schott). For the registration of the photoinduced changes in PSII chlorophyll fluorescence yield (F V ), the dark-adapted samples were illuminated by the light of saturating intensity (λ = 490 nm; 1000 µmol m -2 s -1 , BG39, Schott).

Interaction between PSII and PSI through the Electron Transport Chain Measurements
The effect of the [CuL 2 ]Br 2 complex on the interaction between PSII and PSI through the electron transport chain was studied on thylakoids by a characteristic decrease in the yield of PSII chlorophyll fluorescence due to the oxidation of Q A − upon the additional excitation of PSII by the so-called Light 1, which predominantly excites PSII, as in [58]. Light 1 was obtained by combining two light glass filters, SZS20 and KS19. The transmission spectrum of the light filters combining (Light 1) is shown on the corresponding Figure lower in paragraph 3.7. The sequence of actions was as follows. After the F M level was reached (Q A was completely reduced) as a result of the illumination of the thylakoids, with Light 2 predominantly exciting PSII, Light 2 was switched off, and during the dark decline of F M (dark oxidation of Q A − ), Light 1 was switched on. In this case, when the interaction between PSII and PSI (control) is not inhibited, an acceleration of the dark decay of F M (oxidation of Q A − ) was observed due to the additional predominant excitation of PSI by Light 1.

Aromatic Amino Acids Fluorescence Measurements
Possible changes in the state, structure, and the environment of protein(s) (including PSII proteins) as a result of the binding caused by an inhibitory agent can be detected by changes in the intrinsic fluorescence (mainly fluorescence intensity changes) of amino acids with aromatic ring side chains (tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)), predominantly Trp and Tyr [8,9,59]. The probable interaction of [CuL 2 ]Br 2 with PSII membrane proteins was investigated by monitoring the changes in the intrinsic fluorescence of the aromatic amino acids of these proteins according to the methods described previously in [8,9,59]. The fluorescence emission spectra of the aromatic amino acids suspended in the PSII membranes were recorded on an Agilent Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA, USA). The samples of the PSII membrane suspension with and without the studied complex, [CuL 2 ]Br 2 , were excited at a wavelength of 275 nm, with an excitation spectral slit width of 20 nm, and fluorescence emission was registered in a range from 290 nm to 420 nm, with an emission spectral slit width of 10 nm. Before the measurements, the samples were incubated in the presence of the studied complex in the dark for 3 min (or 21 min). The complex was added to the PSII membrane suspension in the DMSO solution. The DMSO concentration in all of the samples was the same as in the control (not more than 5%). The PSII-membrane concentration was equivalent to 10 µg mL −1 of chlorophyll. The new copper(II) complexes [CuL 2 ]Br 2 were synthesized using an electrochemical method [60], using a Pt wire as the cathode (1 cm × 1 cm) and a copper plate as the sacrificial anode. Thus, a methanol solution (30 mL) of the ligand (~1 mmol) containing about 15 mg of tetrabutylammonium bromide as the supporting electrolyte in a beaker (100 mL) was electrolyzed at room temperature for 3 h. An applied voltage of 5 V allowed for sufficient current flow for the smooth dissolution of the copper. The obtained solid was isolated, washed with water, and dried under a vacuum. The structure of the tested ligand (4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)) and Cu(II)-complex (Bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide) is shown in Figure 1A,B, the expanded structural formulas; C, the optimized molecular structures.

Effects of [CuL2]Br2 on the Photosynthetic Oxygen Evolution
Because the aqua ions of Cu(II) (1) can oxidize many inorganic and organic compounds (shown upper) (2), stimulate photosynthetic oxygen evolution at low concentrations [61,62]

Effects of [CuL 2 ]Br 2 on the Photosynthetic Oxygen Evolution
Because the aqua ions of Cu(II) (1) can oxidize many inorganic and organic compounds (shown upper) (2), stimulate photosynthetic oxygen evolution at low concentrations [61,62]

Effects of [CuL 2 ]Br 2 on the Photoinduced Changes of the PSII Chlorophyll Fluorescence Yield
The suppression of photosynthetic oxygen evolution is one of the most significant indications that the present agent is indeed an inhibitor of PSII functional activity. However, the results obtained in our study do not make it possible to judge whether (1) this agent acts on the donor or acceptor side or at the RC level, or (2) which intermediate of the PSII electron transport chain is affected by this agent, and (3) whether this agent acts as an electron transfer inhibitor or as an exogenous electron donor (acceptor) capable of effectively competing with native endogenous components and thus disrupting the normal course of PSII photochemical reactions. Information (a) on the localization of the inhibition site of this inhibitor in PSII, (b) on the number of sites of action and location of the main and/or additional site(s) of inhibition, and (c) on the possible mechanism of action of the inhibitor can be obtained using the photoinduced changes of the PSII chlorophyll fluorescence yield (F V ) related to the photoreduction of the primary quinone electron acceptor, Q A , including the using the exogenous electron donors (acceptors) and/or known inhibitors with well-studied effects on PSII. Therefore, we decided to use this method in order to obtain more detailed information about the site(s) and mechanism of the inhibitory effect of [CuL 2 ]Br 2 on PSII. The results of these studies are presented in Figure 3.      Figure 4 shows the dependence of the PSII membranes photochemical activity inhibition on [CuL 2 ]Br 2 concentration, measured as both photosynthetic oxygen evolution (black squares) and as light-induced changes in the PSII chlorophyll fluorescence yield (red circles). Both of the presented dependences were measured at a PSII membranes concentration equivalent to 20 µg mL -1 of chlorophyll. The dependence of the oxygen evolution inhibition almost completely coincides with that of the suppression of the photoinduced changes in the PSII chlorophyll fluorescence yields over the entire range of the investigated agent concentrations. Usually, a special indicator is used to quantitatively assess inhibitor potency. It is IC 50 (or pIC 50 -the IC 50 value converted to a negative decadic logarithm of IC 50 (-logIC 50 ) for unification)-the concentration of agent needed to inhibit the investigated activity to half that of the initial value (control) [65,66]. IC 50 is an operational parameter to assess the effective strength of a particular inhibitory compound, and it is strongly dependent on PSII membrane concentration, and is usually expressed as the concentration of chlorophyll [67,68]. In 1972 Cedeno-Maldonado et al. [69] also showed that the inhibition of uncoupled electron transport in chloroplasts by copper ions Cu 2+ occurs at the PSII level and depends on the inhibitor/chlorophyll ratio. At high concentrations of chlorophyll, more Cu 2+ ions are required to achieve the same degree of inhibition [69]. logarithm of IC50 (-logIC50) for unification)-the concentration of agent needed to inhibit the investigated activity to half that of the initial value (control) [65,66]. IC50 is an operational parameter to assess the effective strength of a particular inhibitory compound, and it is strongly dependent on PSII membrane concentration, and is usually expressed as the concentration of chlorophyll [67,68]. In 1972 Cedeno-Maldonado et al. [69] also showed that the inhibition of uncoupled electron transport in chloroplasts by copper ions Cu 2+ occurs at the PSII level and depends on the inhibitor/chlorophyll ratio. At high concentrations of chlorophyll, more Cu 2+ ions are required to achieve the same degree of inhibition [69]. Figure 4. Dependence of the PSII membranes photochemical activity inhibition on [CuL 2 ]Br 2 concentration, measured both as the photosynthetic oxygen evolution (black squares) and as light-induced changes of the PSII chlorophyll fluorescence yield (red circles). Data from four independent experiments are represented as mean ± SD. The PSII membranes concentration was equivalent to 20 µg mL -1 of chlorophyll.

Dependence of the PSII Membranes Photochemical Activity Inhibition on [CuL 2 ]Br 2 Concentration
As can be seen from the data presented in Figure 4, for both of the measured characteristics of the PSII photochemical reactions, the values of the [CuL 2 ]Br 2 concentrations induce the suppression of half of the maximal studied activities (IC 50 ), are about 30 µM. [CuL 2 ]Br 2 in concentrations of a little bit more than 0.1 µM only induces a very weak suppression of these PSII photochemical activities. An almost complete (more than 90% versus control) suppression of both reactions was observed at an agent concentration of 10 mM. It should be noted that all of the presented data were obtained at a concentration of PSII membrane of 20 µg mL −1 chlorophyll.

Graphic Determination of Ki Value
Because IC 50 depends on PSII membranes concentration, it cannot be used to compare the results obtained from different inhibitors or for the same inhibitors obtained by various scientific groups without specifying the target concentration for which that particular IC 50 value was determined. In contrast to IC 50 , another parameter, Ki (the inhibition constant characterizing the intrinsic measure of binding affinity between the inhibitor and its target site), does not depend on the concentration of the PSII membranes. It is a known method for determining Ki based on the IC 50 values data obtained for different concentrations of the target (in our case, PSII membranes) [67]. We also applied this method ( Figure 5). We determined the IC 50 values for the suppression of light-induced oxygen evolution by [CuL 2 ]Br 2 for nine different concentrations of PSII membranes. We then plotted IC 50 as a function of chlorophyll concentration as a line plot and extrapolated the resulting line to a zero chlorophyll concentration, as described previously [67]. The resulting IC 50 value that corresponds to a zero chlorophyll concentration is Ki ( Figure 5). In our case, Ki is about 16 µM (16.2 ± 0.2), and -logKi is 4.8. Based on the explanation and equation (I 50 = Ki + 1/2 xt) given by [67], it is possible to estimate the concentration of the [CuL 2 ]Br 2specific binding sites (xt) to the chlorophyll concentration. In the case of the PSII membrane concentrations used by us, for example, equal to 20 µg ml −1 or about 22 µM, we determined that xt equals 1, i.e., one [CuL 2 ]Br 2 molecule on one chlorophyll molecule.

Graphic Estimation of the [CuL2]Br2 Binding Sites Number
It is known [68] that if the available inhibition data present a so-called Hill plot (dependence of log(inhibition/(1-inhibition)) on the log (inhibitor concentration), then it will be possible to estimate the number of binding sites on the acceptor molecule based on the slope of the obtained line. We used this method for the inhibition data of the PSII lightinduced oxygen evolution by various [CuL2]Br2 concentrations. Figure 6 shows such a Hill plot of these data. As follows from Figure 6, the slope of the obtained line is about 1 (0.87 ± 0.03).

Graphic Estimation of the [CuL 2 ]Br 2 Binding Sites Number
It is known [68] that if the available inhibition data present a so-called Hill plot (dependence of log(inhibition/(1-inhibition)) on the log (inhibitor concentration), then it will be possible to estimate the number of binding sites on the acceptor molecule based on the slope of the obtained line. We used this method for the inhibition data of the PSII light-induced oxygen evolution by various [CuL 2 ]Br 2 concentrations. Figure 6 shows such a Hill plot of these data. As follows from Figure 6, the slope of the obtained line is about 1 (0.87 ± 0.03).

Effects of [CuL2]Br2 on Interaction between PSII and PSI through the Electron Transport Chain Measurements
A significant number of known inhibitors that affect PSII, act on its acceptor side, blocking the re-oxidation of reduced QA by PSI [70]. In addition to the main site of action, many of them have at least one other additional site of action. The effects of the inhibitor on the main and additional sites of inhibition are observed at different concentrations of the inhibitor. The inhibition at the additional site occurs at higher concentrations of the

Effects of [CuL 2 ]Br 2 on Interaction between PSII and PSI through the Electron Transport Chain Measurements
A significant number of known inhibitors that affect PSII, act on its acceptor side, blocking the re-oxidation of reduced Q A by PSI [70]. In addition to the main site of action, many of them have at least one other additional site of action. The effects of the inhibitor on the main and additional sites of inhibition are observed at different concentrations of the inhibitor. The inhibition at the additional site occurs at higher concentrations of the suppressor [70]. Given the above information, we decided to test how [CuL 2 ]Br 2 affects the efficiency of light-induced oxidation by PSI of pre-reduced Q A .
The light-induced interaction of PSI with PSII is possible to estimate when measuring the dark relaxation of the photoinduced changes in the PSII chlorophyll fluorescence yield (variable F) related to the dark oxidation of the reduced Q A (Q A − ) from the F M -level to level F 0 and to switch it on at a determined moment of actinic Light 1 (L1), predominantly exciting PSI (λ > 700 nm). The transmission spectrum of Light 1 is shown in Figure 7B. In this case, induced by Light 1, the acceleration of Q A − re-oxidation is observed. For comparison, we used DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and DBMIB (2,5dibromo-3-methyl-6-isopropyl-p-benzoquinone). DCMU and DBMIB are available, well studied, and the most often and ubiquitously used in scientific research inhibitors. The mechanisms of the inhibitory actions of these agents are well studied. DCMU blocks Q A − , the oxidation by plastoquinone (PQ) from the membrane pool by binding instead of PQ with the Q B -plastoquinone-herbicide-binding protein (Q B -site of D1-protein) of the PSII reaction center [70]. DBMIB blocks oxidation driven by PSI through the cytochrome (cyt) b6f of plastoquinol (PQH2) from the membrane PQ pool [70]. DBMIB binds to the Qo (Qp) site of the cytochrome b6f complex or very close to it [70,71].
The results of such experiments on thylakoids are shown in Figure 7A. In the absence of any inhibitors (control, kinetics 1), actinic Light 1 induced an appreciable acceleration of Q A − oxidation. In the presence of DCMU (0.5 µM), actinic Light 1 did not induce the acceleration of Q A − oxidation. Instead of this, Light 1 induced an increase in PSII chlorophyll fluorescence (kinetics 2). This does not indicate the oxidation of Q A − , but, on the contrary, the reduction of those Q A molecules that had time to oxidize in the process of the dark relaxation of fluorescence before the moment actinic Light 1 was turned on. Why is this occurring? Light 1 excites not only PSI but also PSII, but to a much lesser extent because Light 1 contains a negligible fraction of the light that is absorbed by the main PSII pigments ( Figure 7B). On the other hand, it has been shown that the far-red light limits of PSII photochemistry in the PSII membranes from spinach expend up to 800 nm, but this light has a substantially decreased efficiency for PSII [72]. In our case (when Q A − oxidation is blocked by DCMU), the substantially decreased efficiency of PSII actinic Light 1 is enough to induce the photoaccumulation of reduced Q A , and it is revealed as an increase in the PSII chlorophyll fluorescence yield. The effect of DBMIB (7 µM) on the interaction of PSI with PSII, revealed by the changes in the PSII chlorophyll fluorescence yield, is practically similar to the effect of DCMU ( Figure 7A kinetics 3). As in the case of DCMU, the presence of DBMIB Light 1 induces an increase in PSII chlorophyll fluorescence yield, but in this case, the increase in fluorescence occurs at a slightly slower rate than in the case of DCMU. These comparative experiments with DCMU and DBMIB showed that the appearance of the increase in the PSII chlorophyll fluorescence yield under Light 1 illumination during the dark relaxation of F M is a reliable pointer to the fact that Q A − oxidation by PSI was inhibited at least at the Q B − or Qo site and we could use the pointer to check the [CuL 2 ]Br 2 results. The investigations of the [CuL 2 ]Br 2 effect on the interaction of PSI with PSII are shown in Figure 7A   b6f of plastoquinol (PQH2) from the membrane PQ pool [70]. DBMIB binds to the Qo (Qp) site of the cytochrome b6f complex or very close to it [70,71].

Effects of [CuL 2 ]Br 2 on the Aromatic Amino Acids Intrinsic Fluorescence
Many organic and inorganic substances can affect the structural and functional properties of proteins. Damage and/or changes in the native structure of the proteins that make up photosystem complexes or the properties of their surrounding environment may be one of the causes of disturbances in the photosynthetic electron transfer and the inhibition of photosynthesis in general [8][9][10]13]. Heavy metal ions and their complexes, including Cu ions and Cu complexes, can also act in a similar way. Copper(II) has been shown to inhibit photosynthetic oxygen evolution by binding to a non-protonated protein residue of a component very close to the PSII water-splitting system [73]. At the same time, such changes in PSII proteins can be caused both by the action of metal ions in the composition of the organometallic complex (Cu) [15] and by the action of the ligand, which is the organic part of such a complex [8][9][10]13].
Three amino acids with aromatic circular side chains-phenylalanine, tyrosine, and especially tryptophan-are sensitive to the local environment in the proteins, and these AAA undergo changes in the wavelength and/or intensity of their intrinsic fluorescence upon any impact on the protein [74]. One of the most accessible, and at the same time, quite reliable and informative methods for detecting possible similar effects on photosystem proteins, is the method of recording the intrinsic fluorescence of aromatic amino acids (AAA) that make up proteins, but mainly the intrinsic tryptophan and tyrosine fluorescence [59]. It was shown that some of the previously studied organic [8][9][10] and organometallic complexes [15], as well as free metal cations [13], which have an inhibitory effect on the photochemical activity of PSII, also caused a significant concentration-dependent decrease in the intensity of the intrinsic fluorescence of the PSII protein aromatic amino acids. It could be assumed that some negative effect(s) of the studied [CuL 2 ]Br 2 on the native state and/or structure of the PSII protein(s) is the reason for, or at least contribute to, the suppression of photosynthetic oxygen evolution and the magnitude of the photoinduced changes in PSII chlorophyll fluorescence yield. Therefore, we decided to test whether [CuL 2 ]Br 2 affects the structure of the PSII proteins by comparing the parameters of the AAA intrinsic fluorescence in the suspension of the PSII membranes without and with [CuL 2 ]Br 2 . The results of these studies are presented in Figure 8. Figure 8 shows the fluorescence emission spectra of the AAA of the untreated PSII membranes (spectrum 1) and the PSII membranes treated with [CuL 2 ]Br 2 at various times (spectra 2, 3). Upon excitation with light at a wavelength of 275 nm, a broad band was observed in the emission spectrum with a broad maximum at 330 nm and a shoulder at 350 nm. This is the emission of the intrinsic fluorescence of tyrosine, tryptophan, and phenylalanine [15,75]. Since this fluorescent emission is sensitive to changes in the local environment of the above aromatic amino acids, and if we assume that [CuL 2 ]Br 2 has any effect on PSII proteins, then we could expect any changes in the intensity and/or any shift in the maxima of this fluorescence if the native configuration of the PSII proteins will be disturbed due to the action of [CuL 2 ]Br 2 or as a result of binding to the PSII proteins of the [CuL 2 ]Br 2 complex or the Cu2+ ions released from [CuL 2 ]Br 2 . It is known that, in parallel with a decrease in the intensity of the intrinsic fluorescence of AAA in the protein, a shift in the F maximum is also possible. This indicates a change in the fluorophore microenvironment as a result of the effector impact [76].
However, in all of the cases (3 min and 21 min incubation of the PSII membranes with [CuL 2 ]Br 2 at a concentration of 30 µM), the intensity of the intrinsic fluorescent emission of PSII proteins did not decrease in the presence of [CuL 2 ]Br 2 at any of the indicated wavelengths (Figure 8 spectra 2, 3). Furthermore, no pronounced shifts of these emission maxima or changes in the shape of these spectra were observed (Figure 8 spectra 2 sine fluorescence [59]. It was shown that some of the previously studied organic [8][9][10] and organometallic complexes [15], as well as free metal cations [13], which have an inhibitory effect on the photochemical activity of PSII, also caused a significant concentration-dependent decrease in the intensity of the intrinsic fluorescence of the PSII protein aromatic amino acids. It could be assumed that some negative effect(s) of the studied [CuL2]Br2 on the native state and/or structure of the PSII protein(s) is the reason for, or at least contribute to, the suppression of photosynthetic oxygen evolution and the magnitude of the photoinduced changes in PSII chlorophyll fluorescence yield. Therefore, we decided to test whether [CuL2]Br2 affects the structure of the PSII proteins by comparing the parameters of the AAA intrinsic fluorescence in the suspension of the PSII membranes without and with [CuL2]Br2. The results of these studies are presented in Figure 8.  (3). Excitation wavelength 275 nm. Fluorescence emission was recorded in the range from 290 nm to 420 nm. The DMSO concentration in all samples was the same as in the control (not more than 1%). The PSII membranes concentration was equivalent to 10 µg mL −1 of chlorophyll. Figure 8 shows the fluorescence emission spectra of the AAA of the untreated PSII membranes (spectrum 1) and the PSII membranes treated with [CuL2]Br2 at various times (spectra 2, 3). Upon excitation with light at a wavelength of 275 nm, a broad band was observed in the emission spectrum with a broad maximum at 330 nm and a shoulder at 350 nm. This is the emission of the intrinsic fluorescence of tyrosine, tryptophan, and phenylalanine [15,75]. Since this fluorescent emission is sensitive to changes in the local environment of the above aromatic amino acids, and if we assume that [CuL2]Br2 has any effect on PSII proteins, then we could expect any changes in the intensity and/or any shift in the maxima of this fluorescence if the native configuration of the PSII proteins will be disturbed due to the action of [CuL2]Br2 or as a result of binding to the PSII proteins of the [CuL2]Br2 complex or the Cu2+ ions released from [CuL2]Br2. It is known that, in parallel with a decrease in the intensity of the intrinsic fluorescence of AAA in the protein, a shift  (3). Excitation wavelength 275 nm. Fluorescence emission was recorded in the range from 290 nm to 420 nm. The DMSO concentration in all samples was the same as in the control (not more than 1%). The PSII membranes concentration was equivalent to 10 µg mL −1 of chlorophyll.

Discussion
As there is no photosynthetic oxygen evolution due to the PSII membranes observed in the absence of artificial electron acceptors, but in the presence of the studied agent at a concentration of 0.1 mM (Figure 2, kinetics 6), it can be concluded that the studied agent [CuL 2 ]Br 2 (or, for example, its putative oxidized form), obviously, is not able to act as an exogenous PSII electron acceptor. These results could be taken as an indication that the copper cation in the complex is sufficiently strongly bound to the ligand, and the charge on the copper cation is balanced by a total charge on the ligand sufficient to prevent the possible interaction of the copper cation with other components of the environment as electron acceptor. The previously observed stimulation of the photosynthetic oxygen evolution by the Cu 2+ at low concentrations [61,62] could be due to Cu 2+ functioning as an uncoupler at this concentration, as was proposed earlier [35] based on the stimulation of the overall electron transfer rate by low Cu 2+ concentration at least in the case of Lemna fronds [61]. In thylakoids (closed membrane vesicles-organelles of chloroplasts, plant cells), electron transfer is coupled with proton transfer from outside (stroma) inside the thylakoid (lumen) with the formation of a proton gradient. Uncouplers disturb the conjugation between electron transfer and proton gradient formation, and all of the energy is only used for electron transport. The PSII membranes are open grana membrane fragments and not whole-membrane vesicles. Therefore, in PSII membranes, a proton gradient does not form. Consequently, uncouplers do not affect the efficiency of electron transfer in PSII membranes, as this one has a place in thylakoids. The decrease in the photosynthetic oxygen evolution rate in the PSII membranes in the presence of exogenous electron acceptors is observed if [CuL 2 ]Br 2 is present in the measurement medium; it may indicate that [CuL 2 ]Br 2 inhibits the functional activity of some PSII electron transport chain components. However, it can also be assumed that this observed effect is the result of a direct chemical interaction between AEA-s and [CuL 2 ]Br 2 (for example, oxidation of AEA-s by [CuL 2 ]Br 2 ) leading to the depletion of AEA-s concentrations and, as a consequence, to the decrease in the rate of oxygen evolution. It can be assumed that such an AEA in the used pair of AEA-s can be each of these agents or one of them: DCBQ or FeCN. If we compare the potentials of substances oxidized by the Cu(II) cation: ascorbate E m,7 = +60 mV [77]; 1,5-diphenylcarbazide E m,7 = −300 mV [78]; glutathione E m,7 = −240 mV [79]; NADH E m,7 = −320 mV [77]; DCBQ E m,7 = +309 mV [80] and FeCN E m,7 = +420 mV [81], it is obvious that such an AEA can be DCBQ and not FeCN. On the other hand, it was shown that the redox potential of Cu(II) in the complex could differ from the reduction potential of the Cu(II) Cu(I) aqua couple (164 mV) [82] and be significantly more positive [83], for example, including a number of copper proteins: Rusticyanin (bacteria) 680 mV; Plastocyanin (plants) 370 mV; Amicyanin (bacteria) 294 mV; Stellacyanin (plants) 285 mV; and Azurin (bacteria) 276 mV [84]. Taking these data into account, it can be assumed that FeCN can also be depleted due to direct chemical interaction with [CuL 2 ]Br 2 . However, the following experimental facts testify against this assumption: (1) The absence of changes in the absorption spectra of both FeCN and [CuL 2 ]Br 2 upon mixing and co-incubation for a sufficient time under the conditions of measuring the photosynthetic oxygen evolution rate but in the absence of the PSII membranes (not shown); (2) [CuL 2 ]Br 2 does not have any absorption bands in the red region of the spectrum (λ > 650 nm) (not shown). Therefore, a [CuL 2 ]Br 2 concentration-dependent decrease in the rate of the photosynthetic oxygen evolution is not result of the actinic light screening by [CuL 2 ]Br 2 . The hypothesis that the decrease in the photosynthetic oxygen evolution by PSII membranes is a consequence of the direct interaction of [CuL 2 ]Br 2 with DCBQ or FeCN could be tested by increasing the concentration of FeCN and/or DCBQ. If the inhibition efficiency did not change, then this would testify against the above proposal. However, the concentration of FeCN is already quite high (1 mM), above which it is quite possible to expect non-specific effects of FeCN on the PSII-membranes. On the other hand, it is not recommended to use DCBQ at concentrations above 250-300 µM. Based on the experimental data, it is proposed that DCBQ taking an electron from the reduced species in PSII may then donate it to P 680 +ȯ r Tyr Z˙, while ferricyanide prevents this process through the oxidation of the reduced DCBQ [85]. Therefore, the addition of FeCN is required to prevent the accumulation of reduced DCBQ [56,86]. At the same time, other authors, for example [38], measured the oxygen evolution activity of PSII membranes (500 µmol O 2 mg Chl −1 h −l ) in the presence of DCBQ (without FeCN). It can be assumed that [CuL 2 ]Br 2 acts as an exogenous electron donor capable of competing with the endogenous OEC for positive equivalents from RC and, therefore, for the ability to supply electrons for the reduction of the DCBQ + FeCN pair, not directly as a result of a direct chemical reaction, but through the components of RC. This could be manifested as a decrease in the rate of light-induced oxygen evolution with an increase in the concentration of [CuL 2 ]Br 2 . However, the above indirect data on the redox properties of Cu 2+ aqua ions and the effect of organic ligands on them makes this assumption unlikely. The fact that the degree of inhibition of the photosynthetic oxygen evolution rate by PSII membranes does not depend on the time of incubation in the presence of [CuL 2 ]Br 2 in the dark (effect measured after 3 min is equal to that after 21 min) indicates that [CuL 2 ]Br 2 sufficiently reaches the target quickly, apparently localized in the hydrophobic region of the PSII pigment-protein complex. This assumption is also supported by the fact that [CuL 2 ]Br 2 is very poorly soluble in an aqueous media and, therefore, a stock solution of [CuL 2 ]Br 2 was prepared in DMSO. Based on the above reasoning, we are inclined to assume that the decrease in the photosynthetic oxygen evolution rate by PSII membranes observed in the presence of [CuL 2 ]Br 2 is the result of inhibition by [CuL 2 ]Br 2 of the functional activity of some component of the PSII electron transport chain. Earlier [87], it was shown that PSII oxygen evolution inhibition induced by Cu(II) is reversible in the presence of a Cu(II)-chelating agent (EDTA), but the reversibility is strongly dependent on the incubation time with Cu(II) in light. Only 30 s of illumination in the presence of Cu(II) is enough for the inhibition of PSII by the cation and would be totally irreversible by EDTA.
The PSII maximum quantum photochemical yield of the dark-adapted samples ϕPo = Fv/F M being equal to 0.79 ± 0.02 testifies to the fact that the photochemical activity of PSII in the used PSII membranes is rather high, and these PSII membranes can be used to explore the [CuL 2 ]Br 2 inhibitory effects on PSII. The effective decrease in F M caused by [CuL 2 ]Br 2 may be due to the fact that (a) no separation and stabilization of charges in the RC occurs; (b) due to the inhibition of the functioning of some components of the OEC, electrons from the OEC through the RC to Q A are not supplied, which are necessary for the photoaccumulation of the reduced Q A and, therefore, F M cannot reach a level as in the control; (c) [CuL 2 ]Br 2 acts as an exogenous electron acceptor that removes an electron from Q A more efficiently than the OEC supplies electrons to Q A and therefore photoaccumulation of the reduced Q A does not occur, and it is revealed as a decrease in F M . (c) [CuL 2 ]Br 2 is a quencher of the excited states of chlorophyll, and this manifests itself as the quenching of chlorophyll fluorescence. Let us consider these assumptions in reverse order.
(d) [CuL 2 ]Br 2 only selectively reduces the intensity of the variable chlorophyll fluorescence (Fv) and does not affect the level of F 0 (Figure 3). Taking into account the fact that [CuL 2 ]Br 2 does not decrease the F 0 level, as well as the absence of any [CuL 2 ]Br 2 absorption bands in the range of the chlorophyll fluorescence emission spectrum (not shown), the assumption that [CuL 2 ]Br 2 acts as a fluorescence quencher seems unlikely.
(c) If [CuL 2 ]Br 2 acted as a very efficient exogenous electron acceptor, then (1) the dark decay of F V reflecting the rate of reoxidation of reduced Q A could be greatly accelerated; (2) the photoinduced growth rate of F M could be decreased; (3) we could observe lightinduced oxygen evolution in the absence of exogenous electron acceptors; (4) there could be spectral changes in the absorption spectrum of [CuL 2 ]Br 2 , indicating the formation of the reduced form of this agent. However, none of the abovementioned occurs (Figures 2 and 3). So [CuL 2 ]Br 2 does not act as an electron acceptor.
(b) It could be assumed that [CuL 2 ]Br 2 is incorporated somewhere on the donor side or between its components and blocks and/or slows down normal electron donation and/or acts as an exogenous "poor" electron donor to the RC, unable to provide efficient photoaccumulation of reduced Q A . However, the lack of slowdown in the rate of F M rise in the presence of [CuL 2 ]Br 2 (Figure 3 kinetics 2-4), and especially the inability of exogenous electron donors to overcome the inhibition by [CuL 2 ]Br 2 and thus restore F M to its original level (not shown) is the evidence against the fact that the donor side is the site of the inhibitory action of this agent. In addition, the indirect data on the redox properties of [CuL 2 ]Br 2 discussed above also testify against this assumption.
(a) Thus, the main site of the inhibitory effect of [CuL 2 ]Br 2 most likely is the components of the PSII reaction center. This assumption is quite convincingly confirmed by all the considerations discussed above, which reject other sites of action of this agent on PSII. In addition, as it was considered in the introduction, the most likely and the most experimentally substantiated main site of action among several auxiliary sites of action of both Cu 2+ aqua ions and organometallic complexes based on Cu 2+ seems to be components of the PSII reaction center.
Comparing the effects of DCMU and [CuL 2 ]Br 2 on the interaction of PSI with PSII ( Figure 7A), it can be roughly estimated that the [CuL 2 ]Br 2 effect is no more than 5-10% of the DCMU effect. Comparing the effects of [CuL 2 ]Br 2 on F M (50%) and on the interaction of PSI with PSII (see above), it is reasonable to propose that the latter effect is probably additional and not main. The existence of additional sites (effects) to the main known that is assumed and experimentally shown for many inhibitors of photosynthesis [6,70], including the aqua ions of Cu 2+ [50,89]. Using different methods, the additional effects of diuron (1,1-dimethyl, 3-(3',4'-dichlorophenyl) urea) [90][91][92][93][94], dinoseb (6-sec-butyl-2,4-dinitrophenol) [58,95], and other phenolic TNP inhibitor (2,4,6-trinitrophenol) [96] on the donor side of PSII, have been noted. DBMIB (2,5-dibromo-3-methyl-6-isopropylbenzoquinone), as a strong quencher of the excited state of chlorophyll in the PSII antenna [97], has been identified. Moreover, different authors often indicate completely different loci as the main loci of all of the identified binding sites (effects and mechanisms of action) for the same inhibitor. The dominance of any effects (both on the donor and acceptor side of PSII) depends on the concentration of both the inhibitor and the biological sample [67].
The absence of any change in the AAA fluorescence spectra in the presence of [CuL 2 ]Br 2 compared to the control (including [CuL 2 ]Br 2 concentrations at which the photoinduced electron transfer in PSII is almost completely suppressed, especially during prolonged incubation in the presence of [CuL 2 ]Br 2 ) indicates that (1) neither the whole [CuL 2 ]Br 2 complex nor the copper ion that could be released from this complex had no significant effect on the AAA PSII proteins native state; (2) and, therefore, there evidently is no release of the copper ion from the [CuL 2 ]Br 2 complex. Previously, it was shown that a number of water-soluble organometallic complexes based on Cu(II) effectively quench the intrinsic fluorescence of AAA of PSII proteins. Based on these and other data, it was proved that copper ions are released from these complexes and bind to tryptophan and tyrosine, thus inhibiting the functioning of the components of the PSII donor side [15]. These complexes, in their case, served as a kind of means of delivering copper ions to the site of action. This is one of the most likely common tasks and roles of the corresponding organic ligands in the composition of organometallic complexes. The combination of metal ions with the corresponding organic ligands makes it possible to obtain complexes that not only maintain the initial biological activity of the metal ions but also have high solubility in an appropriate media and the ability to deliver the metal ions to the site of its action practically in the initial biological activity state.
On the basis of the experimental data obtained, it could be assumed that the site of action of [CuL 2 ]Br 2 is a component of the PSII reaction centre. These data are consistent with the fact that Cu(II) aqua ions also act on PSII RC components [45]. At first glance, it is not easy to imagine how a relatively large [CuL 2 ]Br 2 molecule can penetrate into RC components located in a relatively densely packed and hydrophobic RC region. However, on the one hand, [CuL 2 ]Br 2 itself is a highly hydrophobic compound (see above), and, on the other hand, it has been theoretically substantiated and experimentally shown that the RC as a whole and its individual protein components undergo numerous conformational changes in the process of functioning, as well as at different reversible and irreversible impacts [98][99][100][101][102][103][104][105]. Taking these data into account, the assumption that the RC components are the object of [CuL 2 ]Br 2 inhibition seems to be quite realistic. The data on the absence of quenching the intrinsic fluorescence of AAA by [CuL 2 ]Br 2 do not in the least contradict the assumption that the site of action of [CuL 2 ]Br 2 is RC. There are circular dichroism data that indicate that even in the case of a significant quenching of AAA intrinsic fluorescence by Cu 2+ aqua ions due to their binding to a red fluorescent protein (DsRed) or to its derivatives, no effect of the Cu 2+ -binding on the secondary structure or on the conformation of these proteins was revealed [106].

Conclusions
According to the purpose of the work, to investigate the possible effects of a new organometallic complex, (CuL 2 ]Br 2 ) PSII, (1) several different interesting effects were identified and studied at the level of different PSII regions. (2) The assumption was confirmed that the presence of a ligand significantly changes the possible site and mechanism of the inhibitory action of copper ions. The experimental data at the time of writing this article can be explained by the fact that a possible mechanism of inhibition could be some conformational changes in the structure of the reaction center due to its interaction with the inhibitory agent, especially during photoaccumulation. To elucidate the exact mechanism and site of the inhibitory action of this agent, it is planned to significantly expand the list of research methods and the types of biological samples and apply the method of stabilizing the structure of the reaction center. The results obtained can be used in the development of methods for the guaranteed delivery of the active element to the site of its main activity in the organic complex.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.