Probing the Influence of Novel Organometallic Copper(II) Complexes on Spinach PSII Photochemistry Using OJIP Fluorescence Transient Measurements

Modern agricultural cultivation relies heavily on genetically modified plants that survive after exposure to herbicides that kill weeds. Despite this biotechnology, there is a growing need for new sustainable, environmentally friendly, and biodegradable herbicides. We developed a novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino[2,1-b]benzothiazole-2-amine,4-(2-imidazole) that is active on PSII by inhibiting photosynthetic oxygen evolution on the micromolar level. [CuL2]Br2 reduces the FV of PSII fluorescence. Artificial electron donors do not rescind the effect of [CuL2]Br2. The inhibitory mechanism of [CuL2]Br2 remains unclear. To explore this mechanism, we investigated the effect of [CuL2]Br2 in the presence/absence of the well-studied inhibitor DCMU on PSII-containing membranes by OJIP Chl fluorescence transient measurements. [CuL2]Br2 has two effects on Chl fluorescence transients: (1) a substantial decrease of the Chl fluorescence intensity throughout the entire kinetics, and (2) an auxiliary “diuron-like” effect. The initial decrease dominates and is observed both with and without DCMU. In contrast, the “diuron-like” effect is small and is observed only without DCMU. We propose that [CuL2]Br2 has two binding sites for PSII with different affinities. At the high-affinity site, [CuL2]Br2 produces effects similar to PSII reaction center inhibition, while at the low-affinity site, [CuL2]Br2 produces effects identical to those of DCMU. These results are compared with other PSII-specific classes of herbicides.


Introduction
Currently, new approaches in agricultural economics are being intensively developed and put into practice, which selectively endow economically significant (genetically modified) crops with survival when treated with modern chemical agents that effectively suppress the growth and development of unwanted plant species [1]. The other side of these approaches (no less significant than the creation of genetically modified plant species) are studies aimed at identifying and studying the mechanism(s) of action of the widest possible range and the largest number of different chemical agents-potential prototypes Biomolecules 2023, 13, 1058 3 of 20 sites of inhibitor action are detected by JIP-testing of PSII, one may determine which of these sites of action (and/or effects) of the inhibitor are of a primary or secondary nature. It may be revealed by differences in the dependence of changes in the magnitude of different peaks of OJIP kinetics on the concentration of the added inhibitor, and/or by identifying additional peaks after appropriate normalization procedures and the subtraction of control kinetics OJIP from the OJIP-kinetics in the presence of the inhibitor [27,28]. on copper (Cu(II)) [16,17].
Many of these metals and semimetals in their free form have a very high ability to enter into various reactions, but exhibit low solubility in hydrophobic media and, as a result, may not achieve their intended targets as plant growth inhibitors. This also applies to copper cations, which are efficiently bound by organic buffer solutions [18], e.g., diphenylcarbazide, an exogenous electron donor widely used in photosynthesis studies [19,20], that chemically interact in solution with sodium ascorbate [21], hydroxylamine [22], dithionite [23], quinones [24], ferricyanide [25].
The several effects of a new organometallic complex based on Cu(II) ions with organic ligand (L = bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex)-[CuL2]Br2 on photochemical activity in PSII-containing membranes have recently been described [26]. To relieve the reader of the need to refer to our previous publication, we present the structure of the ligand (A) and the Cu(II)-complex (B) here again in Figure 1.  [26]. Structure of the ligand (A) and the Cu(II)-complex (B) are given in Figure 1. The Cu(II)-complex is a [CuL 2 ] 2+ mononuclear cationic complex, with two bromide counterions to achieve neutrality, based on MS spectrum corresponding to [CuL 2 ] 2 cation, a 1:2 electrolyte matching molar conductivity measurement, and elemental analysis values. The neutral bidentate ligand is bound to a copper(II) atom with an imidazole nitrogen atom and benzothiazol nitrogen atom. Geometrical optimization calculation with DFT/B3LYP/6-31G(d,p) method showed that it has distorted tetrahedral geometries around Cu(II) atom.
Here, we used the JIP test to elucidate inhibitory impacts of [CuL 2 ]Br 2 on PSIIcontaining membranes in more detail. Furthermore, we studied the effects of [CuL 2 ]Br 2 on PSII-containing membranes in the presence of DCMU, a known inhibitor of electron transfer on the acceptor side of PSII, blocking the oxidation of the reduced primary electron acceptor Q A (Q A − ) by the plastoquinone molecules from the membrane pool [4,29,30]. Investigation of the effects of [CuL 2 ]Br 2 in the presence of DCMU is a constructive experimental approach, since it allows using DCMU to exclude the possible influence of the remaining (in the PSII-membranes after their isolation) molecules of plastoquinone electron acceptors (PQ-9), approximately two molecules of PQ-9 per PSII RC [31,32]. On the other hand, if it turns out that the effects of [CuL 2 ]Br 2 and DCMU manifest themselves dependently or independently of each other, then these data will make it possible to more clearly judge the possible site of action and/or binding of the new Cu(II)-complex, as was shown, for example, in a study evaluating the site of action and/or binding of perfluoroisopropyldinitrobenzene derivatives, inhibitors of the K-15 type [33], and a protein synthesis inhibitor, chloramphenicol [34].

Fast Induction Kinetics of Chlorophyll Fluorescence
Fast induction kinetics of chlorophyll fluorescence associated with photoreduction of the PSII primary electron acceptor, plastoquinone Q A , were recorded using a MULTI-COLOR-PAM fluorimeter (Heinz Walz GmbH, Pfullingen, Germany) in a quartz cuvette (optical path length, 1 cm), at room temperature and constant stirring, after adaptation in the dark for at least 15 min. The final concentration of PSII-containing membranes in terms of chlorophyll was 4 µg mL −1 . The conditions for measuring fast induction curves of chlorophyll fluorescence using this fluorimeter are described in detail in [39]. Each kinetic result represents are average of 5 independent experiments. The measurements were carried out as follows. A volume of the initial solution of PSII-containing membranes was prepared in medium containing: 50 mM MES-NaOH (pH 6.5), 300 mM sucrose, 15 mM NaCl, and then either an inhibitor solution or the same volume of solvent (in which this inhibitory agent was prepared) was added to an aliquot taken from this volume. This guaranteed the same chlorophyll concentration in all measurements. Based on the chlorophyll fluorescence fast induction curves, a number of fluorescence parameters of PSII chlorophyll were determined and/or calculated.

Spectrophotometric Measurements
The absorption spectra of the [CuL 2 ]Br 2 complex were recorded in a standard quartz cell (Hellma, Müllheim, Germany) with an optical path length of 10 mm on a two-beam Shimadzu spectrophotometer, model UV-1800 (Shimadzu UV-1800, Shimadzu Europa GmbH, Duisburg, Germany) in the wavelength range 200-700 nm (optical slit width 2 nm, write speed 2 nm s −1 ) at room temperature, in measurement medium used for OJIP kinetics. The concentration of the [CuL 2 ]Br 2 complex was 0.1 mM and corresponded to the maximum concentration used in all experiments.

Solutions of Inhibitory Agents
Stock solutions of (3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU,) and [CuL 2 ]Br 2 and subsequent dilute solutions were prepared in dimethyl sulfoxide (DMSO). In all measurements, the final concentration of DMSO did not exceed 1%. In separate experiments, we have shown that DMSO at this concentration has no effect on either the intensity or the shape of the OJIP-kinetic. Figure 2 shows the original OJIP kinetics measured on PSII-containing membranes in the absence of other additives (control-kinetic 1) or in the presence of: 3.6 µM [CuL 2 ]Br 2 (kinetic 2); 14.5 µM [CuL 2 ]Br 2 (kinetic 3); 4 µM DCMU (kinetic 4); 3.6 µM [CuL 2 ]Br 2 + 4 µM DCMU (kinetic 5); 14.5 µM [CuL 2 ]Br 2 + 4 µM DCMU (kinetic 6). The F V /F M ratio is a generally accepted, widely used measure characterizing the quantum yield of the primary photochemical reaction of PSII [28]. Based on the analysis of the original kinetics presented in Figure 2, it can be seen that all the studied agents and their combinations cause significant decreases in the Chl "a" fluorescence intensity, especially noticeable at the F M level leading to a decrease in the variable fluorescence (F V ). Furthermore, 3.6 µM [CuL 2 ]Br 2 (kinetics 2); 14.5 µM [CuL 2 ]Br 2 (kinetic 3); and 4 µM DCMU (kinetic 4) also induced insignificant increases in the F 0 level (inset to Figure 2). In the presence of DCMU, such increases of F 0 by [CuL 2 ]Br 2 are not evident. It is important to especially note that the decrease in the F M value caused by both [CuL 2 ]Br 2 concentrations is at list in ten times more significant than the increase in the F 0 value induced by [CuL 2 ]Br 2 without DCMU. An increase in the F 0 level in the presence of DCMU has been repeatedly noted earlier on leaves [40], thylakoids [41], and PSII-containing membranes [42][43][44]. Both types of these changes (F M and F 0 levels) lead to a decrease in F V /F M ratio. that the decrease in the FM value caused by both [CuL2]Br2 concentrations is at list in ten times more significant than the increase in the F0 value induced by [CuL2]Br2 without DCMU. An increase in the F0 level in the presence of DCMU has been repeatedly noted earlier on leaves [40], thylakoids [41], and PSII-containing membranes [42][43][44]. Both types of these changes (FM and F0 levels) lead to a decrease in FV/FM ratio.

Original OJIP Kinetics Normalized Relative to F0 (F0.02ms)
In order to make it easier to analyze and more clearly represent the possible changes caused by the agents added to the control (in the absence of other additives); in comparison with the control, normalization is carried out to the initial level of fluorescence F0, as a rule, by the value of F20µs or F50µs measured at 20 µs or 50 µs, respectively [28], but sometimes by the F0 value measured at time t = 0 [45,46]. In recent years, normalization to F0.05ms has been favored, although normalization to F0.02ms is acceptable and still quite common [47,48]. In addition, it is shown that the possible errors in the calculation of the parameters of the JIP test in the case when F50µs is used as F0 is higher than for F20µs and Ft→0 [45].
The original OJIP-kinetics normalized relative to F0 are presented as Ft − F0 versus time in Figure 3 (where F0 is the fluorescence at time 0.02 ms; Ft is the fluorescence at time t). The analysis of the presented kinetics shows the following main properties of the obtained kinetics and their changes caused by the studied agents and their combinations.  In order to make it easier to analyze and more clearly represent the possible changes caused by the agents added to the control (in the absence of other additives); in comparison with the control, normalization is carried out to the initial level of fluorescence F 0 , as a rule, by the value of F 20µs or F 50µs measured at 20 µs or 50 µs, respectively [28], but sometimes by the F 0 value measured at time t = 0 [45,46]. In recent years, normalization to F 0.05ms has been favored, although normalization to F 0.02ms is acceptable and still quite common [47,48]. In addition, it is shown that the possible errors in the calculation of the parameters of the JIP test in the case when F 50µs is used as F 0 is higher than for F 20µs and F t →0 [45].
The original OJIP-kinetics normalized relative to F 0 are presented as F t − F 0 versus time in Figure 3 (where F 0 is the fluorescence at time 0.02 ms; F t is the fluorescence at time t). The analysis of the presented kinetics shows the following main properties of the obtained kinetics and their changes caused by the studied agents and their combinations.  Kinetics measured in the absence of additions (control) are completely identical to those recorded on PSII-containing membranes [42][43][44]49]. There is no peak I in the kinetic (plateau J-I), the main feature characterizing the kinetics of fast chlorophyll fluorescence induction measured on PSII-containing membranes [42][43][44]49] and therefore the kinetics will be designated below as OJP kinetics [42]. The absence of peak I (plateau J-I) in the OJP kinetics of PSII-containing membranes has been substantiated previously [42].
In the presence of both studied concentrations (3.6 µM and 14.5 µM) of [CuL2]Br2, a significant simultaneous almost synchronous decrease in the chlorophyll fluorescence intensity (F) is observed along the entire length of the OJP kinetics. The decrease also includes the FJ level (2-3 ms), and it is in greater extent in the presence of 14.5 µM [CuL2]Br2. The chlorophyll fluorescence decrease is especially significant at the FM level-in the presence of 3.6 µM and 14.5 µM [CuL2]Br2 by 22% and 45%, respectively, kinetics 2 and 3, Table  1 compared with the control (kinetic 1). The FM decrease is especially pronounced at 14.5 µM [CuL2]Br2 (kinetic 3). Let us designate these decreases in F (including FJ and FM) as described above as the "effect of [CuL2]Br2".  Kinetics measured in the absence of additions (control) are completely identical to those recorded on PSII-containing membranes [42][43][44]49]. There is no peak I in the kinetic (plateau J-I), the main feature characterizing the kinetics of fast chlorophyll fluorescence induction measured on PSII-containing membranes [42][43][44]49] and therefore the kinetics will be designated below as OJP kinetics [42]. The absence of peak I (plateau J-I) in the OJP kinetics of PSII-containing membranes has been substantiated previously [42].
In the presence of both studied concentrations (3.6 µM and 14.5 µM) of [CuL 2 ]Br 2 , a significant simultaneous almost synchronous decrease in the chlorophyll fluorescence intensity (F) is observed along the entire length of the OJP kinetics. The decrease also includes the F J level (2-3 ms), and it is in greater extent in the presence of 14.5 µM [CuL 2 ]Br 2 . The chlorophyll fluorescence decrease is especially significant at the F M level-in the presence of 3.6 µM and 14.5 µM [CuL 2 ]Br 2 by 22% and 45%, respectively, kinetics 2 and 3, Table 1 compared with the control (kinetic 1). The F M decrease is especially pronounced at 14.5 µM [CuL 2 ]Br 2 (kinetic 3). Let us designate these decreases in F (including F J and F M ) as described above as the "effect of [CuL 2 ]Br 2 ".
Thus, these experimental data suggest that out of the total number of PSII-containing membranes, 22% and 45%, PSII-containing membranes (respectively, in the presence of 3.6 µM and 14.5 µM [CuL 2 ]Br 2 ) are no longer capable of photochemical reduction of the corresponding components of the acceptor side of PSII. This effect is a consequence of a certain suppressive effect of [CuL 2 ]Br 2 on the components providing either charge separation or the source of electrons from the components of the donor side of PSII, and onward can be excluded from further consideration because they no longer produce JIP kinetics due to the action of [CuL 2 ]Br 2 . Therefore, the remainder of the total number of PSII-containing membranes that retained photochemical activity in the presence of 3.6 µM and 14.5 µM [CuL 2 ]Br 2 , respectively, should be considered, namely 78% and 55%. And using these data, it will be possible to find out by what mechanisms [CuL 2 ]Br 2 disrupts the functioning of PSII and in what sequence these mechanisms function. In addition, F M is reduced in the presence of 4 µM DCMU and especially in the presence of its combinations with both concentrations of [CuL 2 ]Br 2 ( Figure 3, Table 1). Moreover, in the case of a combination of 14.5 µM [CuL 2 ]Br 2 + 4 µM DCMU, an almost synchronous decrease in the chlorophyll fluorescence intensity (F) occurs along the entire length of the OJP kinetics, which are similar to described above.
In the presence of DCMU (without [CuL 2 ]Br 2 ), changes in the OJP kinetics characteristic of DCMU are observed (the so-called "DCMU effect")-namely, an increase in the F J peak to the so-called F M peak (kinetic 4), the intensity of which is less than the F M peak of control. The effects of DCMU have been repeatedly shown and explained previously by other authors [41][42][43][44]50]. In the presence of DCMU, all the amount of Q A present in the sample is restored, which is expressed in an increase of the J peak to the highest possible level. At the same time, there is a decrease in the F M value to a value that is 62% from the control F M . This decrease is due to the quenching of F by oxidized PQ-9 molecules [41][42][43][44]50]. A further decrease in the F M intensity by above reason seems unlikely, since in a preliminary experiment, we showed that 4 µM DCMU inhibited the oxidation of all reduced Q A molecules at the concentration of PSII-containing membranes we used.
Of particular interest and significance are the changes in OJP kinetics that occur in the presence of simultaneously both DCMU and [CuL 2 ]Br 2 (kinetics 5 and 6). Both kinetics are similar to the kinetics recorded in the presence of only DCMU ("DCMU effect" (kinetic 4)), but at the same time, the intensity of chlorophyll fluorescence decreases even more significantly over the entire OJP kinetics ("[CuL 2 ]Br 2 ) effect"). This decrease is especially evident in the case of 14.5 µM [CuL 2 ]Br 2 )+ 4 µM DCMU (kinetic 6). The intensity of F at the F M level decreases in the case of these combinations of inhibitors (3.6 µM [CuL 2 ]Br 2 ) + 4 µM DCMU) and (14.5 µM [CuL 2 ]Br 2 ) + 4 µM DCMU), by 50% and 66%, respectively, relative to the control F M .
In this case, in the presence of both combinations of DCMU with 3.6 µM and 14.5 µM [CuL 2 ]Br 2 (similar to situation without DCMU described above), there is for further research only part from the total number of PSII-containing membranes that retained photochemical activity, namely 50% and 34% in this case relative to F M in the presence of 4 µM DCMU alone. In such case, in the presence of both combinations of DCMU with 3.6 µM and or 14.5 µM [CuL 2 ]Br 2 , the remaining parts of the total number of PSII-membranes that retained photochemical activity, namely 50% and 34%, should be further considered.
Thus, DCMU induces a "DCMU effect" regardless of the presence of [CuL 2 ]Br 2 . At the same time, [CuL 2 ]Br 2 effectively suppresses the F M value both in the absence and in the presence of DCMU.
In the presence of DCMU, it is important to correctly estimate how much [CuL 2 ]Br 2 reduces the F M value. Since a further decrease due to quenching of F by oxidized PQ-9 molecules remaining in PSII-membrane is unlikely, since oxidation of all available Q A molecules is blocked by DCMU, then the observed decrease caused by both concentrations of [CuL 2 ]Br 2 in the presence of DCMU is based on another reason, and the percentage of decrease in F M in this case should be calculated by taking as 100% the value of F M measured in the presence of 4 µM DCMU. In this case, a further F M reduction due to quenching of F by oxidized PQ-9 molecules remaining in PSII-containing membranes is unlikely, since oxidation of all available Q A molecules is blocked by DCMU. Consequently, the observed F M reduction caused by both concentrations of [CuL 2 ]Br 2 in the presence of DCMU is based on another reason. The percentage of decreased F M reduction in this case should be calculated by taking as 100% the value of F M measured in the presence of 4 µM DCMU. These calculated data are shown in Table 1

OJIP Kinetics Normalized Relative to F 20µs and F M
Many stresses, including high or low temperature stress; high light intensities; UV-B; inhibitors of PSII photochemical activity, etc., affect the photoinduced redox state of Q A , and this is reflected in the form of changes in the intensity of the F J peak of OJIP kinetics and/or time to J-peak [40,41,[51][52][53].
In Figure 3, it is not easy to understand how the intensity F changes at the level of peak J for almost every kinetic compared to the control, with the exception of kinetics 4 (4 µM DCMU) and 5 (3.6 µM [CuL 2 ]Br 2 + 4 µM DCMU) in which an increase in F J intensity is clearly shown. Normalization of the original OJP kinetics simultaneously relative to the value of F 0 and the value of F M makes it possible to reveal in more detail possible changes, including intermediate peaks, in the case of PSII-containing membranes-peak J. It was of interest to clarify more clearly how [CuL 2 ]Br 2 affects the properties of the J peak in the absence and the presence of DCMU. Figure 4 shows the original OJP kinetics normalized relative to F 0.02ms and to F M . After such normalization, it became obvious that, in addition to the simultaneous decrease in the chlorophyll fluorescence intensity over the entire OJP kinetics (slightly at the F J level (2-3 ms) and especially pronounced at the F M level), which was clearly pronounced after normalization original OJP kinetics relative only to F 0.02ms , now there are significant changes in OJP kinetics compared with the control in the presence of both concentrations of [CuL 2 ]Br 2 , as well as their combinations with DCMU, which in this case became especially pronounced in the region of peak J (Figure 4).
From the data presented in Figure 4, it is evident that: (1) without DCMU in the presence of 3.6 µM [CuL 2 ]Br 2 (kinetic 2), the intensity of the J peak increases compared to the control (kinetic 1), but at a higher concentration of [CuL 2 ]Br 2 (14.5 µM) (kinetic 3), this effect, which is expressed in an increase in the J peak, already becomes significantly less; (2) in the case of a combination of 3.6 µM [CuL 2 ]Br 2 and 4 µM DCMU (kinetic 5), the J peak becomes a little bit higher compared to 4 µM DCMU (kinetic 4), however, at a higher concentration of [CuL 2 ]Br 2 (14.5 µM) in this combination inhibitors (kinetic 6), a significant decrease in the J peak is already observed.  From the data presented in Figure 4, it is evident that: (1) without DCMU in the presence of 3.6 µM [CuL2]Br2 (kinetic 2), the intensity of the J peak increases compared to the control (kinetic 1), but at a higher concentration of [CuL2]Br2 (14.5 µM) (kinetic 3), this effect, which is expressed in an increase in the J peak, already becomes significantly less; (2) in the case of a combination of 3.6 µM [CuL2]Br2 and 4 µM DCMU (kinetic 5), the J peak becomes a little bit higher compared to 4 µM DCMU (kinetic 4), however, at a higher concentration of [CuL2]Br2 (14.5 µM) in this combination inhibitors (kinetic 6), a significant decrease in the J peak is already observed.
Thus, in both above cases (namely in the absence and in the presence of DCMU), the differently directed effect on the F intensity of the J peak of these two concentrations of [CuL2]Br2 (3.6 µM and 14.5 µM) is clearly visible. It should emphasize that in the presence of DCMU the difference in the above effects between these concentrations is much greater. Despite the fact that after this normalization it is possible to identify additional changes in the OJP kinetics, nevertheless, in this case, these changes are not yet clearly expressed, and it is not possible to quantify the degree of these changes.
Since there is no I peak in PSII-containing membranes, in order to more conveniently analyze and visualize possible changes at the level of the J peak, which are induced by the studied inhibitory agents and their combinations, we first double normalized the original kinetics relative to both the F0 level (F0.02ms) and to the level of finding the peak I (30ms), i.e., to the level F30ms, according to the formula: Thus, in both above cases (namely in the absence and in the presence of DCMU), the differently directed effect on the F intensity of the J peak of these two concentrations of [CuL 2 ]Br 2 (3.6 µM and 14.5 µM) is clearly visible. It should emphasize that in the presence of DCMU the difference in the above effects between these concentrations is much greater. Despite the fact that after this normalization it is possible to identify additional changes in the OJP kinetics, nevertheless, in this case, these changes are not yet clearly expressed, and it is not possible to quantify the degree of these changes.
Since there is no I peak in PSII-containing membranes, in order to more conveniently analyze and visualize possible changes at the level of the J peak, which are induced by the studied inhibitory agents and their combinations, we first double normalized the original kinetics relative to both the F 0 level (F 0.02ms ) and to the level of finding the peak I ( 30ms ), i.e., to the level F 30ms , according to the formula: The resulting kinetics V 0I = (F t − F 0.02ms )/(F 30ms − F 0.02ms ) are shown in Figure 5A. Next, we subtracted the kinetic obtained in the absence of any additions (control) from the kinetics obtained in the presence of inhibitory agents, for each of the studied inhibitory agents and their combinations. The obtained difference kinetics W 0I = V 0I experiment − V 0I control are shown in Figure 5B. The resulting kinetics V0I = (Ft − F0.02ms)/(F30ms − F0.02ms) are shown in Figure 5A. Next, we subtracted the kinetic obtained in the absence of any additions (control) from the kinetics obtained in the presence of inhibitory agents, for each of the studied inhibitory agents and their combinations. The obtained difference kinetics W0I = V0I experiment − V0I control are shown in Figure 5B. It is known that DCMU blocks electron transfer from the reduced primary PSII electron acceptor, plastoquinone QA, into the membrane pool of plastoquinones (PQ-9), competing with the PSII secondary electron acceptor, plastoquinone QB for the binding site on the so-called QB herbicide-binding site of the D1 protein. Therefore, in the presence of DCMU, the so-called "diuron effect" is observed, which is expressed on the original OJIP kinetics as a significant increase in fluorescence intensity at a level of 2-3 ms (peak J) compared to the control [40,41,[51][52][53]. This can be especially clearly seen in the difference OJPkinetics obtained by subtracting from OJP-kinetics measured in the presence of DCMU, the kinetics obtained in the absence of any additions (control) [52,53].
Preliminarily, for the conditions of our measurements (the concentration of PSII-con- It is known that DCMU blocks electron transfer from the reduced primary PSII electron acceptor, plastoquinone Q A , into the membrane pool of plastoquinones (PQ-9), competing with the PSII secondary electron acceptor, plastoquinone Q B for the binding site on the so-called Q B herbicide-binding site of the D1 protein. Therefore, in the presence of DCMU, the so-called "diuron effect" is observed, which is expressed on the original OJIP kinetics as a significant increase in fluorescence intensity at a level of 2-3 ms (peak J) compared to the control [40,41,[51][52][53]. This can be especially clearly seen in the difference OJP-kinetics obtained by subtracting from OJP-kinetics measured in the presence of DCMU, the kinetics obtained in the absence of any additions (control) [52,53].
Preliminarily, for the conditions of our measurements (the concentration of PSIIcontaining membranes, expressed as the concentration of chlorophyll contained in them, is 4 µg mL −1 ), we found that the concentration of DCMU used by us (4 µM) causes practically maximal "diuron effect" on the chlorophyll fluorescence of PSII.
In addition, the use of higher concentrations of DCMU may be accompanied by the effects of DCMU on other sites of the PSII electron transport chain, as described earlier [54][55][56][57]. In order to quantify the "diuron effect" of other studied inhibitory agents or their combined use with DCMU, we evaluated the F J values for the other difference OJP-kinetics (W 0I = V 0I exp − V 0I control ) presented in Figure 5B in % from that with DCMU. The magnitude of the "diuron effect" F J measured in the presence of 4 µM DCMU, which indicates amount of reduced Q A (Q A − ), we took as 100%. And the effects of other supplements were evaluated in % relative to this effect of DCMU. The data obtained are presented in Table 2.  Table 2, it can be seen that in the absence of DCMU, low concentrations (3.6 µM) of [CuL 2 ]Br 2 cause a "diuron effect" of approximately 38% of that caused by DCMU. With an increase in the [CuL 2 ]Br 2 concentration to 14.5 µM, the "diuron effect" increases and is already 71% of the "diuron effect" caused by DCMU.

Effects of [CuL 2 ]Br 2 in the Presence of DCMU
A completely different effect of the [CuL 2 ]Br 2 complex on the photochemical activity of PSII is observed when [CuL 2 ]Br 2 complex is added in the presence of DCMU. In this case, both concentrations (3.6 µM and 14.5 µM) of [CuL 2 ]Br 2 significantly reduced the "diuron effect" of DCMU from 100%, respectively, to 59% and to about 3%.
Thus, from the data presented in Table 2, it is obvious that in the absence of DCMU, the amount of reduced Q A increases with increasing concentration of the [CuL 2 ]Br 2 complex. However, in the presence of DCMU, on the contrary, the amount of reduced Q A decreases significantly with an increase in the concentration of the [CuL 2 ]Br 2 .
We evaluated the potency of these effects of [CuL 2 ]Br 2 , namely (1) the effect of increasing the amount of reduced Q A in the absence of DCMU and (2) the effect of decreasing the amount of reduced Q A in the presence of DCMU on the concentration of the [CuL 2 ]Br 2 complex from the slope of the corresponding fitted curves. It turned out that the second mechanism of action of the [CuL 2 ]Br 2 complex, which manifests itself in a decrease in the amount of reduced Q A in the presence of DCMU, is about two times more effective than the first one, the accumulation of the amount of reduced Q A in the absence of DCMU.
Based on the comparison of the positions of the J peaks on the time scale, it can be roughly assumed that in the presence of 4 µM DCMU, the time to reach the maximum value of the fluorescence intensity of the J peak (F J ) on the difference kinetics W OI = V OI exp − V OI control (Figure 5B), which characterizes the rate of Q A reduction with increasing concentration of the [CuL 2 ]Br 2 complex, also increases-as can be seen when comparing the difference kinetics for (3. However, there is a more reliable way to quantify the rate of photoinduced Q A reduction.

Estimation of the Rate of Photoinduced Reduction of Q A
Graphical or computational determination of the initial slope (M 0 ) of the JIP kinetics makes it possible to estimate the rate of photoinduced reduction of Q A and its changes as a result of various influences [53,[58][59][60][61][62]. We have used both of these approaches.
The graphical data presented in Figure 6 allow you to see much more clearly what changes are induced by the studied agents in PSII photochemical reactions; the graphical approach is also used by other researchers [53,58,60,62]. In addition, we calculated the values of M 0 using the corresponding formula M 0 = 4 (F 300 − F 0 )/(F M − F 0 ). The results of the calculations are presented in the Table 3. The graphical data presented in Figure 6 allow you to see much more clearly what changes are induced by the studied agents in PSII photochemical reactions; the graphical approach is also used by other researchers [53,58,60,62]. In addition, we calculated the values of M0 using the corresponding formula M0 = 4 (F300 − F0)/(FM − F0). The results of the calculations are presented in the Table 3.    It should be noted that the values (M 0 ) determined on the basis of the data presented in Figure 6 almost coincide with those obtained as a result of calculations. However, since the "first" ones were determined as a result of approximating real values, the data obtained in the calculations should be considered more accurate.
From the data presented in the form of kinetics in Figure 6 and the corresponding values of M 0 in the Table 3, it follows that in all variants in the presence of the studied agents (both concentrations of [CuL 2 ]Br 2 without DCMU, 4 µM DCMU, both combinations of [CuL 2 ]Br 2 with DCMU), the rate of accumulation of reduced Q A (Q A − ), compared to the control is above ( Figure 6 and Table 3).
If we evaluate the rate of accumulation of reduced Q A (Q A − ), compared with DCMU, then in the absence of DCMU, both concentrations of [CuL 2 ]Br 2 increase the rate of accumulation of reduced Q A (Q A − ), compared with the control, as well as in the presence of DCMU alone, however, with a significantly lower efficiency compared to DCMU (46% and 32% of that of DCMU) (kinetics 4 and 5). Interestingly, in the presence of a lower concentration of [CuL 2 ]Br 2 (3.6 µM), this effect is greater (46%) compared with a higher concentration (14.5 µM) of this agent (32%), i.e., without DCMU, the ability to cause an increase in the rate of Q A reduction decreases with increasing concentration of [CuL 2 ]Br 2 .
In the presence of DCMU, [CuL 2 ]Br 2 also reduces the rate of accumulation of reduced Q A (Q A − ), respectively, to 69% and 56%, relative to DCMU (Table 3 of kinetics 2 and 3), and this effect of [CuL 2 ]Br 2 also increases with increasing concentration [CuL 2 ]Br 2 .
We evaluated the effectiveness of the impact of [CuL 2 ]Br 2 on the rate of Q A reduction in the absence and presence of DCMU by the slope of the approximated lines plotted using the corresponding experimental data from Table 3. It turned out that both in the absence of DCMU and with DCMU, the rate of Q A reduction with increasing concentration of [CuL 2 ]Br 2 goes down. However, in the presence of DCMU, the rate of Q A reduction with increasing concentration of [CuL 2 ]Br 2 decreases approximately three times faster than in the absence of DCMU.

Absorption Spectrum of [CuL 2 ]Br 2
It could be assumed that the observed simultaneous synchronous decrease in the intensity of the fast chlorophyll fluorescence induction curves and almost all its peaks in the presence of [CuL 2 ]Br 2 , which enlarges with an increase in the concentration of this organometallic complex added to the measuring medium, could be due to (1) a decrease in the intensity of the measuring and/or acting light due to the screening effect-absorption of light quanta by [CuL 2 ]Br 2 or (2) a decrease in the intensity of fluorescence emitted by chlorophyll molecules due to its absorption by the molecules of the [CuL 2 ]Br 2 -the effect of chlorophyll fluorescence reabsorption. To test this assumption, we studied the absorption spectrum of the [CuL 2 ]Br 2 complex. As shown in Figure 7, the [CuL 2 ]Br 2 complex has no absorption bands either in the region of wavelengths of both types of light or in the region of emission wavelengths of chlorophyll fluorescence. Therefore, the above assumption is erroneous.

Main Inhibitory Impact of [CuL2]Br2 on OJP Transients
The strongest and, therefore, undoubtedly, the main effect of the studied complex [CuL2]Br2 on the photochemical activity of PSII-containing membranes is a total synchronous decrease in the F intensity along the entire JIP kinetics (Figures 2 and 3, kinetics 2, 3 as well as Table 1). [CuL2]Br2 causes some changes in PSII when it becomes no longer capable of photoinduced QA reduction. Already at a concentration of 3.6 µM, [CuL2]Br2 is able to completely exclude 22% PSII-containing membranes from the total number of photochemically active PSII-containing membranes, but when at a concentration of 14.5 µM [CuL2]Br2 totally disables already 45% PSII-containing membranes (in the absence of DCMU). It is important to note that [CuL2]Br2 also demonstrates this effect on PSII in the presence of DCMU, with an efficiency that is well comparable to that estimated in the absence of DCMU (Figures 2 and 3 (kinetics 5, 6) and Table 1, data highlighted in green). Based on these data, we can make an experimentally substantiated conclusion that the main effect of [CuL2]Br2 on PSII does not depend on DCMU. Fairly well-comparable values of FM reduction by both concentrations of [CuL2]Br2 in the absence and presence of DCMU ( Figure 3, Table 1) suggest that in both cases, PSII inhibition by the [CuL2]Br2 complex may be based on the same mechanism of action. This in turn suggests that [CuL2]Br2 does not need to bind to the DCMU binding site to exert this effect. The fact that [CuL2]Br2 suppresses FM regardless of the presence of diuron suggests that the site of action and/or binding of [CuL2]Br2 on PSII is prior to the site of action and/or binding of diuron. Similarly, based on the obtained data about the independent manifestation of the effects of diuron and chloramphenicol on the OJIP kinetics of PSII-containing membranes, an experimentally substantiated conclusion was made that the site of action of chloramphenicol

Main Inhibitory Impact of [CuL 2 ]Br 2 on OJP Transients
The strongest and, therefore, undoubtedly, the main effect of the studied complex [CuL 2 ]Br 2 on the photochemical activity of PSII-containing membranes is a total synchronous decrease in the F intensity along the entire JIP kinetics (Figures 2 and 3, kinetics 2, 3 as well as Table 1). [CuL 2 ]Br 2 causes some changes in PSII when it becomes no longer capable of photoinduced Q A reduction. Already at a concentration of 3.6 µM, [CuL 2 ]Br 2 is able to completely exclude 22% PSII-containing membranes from the total number of photochemically active PSII-containing membranes, but when at a concentration of 14.5 µM [CuL 2 ]Br 2 totally disables already 45% PSII-containing membranes (in the absence of DCMU). It is important to note that [CuL 2 ]Br 2 also demonstrates this effect on PSII in the presence of DCMU, with an efficiency that is well comparable to that estimated in the absence of DCMU (Figures 2 and 3 (kinetics 5, 6) and Table 1, data highlighted in green). Based on these data, we can make an experimentally substantiated conclusion that the main effect of [CuL 2 ]Br 2 on PSII does not depend on DCMU. Fairly well-comparable values of F M reduction by both concentrations of [CuL 2 ]Br 2 in the absence and presence of DCMU ( Figure 3, Table 1) suggest that in both cases, PSII inhibition by the [CuL 2 ]Br 2 complex may be based on the same mechanism of action. This in turn suggests that [CuL 2 ]Br 2 does not need to bind to the DCMU binding site to exert this effect. The fact that [CuL 2 ]Br 2 suppresses F M regardless of the presence of diuron suggests that the site of action and/or binding of [CuL 2 ]Br 2 on PSII is prior to the site of action and/or binding of diuron. Similarly, based on the obtained data about the independent manifestation of the effects of diuron and chloramphenicol on the OJIP kinetics of PSII-containing membranes, an experi-mentally substantiated conclusion was made that the site of action of chloramphenicol in PSII is located before the site of action of diuron [34].
What are the reasons for the revealed total synchronous decrease in the intensity F over the entire kinetics of JIP induced by [CuL 2 ]Br 2 ?
The corresponding decrease in the intensity F may be due to electron acceptance if this Cu(II)-complex acts as artificial electron acceptor. A similar decrease in fluorescence intensity is observed in the presence of known PSII electron acceptors, such as DCBQ [43,63]. The similar effect was observed in the case of chloramphenicol capable of effectively oxidizing pheophytin [34]. However, we have previously shown that [CuL 2 ]Br 2 is not an artificial electron acceptor, because it does not support photosynthetic oxygen evolution [26].
A significant simultaneous almost synchronous decrease in the fluorescence intensity of chlorophyll along the entire length of the OJP kinetics, especially at the F M level, increasing with increasing concentration of [CuL 2 ]Br 2 , which we designated as the "[CuL 2 ]Br 2 effect", may be the result of a violation of the donor side or the reaction PSII center itself. Similar changes in OJIP kinetics were observed when the PSII donor side becomes nonfunctional [42,44]. However, artificial electron donors do not eliminate the inhibitory action of [CuL 2 ]Br 2 [26].
Based on experimental data obtained earlier [26], now we propose that [CuL 2 ]Br 2 probably acts directly on the reaction center of PSII, and it is concerning to its main impact. It was shown that single-walled carbon nanotubes (SWCNT) at concentration of 300 mg/L influence the fast chlorophyll fluorescence induction curve [64]. The effect is very similar to that of the [CuL 2 ]Br 2 . In the presence of 300 mg/L SWCNT, a total decrease in fluorescence intensity occurs along the entire length of the OJIP kinetics, which is especially pronounced at the F M level. It was shown that this effect of SWCNT is due to SWCNT inactivation of PSII RC [64]. Furthermore, earlier it was shown that Cu(II) aqua-ions act at the level of reaction centers of PSII [65].  (Figure 2 and inset kinetics 2 and 3) like it is usually induced by DCMU and agents with similar inhibitory mechanism-stopping electron transfer from reduced Q A onto the next mediator of electron transport chain. This is not observed in the presence of DCMU.
[CuL 2 ]Br 2 causes a slight increase in F J levels (Figures 4 and 5B). It is interesting that in these figures, at the first glance, there is opposite dependence of the [CuL 2 ]Br 2 effects on the FJ intensity. On Figure 4, at low [CuL 2 ]Br 2 concentration (3.6 µM), increasing of the FJ intensity seems more expressed than at 14.5 µM). Whereas on Figure 5B, the dependence is opposite.
In fact, a correct picture of the [CuL 2 ]Br 2 influence on the F J level can be obtained by subtracting the control OJP kinetic doubly normalized relative to F 0 and F M from those in the presence of [CuL 2 ]Br 2 , i.e., kinetics 2 and 4 shown on Figure 5B. This figure shows that the auxiliary effect of [CuL 2 ]Br 2 on the difference kinetics is similar to the effect of diuron, i.e., an increase in the amount of reduced Q A . This "diuron-like effect" increases with increasing concentration of [CuL 2 ]Br 2 . Thus, in the absence of DCMU, both of the above results, namely, an increase in the level of F 0 and F J , suggest that in the absence of DCMU, an auxiliary (not main) effect of [CuL 2 ]Br 2 is that [CuL 2 ]Br 2 acts like a DCMU, but with less efficiency than DCMU. Figure 4 shows that in the presence of DCMU, both concentrations of [CuL 2 ]Br 2 seem to cause an increase in the J level comparable to that induced by DCMU (kinetics 5 and 6). However in fact, it must be taken into account that this increase in J is a joint effect of DCMU and [CuL 2 ]Br 2 . The real picture of the influence of both [CuL 2 ]Br 2 concentrations on the magnitude of the J peak in the presence of DCMU is clear only on the difference kinetics ( Figure 5B of kinetics 3 and 5). Figure 5B shows that both [CuL 2 ]Br 2 concentrations in fact decrease the DCMU effect, and the decrease is more at higher [CuL 2 ]Br 2 concentration (respectively, to 59% and to about 3% relative to 100% DCMU effect) ( Table 2).

Impact of [CuL 2 ]Br 2 on J and 0 Peaks in the Presence DCMU
It could be assumed that [CuL 2 ]Br 2 can displace DCMU from its binding site, and this results in the decrease in the effect of DCMU. However, in this case, kinetics 3 and 5 would not be observed, but kinetics 2 and 4, because in the absence of DCMU, [CuL 2 ]Br 2 causes such auxiliary effects (the so-called "diuron effect"). However, this is in fact not the case. This means that in the presence of DCMU, [CuL 2 ]Br 2 causes some changes in PSII when it is no longer capable of photoinduced Q A reduction even in the presence of DCMU, an effect similar to the main effect of [CuL 2 ]Br 2 .

The Rate of Photoinduced Reduction of Q A
The fact that in the absence of DCMU simultaneously with the main effect [CuL 2 ]Br 2 appears to have a much less effective auxiliary effect is also evidenced by the fact that the M 0 values reflecting the rate of photoinduced Q A reduction in the presence of this complex are higher compared to the control ( Figure 6 kinetics 4 and 5, Table 3). That higher M 0 values are the result of an auxiliary effect of [CuL 2 ]Br 2 is evidenced by the fact that the ability to cause an increase in the rate of Q A reduction decreases with increasing concentration of [CuL 2 ]Br 2 . This trend is also observed in the presence of DCMU. The evidence that in the presence of DCMU, the rate of Q A reduction with increasing concentration of [CuL 2 ]Br 2 decreases approximately three times faster than in the absence of DCMU also strongly suggests that in this case the ancillary effect of [CuL 2 ]Br 2 becomes the main one.
It could be assumed that the revealed decrease in the F intensity of OJP kinetics caused by [CuL 2 ]Br 2 is the result of the physical or functional separation of the antenna from the RC [66]. In this case, an increase in the F 0 level can serve as a fairly reliable indication of this effect [66]. In our case, in the absence of DCMU, [CuL 2 ]Br 2 does not cause any increase in the F 0 level, and it is obvious that its effect on PSII is not associated with the separation of the antenna from the RC.
It could be assumed that the decrease in the intensity of the chlorophyll fluorescence through whole OJP kinetic (but not F 0 level) in the presence of our exogenous agent is the result of quenching of the chlorophyll fluorescence. It has been previously shown in thylakoids that DCBQ can act as an artificial electron acceptor and as a chlorophyll fluorescence quencher [63]. In this case, in addition to a decrease in the chlorophyll fluorescence intensity over the entire OJP kinetics, as a result of electron acceptance and an increase in the photochemistry rate, a decrease in the F 0 level is also observed as a result of F antenna quenching. Moreover, it is important that the decrease in F 0 caused by DCBQ is observed even in the presence of DCMU [63]. In our studies in the presence of DCMU, [CuL 2 ]Br 2 does not cause any decrease in the F 0 value and, therefore, it is not a chlorophyll fluorescence quencher (Figures 2 and 3 kinetics 5, 6).
The fact that the [CuL 2 ]Br 2 complex has no absorption bands either in the region of wavelengths of both types of light, or in the region of chlorophyll fluorescence emission wavelengths (Figure 7) gives grounds to suggest that a decrease in the chlorophyll fluorescence intensity of PSII-containing membranes in the presence of the [CuL 2 ]Br 2 complex could be due to its screening effect on the measuring and/or acting light. Another reason-the reabsorption of chlorophyll fluorescence by molecules of the [CuL 2 ]Br 2 could be excluded.

Conclusions
Based on the results obtained, we can assume: (1) The main (dominating in terms of the degree of inhibition of PSII activity) effect of [CuL 2 ]Br 2 on PSII is probably associated with inhibition of the activity of the PSII RC.
(2) The manifestation of auxiliary effects of [CuL 2 ]Br 2 on PSII is determined by the presence of DCMU. In the absence of DCMU, i.e., when the DCMU binding site is free, some part of [CuL 2 ]Br 2 that is involved in the induction of an auxiliary effect on PSII causes a "diuronlike" effect-an increase in the level of F 0 and F J , i.e., blocks electron transfer from the reduced Q A into the electron transport chain, but with less efficiency compared to DCMU. However, in the presence of DCMU, i.e., when the DCMU binding site is occupied by DCMU, this part of [CuL 2 ]Br 2 , which is involved in the induction of the auxiliary effect on PSII, participates in the induction of the main effect, i.e., a total decrease in the intensity F over the entire OJP kinetics. Thus, it can be assumed that in PSII-containing membranes, there are two binding sites for [CuL 2 ]Br 2 with different affinities for [CuL 2 ]Br 2 . At the high affinity site, [CuL 2 ]Br 2 produces effects similar inhibition of the PSII RC activity, while at the low affinity site, [CuL 2 ]Br 2 produces effects similar to those of DCMU. The data obtained can be useful in the development of promising herbicides for use in agricultural economics.