Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors.

Using artificial electron donors and acceptors, it is shown here that the major HCO3- effect in the Hill reaction is after the "primary" electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acdeptor Q- to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q-, show no significant bicarbonate stimulation of electron flow. However, a 6-7 fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3- (3',4'-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts IV. The effects of electron-transport conditions on violaxanthin availability

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electrontransport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than for cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear r flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity.2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (≤ μM) had no effect on availability, whereas low levels of 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50% decrease at 1 μM). Furthermore, availability in 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone.3. Violaxanthin availability was zero in the dark and increased in the light to a maximum of 67% of the total violaxanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.     

Clara cell damage and inhibition of pulmonary mixed-function oxidase activity by naphthalene

The photosystem II electron acceptor 3,6-dichloro-2,5-dimethoxy-p-benzoquinone [DCDMQ] is suggested to replace the second quinone-type two electron acceptor B (or R); the DCDMQ Hill reaction is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is insensitive to dry heptane extraction of thylakoids and other photosystem II inhibitors. Addition of HCO₃^? to CO₂-depleted thylakoids in silicomolybdate, DCDMQ, diaminodurene and ferricyanide Hill reactions brought about 1,3,10 and 10 fold increase in the electron transport rates; these data confirm that HCO₃^? affects both Q^? to B and B^2? to PQ reactions.     

A major site of bicarbonate effect in system II reaction. Evidence from ESR signal IIvf, fast fluorescence yield changes and delayed light emission

In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal II_vf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680⁺; (4) electron flow from Q^? to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Action of bicarbonate and Photosystem 2 inhibiting herbicides on electron transport in pea grana and in thylakoids of a blue-green alga

Bicarbonate (or carbon dioxide) is required for electron transport in isolated broken pea chloroplasts. The site of action of the bicarbonate ion is between the primary electron acceptor of Photosystem 2, Q, and the plastoquinone pool. After trypsin treatment the Hill reaction with ferricyanide does not require bicarbonate. Photosystem 2 inhibiting herbicides act also at this site. Therefore, a possible interaction of bicarbonate and these herbicides in their effect on photosynthetic electron transport was studied.
The reciprocal of the Hill reaction rate in CO₂-depleted chloroplasts was plotted against the reciprocal of added bicarbonate concentration in the absence and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-methoxy-4,6-bis (ethylamino)-1,3,5-triazine (simeton) or 4,6-dinitro- o -cresol (DNOC). From these Lineweaver-Burk plots we concluded that DCMU and simeton inhibit both bicarbonate binding and V_max. There is a purely competitive inhibition of bicarbonate binding by DNOC. We suggest that DNOC may exert its inhibition of electron transport by removing bicarbonate from its binding site.
In isolated thylakoid membranes of Synechococcus leopoliensis we did not find a bicarbonate effect nor inhibition by DNOC after Q, indicating that in the thylakoids of this blue-green alga the binding site for bicarbonate and DNOC between Q and plastoquinone is absent.     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Bicarbonate effect on electron flow in a cyanobacteriumSynechocystis PCC 6803

In this communication, evidence is presented from the kinetics of Q_A ^? decay (where Q_A is the first plastoquinone electron acceptor of photosystem II) and oxygen evolution for the requirement of bicarbonate in the electron transport in a cyanobacteriumSynechocystis (Pasteur Culture Collection 6803). A large slowing down of Q_A ^? oxidation, measured from the variable chlorophylla fluorescence after saturating actinic flashes, was observed in the thylakoids ofSynechocystis 6803 depleted of bicarbonate in the presence of 25 mM formate. Qualitatively similar results were obtained with DCMU-treated thylakoids. This shows that bicarbonate depletion inhibits electron transport on the acceptor side of photosystem II between Q_A and the plastoquinone (PQ) pool in cyanobacteria. Addition of 2.5 mM HCO₃ ^? fully reversed the inhibition of electron flow caused by bicarbonate depletion. Two exponential phases of Q_A ^? decay, a fast one and a slow one, were observed with halftimes of approx. 400 μs (fast) and 26 ms (slow) at pH 6.5. At pH 7.5, these phases were approx. 330 μs (fast) and 21 ms (slow), respectively. The amplitude, but not the halftime, of the fast component decreased by about 70% (pH 6.5) or 50% (pH 7.5); this was accompanied by a concomittant increase in the slow phase. Twenty mM bicarbonate stimulated, by a factor of 4, the Hill reaction in bicarbonate-depletedSynechocystis cells. This effect is independent of CO₂ fixation as it was observed even in the presence of an inhibitor DBMIB.     

The kinetic relationship between the C-550 absorbance change,the reduction of Q(ΔA320) and the variable fluorescence yield change in chloroplasts at room temperature

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolated, envelopefree chloroplasts in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isosbestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differential extinction coefficient Δ?(540–550) is calculated to be approximately 5 mM^?1 · cm^?1. The kinetics of the C-550 absorbance change, occurring upon the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical conditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The light-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers.It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Thus, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at room temperature.     

Shikonin isovalerate: An inhibitor of photosystem II in Chlorogloeopsis fritschi

Shikonin isovalerate, extracted from the roots of the desert plant Arnebia decumbens, was tested for its effect on photosynthetic electron transport system of Chlorogloeopsis fritschii. The ferricyanide-Hill reaction with water and DPC as electron donors was inhibited completely with 10^-5 M shikonin isovalerate. The photoreduction of DCPIP through photosystem II was only slightly inhibited. Photosystem I from durohydroquinone to methyl viologen was not affected using 10^-6 M shikonin isovalerate. The same concentration caused 49% inhibition of cyclic photophosphorylation. These results suggest that shikonin isovalerate inhibits photosynthetic electron flow at the plastoquinone pool.Abbreviations DCMU 3-(3,4-dichlorophenyl)-N,N-dimethyl urea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-P-benzoquinone - DCPIP 2–6-dichlorophenolindophenol - DPC Diphenylcarbazide - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Photosystem I-initiated postillumination CO2 burst in a cyanobacterium,Anabaena variabilis

In Anabaena variabilis, a postillumination CO₂ burst originating from a pool of HCO₃^? is described here. This burst is insensitive to the electron-transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and N,N′-dicyclohexylcarbodiimide (inhibitors of photophosphorylation). The action spectrum for the burst shows that only Photosystem I is involved.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

Evidence from thermoluminescence for bicarbonate action on the recombination reactions involving the secondary quinone electron acceptor of Photosystem II

In this paper, we present the first measurements on thermoluminescence from isolated thylakoids to probe the recombination reactions of S₂ (or possibly S₃) with Q^?_B or Q^?_A, after bicarbonate depletion and its readdition. The effects of bicarbonate depletion on the S₂Q^?_B (or S₃O^?_B) thermoluminescence band was (1) a 6–10°C shift to a higher temperature; (2) a reduction in its intensity upon prolonged depletion; and (3) elimination after the first few flashes of the characteristic period four oscillations in its intensity as a function of the flash number. On the other hand, addition of diuron (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea), which blocks electron flow from Q^?_A to Q_B, produced the same thermoluminescence band, at about + 20°C, assigned to S₂Q^?_A recombination, in both depleted and reconstituted samples. These results suggest (1) the initial effect of bicarbonate depletion is to increase the activation energy for S₂(S₃)Q^?_B recombination; (2) with further depletion, the incidence of this recombination decreases and the cycling of the S₂Q^?_B and S₃Q^?_B recombination is inhibited through effects at the Q_B apoprotein; and (3) the depletion effects are fully reversible. It is suggested that a conformational change of the PS II complex in the region of the Q_B apoprotein is responsible for these effects.     

Fluorescence induction in intact spinach chloroplasts

Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.     

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

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

Binding affinity of bicarbonate and formate in herbicide-resistant D1 mutants of Synechococcus sp. PCC 7942

We examined the effects of mutations at amino acid residues S264 and F255 in the D1 protein on the binding affinity of the stimulatory anion bicarbonate and inhibitory anion formate in Photosystem II (PS II) in Synechococcus sp. PCC 7942. Measurements on the rates of oxygen evolution in the wild type and mutant cells in the presence of different concentrations of formate with a fixed bicarbonate concentration and vice versa, analyzed in terms of an equilibrium activator-inhibitor model, led to the conclusion that the equilibrium dissociation constant for bicarbonate is increased in the mutants, while that of the formate remains unchanged (11±0.5 mM). The hierarchy of the equilibrium dissociation constant for bicarbonate (highest to lowest, ±2 M) was: D1-F255L/S264A (46 M)>D1-F255Y/ S264A (31 M)D1-S264A (34 M)D1-F255Y (33 M)>wild type (25 M). The data suggest the importance of D1-S264 and D1-F255 in the bicarbonate binding niche. A possible involvement of bicarbonate and these two residues in the protonation of Q_B ^-, the reduced secondary plastoquinone of PS II, in the D1 protein is discussed.Abbreviations Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - MES 2-[N-morpholino]ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - Q_A bound plastoquinone, a one-electron acceptor in Photosystem II - Q_B another bound plastoquinone, a two-electron acceptor in Photosystem II This paper is dedicated to the memory of my dear friend Robin Hill-Govindjee.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Optical characterization of Photosystem II electron donors

Detailed absorbance difference spectra are reported for the Photosystem II acceptor Q, the secondary donor Z, and the donor involved in photosynthetic oxygen evolution which we call M. The spectra of Z and Q could be resolved by analysis of flash-induced kinetics of prompt and delayed fluorescence, EPR signal II_f and absorbance changes in Tris-washed system II preparations in the presence of ferricyanide and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The spectrum of Z oxidation consists mainly of positive bands at 260, 300 and 390–450 nm on which a chlorophyll a band shift around 438 nm is superimposed, and is largely pH-independent as is also the case for the spectrum of Q reduction. The re-reduction of Z⁺ occurred in the millisecond time range, and could be explained by a competition between back reaction with Q^? (120 ms at pH 6.0) and reduction by ferrocyanide. When the Tris treatment is omitted the preparations evolve oxygen, and the photoreduction of Q (with DCMU present) is accompanied by the oxidation of M. The Q spectrum being known, the spectrum of the oxidation of M could be determined as well. It consists of a broad, asymmetric increase peaking near 305 nm and of a Chl a band shift, which is about the same as that accompanying Z in Tris-washed system II. Comparison with spectra of model compounds suggests that Z is a bound plastoquinol which is oxidized to the semiquinone cation and that the oxidation of M is an Mn(III) → Mn(IV) transition.     

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-Q_A-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, K_i, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the Q_B niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.Abbreviations BSA bovine seroalbumin - Chl chlorophyll - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2-(N-morpholino)-ethanesulphonic acid - Pheo pheophytin - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - PS Photosystem - RC reaction center - Tricine N-[Tris(hydroxymethyl)-methyl]-glycine