Fluorescence Induction Kinetics in Membrane Preparations of Photosystem II with Heterogeneous Metal Clusters (Mn/Fe) in the Oxygen-Evolving Complex

Transport of electrons in spinach photosystem II (PSII) whose oxygen-evolving complex (OEC) contains heterogeneous metal clusters 2Mn2Fe and 3Mn1Fe was studied by measuring the fluorescence induction kinetics (FIK). PSII(2Mn,2Fe) and PSII(3Mn,1Fe) preparations were produced using Cadepleted PSII membranes (PSII(–Ca)). It was found that FIK in PSII(2Mn,2Fe) membranes is similar in form to FIK in PSII(–Ca) samples, but the fluorescence yield is lower in PSII(2Mn,2Fe). The results demonstrate that, just as in PSII(–Ca) preparations, there is electron transfer from the metal cluster in the OEC to the primary plastoquinone electron acceptor Q_A. They also show that partial substitution of Mn cations with Fe has no effect on the electron transport on the acceptor side of PSII. Thus, these data demonstrate the possibility of water oxidation either by the heterogeneous metal cluster or just by the manganese dimer. We established that FIK in PSII(3Mn,1Fe) preparations are similar in form to FIK in PSII(2Mn,2Fe) membranes but PSII(3Mn,1Fe) is characterized by a slightly higher maximal fluorescence yield, F_max. The electron transfer rate in PSII(3Mn,1Fe) preparations significantly (by a factor of two) increases in the presence of Ca²⁺, whereas Ca²⁺ has hardly any effect on the electron transport in PSII(2Mn,2Fe) membranes. In Mndepleted PSII membranes, FIK reaches its maximum (the so-called peak K), after which the fluorescence yield starts to decrease as the result of two factors: the oxidation of reduced primary plastoquinone Q _A ^? and the absence of electron influx from the donor side of PSII. The replacement of Mn cations by Fe in PSII(?Mn) preparations leads to fluorescence saturation and disappearance of the K peak. This is possibly due to the deceleration of the charge recombination process that takes place between reduced primary electron acceptor Q _A ^? and oxidized tyrosine Y _Z ^+. which is an electron carrier between the OEC and the primary electron donor P680.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Ca2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing,Ca-depleted complexes

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the “blocking effect” since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca²⁺ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca²⁺ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q_A^? and Y_Z^?+ from t_1/2?=?30 ms to 46 ms. However, Ca²⁺ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn₄CaO₅ catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca²⁺ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca²⁺). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca²⁺ and Sr²⁺ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La³⁺, Ni²⁺, Cd²⁺, and Mg²⁺ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn₄CaO₅ cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

     

Rapid degradation of the tetrameric Mn cluster in illuminated, PsbO-depleted photosystem II preparations

A “decoupling effect” (light-induced electron transport without O₂ evolution) was observed in Ca-depleted photosystem II (PSII(-Ca)) membranes, which lack PsbP and PsbQ (Semin et al. (2008) Photosynth. Res., 98, 235–249). Here PsbO-depleted PSII (PSII(-PsbO)) membranes (which also lack PsbP and PsbQ) were used to examine effects of PsbO on the decoupling. PSII(-PsbO) membranes do not reduce the acceptor 2,6-dichlorophenolindophenol (DCIP), in contrast to PSII(-Ca) membranes. To understand why DCIP reduction is lost, we studied light effects on the Mn content of PSII(-PsbO) samples and found that when they are first illuminated, Mn cations are rapidly released from the Mn cluster. Addition of an electron acceptor to PSII(-PsbO) samples accelerates the process. No effect of light was found on the Mn cluster in PSII(-Ca) membranes. Our results demonstrate that: (a) the oxidant, which directly oxidizes an as yet undefined substrate in PSII(-Ca) membranes, is the Mn cluster (not the Y_Z radical or P680⁺); (b) light causes rapid release of Mn cations from the Mn cluster in PSII(-PsbO) membranes, and the mechanism is discussed; and (c) rapid degradation of the Mn cluster under illumination is significant for understanding the lack of functional activity in some PSII(-PsbO) samples reported by others.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.     

pH Dependence of the Efficiency of Binding of Iron Cations to the Donor Side of Photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HA_Z) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK ₁ = 5.8 and pK ₂ = 8.0. The pH dependence of the O₂-evolution mediated by PS II membranes is also bellshaped (pK ₂ = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing ⁵⁷Fe(II) + ⁵⁷Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK ₂ = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH^– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH^– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Blocking of electron donation by Mn(II) to Y(Z*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light

The donation of electrons by Mn(II) and Fe(II) to Y(Z*) through the high-affinity (HA(Z)) site in Mn-depleted photosystem II (PSII) membranes has been studied by flash-probe fluorescence yield measurements. Mn(II) and Fe(II) donate electrons to Y(Z*) with about the same efficiency, saturating this reaction at the same concentration (ca. 5 microM). However, following a short incubation of the membranes with 5 microM Fe(II), but not with Mn(II) in room light, added Mn(II) or Fe(II) can no longer be photooxidized by Y(Z)(*). This blocking effect is caused by specifically bound, photooxidized Fe [> or =Fe(III)] and is accompanied by a delay in the fluorescence yield decay kinetics attributed to the slowing down of the charge recombination rate between Q(a-) and Y(Z*). Exogenously added Fe(III), on the other hand, does not donate electrons to Y(Z*), does not block the donation of electrons by added Mn(II) and Fe(II), and does not change the kinetics of the decay of the fluorescence yield. These results demonstrate that the light-dependent oxidation of Fe(II) by Y(Z*) creates an Fe species that binds at the HA(Z) site and causes the blocking effect. The pH dependence of Mn(II) electron donation to Y(Z*) via the HA(Z) site and of the Fe-blocking effect is different. These results, together with sequence homologies between the C-terminal ends of the D1 and D2 polypeptides of the PSII reaction center and several diiron-oxo enzymes, suggest the involvement of two or perhaps more (to an upper limit of four to five) bound iron cations per reaction center of PSII in the blocking effect. Similarities in the interaction of Fe(II) and Mn(II) with the HA(Z) Mn site of PSII during the initial steps of the photoactivation process are discussed. The Fe-blocking effect was also used to investigate the relationship between the HA(Z) Mn site and the HA sites on PSII for diphenylcarbazide (DPC) and NH2OH oxidation. Blocking of the HA(Z) site with specifically bound Fe leads to the total inhibition of electron donation to Y(Z*) by DPC. Since DPC and Mn(II) donation to PSII is noncompetitive [Preston, C., and Seibert, M. (1991) Biochemistry 30, 9615-9624], the Fe bound to the HA(Z) site can also block the DPC donation site. On the other hand, electron donation by NH2OH to PSII still occurs in Fe-blocked membranes. Since hydroxylamine does not reduce the Fe [> or =Fe(III)] specifically bound to the HA(Z) site, NH2OH must donate to Y(Z*) through its own site or directly to P680+.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

Comparative study of effects of artificial electron donors on the AT-band of photosystem II thermoluminescence

Extraction of the Mn-cluster from photosystem II (PS II) inhibits the main bands of thermoluminescence and induces a new A_T-band at –20°C. This band is attributed to the charge recombination between acceptor Q_A ^– and a redoxactive histidine residue on the donor side of PS II. The effect of Mn(II) and Fe(II) cations as well as the artificial donors diphenylcarbazide and hydroxylamine on the A_T-band of thermoluminescence was studied to elucidate the role of the redoxactive His residue in binding to the Mn(II) and Fe(II). At the Mn/PS II reaction center (RC) ratio of 90 : 1 and Fe/PS II RC ratio of 120 : 1, treatment with Mn(II) and Fe(II) causes only 60% inhibition of the A_T-band. Preliminary exposure of Mn-depleted PS II preparations to light in the presence of Mn(II) and Fe(II) causes binding of the cations to the high-affinity Mn-binding site, thereby inhibiting oxidation of the His residue involved in the AT -band formation. The efficiency of the A_T-band quenching induced by diphenylcarbazide and hydroxylamine is almost an order of magnitude higher than the quenching efficiency of Mn(II) and Fe(II). Our results suggest that the redox-active His is not a ligand of the high-affinity site and does not participate in the electron transport from Mn(II) and Fe(II) to YZ . The concentration dependences of the A_T-band inhibition by Mn(II) and Fe(II) coincide with each other, thereby implying specific interaction of Fe(II) with the donor side of PS II.     

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P₆₈₀⁺Q_A⁻ limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P₆₈₀⁺ by the secondary electron donor Tyrosine Z cannot occur because Y_Z is already oxidized. The 1.4 ms recombination is seen in Y_Z-less mutants of the cyanobacterium Synechocystis. However, the rate of P₆₈₀⁺Q_A⁻ recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P₆₈₀⁺ reduction by Y_Z were obtained, and the rate of the competing P₆₈₀⁺Q_A⁻ recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y_Z^ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P₆₈₀⁺Q_A⁻ recombination. The two P₆₈₀⁺Q_A⁻ lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Molecular interference of Cd with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd²⁺ is known to exchange, with high affinity in a slow reaction, for the Ca²⁺ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd²⁺ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd²⁺-binding to those sites, we have studied how Cd²⁺ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd²⁺ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd²⁺ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y_Z to P₆₈₀⁺ was inhibited; (ii) Q_A⁻ to Q_B electron transfer was slowed down; (iii) the S₂ state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q_A⁻Fe²⁺ was modified but its amplitude was not sensitive to the presence of Cd²⁺. In addition, the presence of both Ca²⁺ and DCMU abolished Cd²⁺-induced effects partially and in different sites. The number of sites for Cd²⁺ binding and the possible nature of these sites are discussed.     

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

Iron bound to the high-affinity Mn-binding site of the oxygen-evolving complex shifts the pK of a component controlling electron transport via Y(Z)

Flash-probe fluorescence spectroscopy was used to compare the pH dependence of charge recombination between Y(Z)(*) and Q(a)(-) in Mn-depleted, photosystem II membranes [PSII(-Mn)] and in membranes with the high-affinity (HA(Z)) Mn-binding site blocked by iron [PSII(-Mn,+Fe); Semin, B. K., Ghirardi, M. L., and Seibert, M. (2002) Biochemistry 41, 5854-5864]. The apparent half-time for fluorescence decay (t(1/2)) in PSII(-Mn) increased from 9 ms at pH 4.4 to 75 ms at pH 9.0 [with an apparent pK (pK(app)) of 7.1]. The actual fluorescence decay kinetics can be fit to one exponential component at pH <6.0 (t(1/2) = 9.5 ms), but it requires an additional component at pH >6.0 (t(1/2) = 385 ms). Similar measurements with PSII(-Mn,+Fe) membranes show that iron binding has little effect on the maximum and minimum t(1/2) values measured at alkaline and acidic pHs but that it does shift the pK(app) from 7.1 to 6.1 toward the more acidic pK(app) value typical of intact membranes. Light-induced Fe(II) blocking of the PSII(-Mn) membrane is accompanied by a decrease in buffer Fe(II) concentration. This decrease was not the result of Fe(II) binding, but rather of its oxidation at two sites, the HA(Z) site and the low-affinity site. M?ssbauer spectroscopy at 80 K on PSII(-Mn,+Fe) samples, prepared under conditions providing the maximal blocking effect but minimizing the amount of nonspecifically bound iron cations, supports this conclusion since this method detected only Fe(III) cations bound to the membranes. Correlation of the kinetics of Fe(II) oxidation with the blocking parameters showed that blocking occurs after four to five Fe(II) cations were oxidized at the HA(Z) site. In summary, the blocking of the HA(Z) Mn-binding site by iron in PSII(-Mn) membranes not only prevents the access of exogenous donors to Y(Z) but also partially restores the properties of the hydrogen bond net found in intact PS(II), which in turn controls the rate of electron transport to Y(Z).     

Formation of tyrosine radicals in photosystem II under far-red illumination

Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P₆₈₀. Tyr_Z, part of the water-oxidizing complex, is a preferential fast electron donor while Tyr_D is a slow auxiliary donor to P₆₈₀ ⁺. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of Tyr_D occurs via equilibrium with Tyr_Z ^? at pH 4.7 and 6.3. At pH 8.5 direct Tyr_D oxidation by P₆₈₀ ⁺ occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P₆₈₀ ⁺) on the Chl_D1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.     

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

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

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

Amino acid residues that modulate the properties of tyrosine YZ and the manganese cluster in the water oxidizing complex of photosystem II

The catalytic site for photosynthetic water oxidation is embedded in a protein matrix consisting of nearly 30 different polypeptides. Residues from several of these polypeptides modulate the properties of the tetrameric Mn cluster and the redox-active tyrosine residue, Y_Z, that are located at the catalytic site. However, most or all of the residues that interact directly with Y_Z and the Mn cluster appear to be contributed by the D1 polypeptide. This review summarizes our knowledge of the environments of Y_Z and the Mn cluster as obtained from the introduction of site-directed, deletion, and other mutations into the photosystem II polypeptides of the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.