Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m⁻²) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA₃₂₅(t), as an indicator of redox changes in Q_A, and of the relative fluorescence quantum yield, F(t)/F_o, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA₃₂₅, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA₃₂₅ dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl₂, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680^+·Q_A ^−● is almost invariant to this UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of TyrD in the electron transfer kinetics in Photosystem II

Redox-active tyrosine (Tyr) D is indirectly involved in controlling the primary electron transfer in PSII. The presence of the oxidized TyrD renders P680+ more oxidizing by localizing the charge more on PD1 and thus facilitates trapping of the excitation energy in PSII. We also conclude that the mechanism of the primary charge separation and stabilization is altered upon QA reduction.     

Evidence for two types of electron transfer processes through Photosystem II

Inhibition of electron flow from H₂O to methylviologen by 3-(34 dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q _A and Q _B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H⁺]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q _A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q _B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.Abbreviations DCMU 3-(34 dichlorophenyl)-1, 1 Dimethyl urea - MV methyl viologen - PS II Photosystem II - Tris tris (hydroxymethyl)aminomethane     

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.     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I⁻, Fe(CN)⁴⁻₆, W(CN)⁴⁻₈) are poor reducing agents for Z⁺· whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors.

A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z⁺·. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I-, Fe(CN)6(4-), W(CN)8(4-) are poor reducing agents for Z.+ whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors. A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z.+. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

Energy transfer between Photosystem II and Photosystem I in chloroplasts

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at ?196 °C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5 mM MgCl₂ which presumably changes the distribution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, α, being the fraction distributed to Photosystem I, and β, the fraction to Photosystem II, and a term for the rate constant for energy transfer from Photosystem II to Photosystem I,k_T(II→I). The data, analyzed within the context of the model, permit a direct comparison of α andk_T(II→I) in the absence (?) and presence (+) of Mg²⁺:α/^?α⁺= 1.2andk/^?_T(II→I)k⁺_T(II→I)= 1.9. If the criterion thatα + β = 1 is applied absolute values can be calculated: in the presence of Mg²⁺,a⁺ = 0.27 and the yield of energy transfer,φ⁺_T(II→I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg²⁺,α^? = 0.32 andφ_T(II→I) varied from 0.12 to 0.28.The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvseting chlorophyll of Photosystem II to Photosystem I,k_T(II→I), and a transfer from the reaction centers of Photosystem II to Photosystem I,k_t(II→I). In that caseα/^?α⁺= 1.3,k/^?_T(II→I)k⁺_T(II→I)= 1.3 andk/^?_t(II→I)k⁺_(tII→I)= 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.     

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.     

Excitation energy transfer and trapping in higher plant Photosystem II complexes with different antenna sizes

We performed picosecond fluorescence measurements on well-defined Photosystem II (PSII) supercomplexes from Arabidopsis with largely varying antenna sizes. The average excited-state lifetime ranged from 109 ps for PSII core to 158 ps for the largest C₂S₂M₂ complex in 0.01% α-DM. Excitation energy transfer and trapping were investigated by coarse-grained modeling of the fluorescence kinetics. The results reveal a large drop in free energy upon charge separation (>700 cm⁻¹) and a slow relaxation of the radical pair to an irreversible state (∼150 ps). Somewhat unexpectedly, we had to reduce the energy-transfer and charge-separation rates in complexes with decreasing size to obtain optimal fits. This strongly suggests that the antenna system is important for plant PSII integrity and functionality, which is supported by biochemical results. Furthermore, we used the coarse-grained model to investigate several aspects of PSII functioning. The excitation trapping time appears to be independent of the presence/absence of most of the individual contacts between light-harvesting complexes in PSII supercomplexes, demonstrating the robustness of the light-harvesting process. We conclude that the efficiency of the nonphotochemical quenching process is hardly dependent on the exact location of a quencher within the supercomplexes.     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Stimulation of electron transport from Photosystem II to Photosystem I in spinach chloroplasts

Electron transport from Photosystem II to Photosystem I of spinach chloroplasts can be stimulated by bicarbonate and various carbonyl or carboxyl compounds. Monovalent or divalent cations, which have hitherto been implicated in the energy distribution between the two photosystems, i.e., spillover phenomena at low light intensities, show a similar effect under high light conditions employed in this study. A mechanism for this stimulation of forward electron transport from Photosystem II to Photosystem I could involve inhibition of two types of Photosystem II partial reactions, which may involve cycling of electrons around Photosystem II. One of these is the DCMU-insensitive silicomolybdate reduction, and the other is ferricyanide reduction by Photosystem II at pH 8 in the presence of dibromothymoquinone. Greater stimulation of forward electron transport reactions is observed when both types of Photosystem II cyclic reactions are inhibited by bicarbonate, carbonyl and carboxyl-type compounds, or by certain mono- or divalent cations.Abbreviations used: DCMU, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea; DCIP, 2,6-dichloroindophenol; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; FeCN, potassium ferricyanide; MV, methylviologen; PS I, photosystem I; PS II, photosystem II; SM, silicomolybdic acid.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

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.

     

Energy transfer and quantum yield in Photosystem II

The photoreduction and dark reoxidation of Q_α and Q_β, the primary electron acceptors of Photosystems (PS) IIα and IIβ, respectively, in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) were studied in tobacco chloroplasts by means of fluorescence and absorbance measurements. The magnitude of a correction for an absorbance change by the oxidizing side of PS II needed in our previous study of the quantum yield of Q reduction (Biochim. Biophys. Acta 635 (1981), 111–120) has been determined. The absorbance change occurs in PS IIα mainly. The maximum fluorescence yield was found to be the same as in the mutant Su/su, which has a 3-fold higher reaction center concentration and a lower PS IIα to PS IIβ ratio. The kinetics of the light-induced fluorescence increase were measured after various pretreatments and the corresponding kinetics of the integrated fluorescence deficit were analyzed into their α and β components. From the results the contribution to the minimum fluorescence level, the degree of energy transfer between units, and the quantum efficiency of Q reduction were calculated for both types of PS II. This led to the following conclusions. The absence of energy between PS IIβ antennae is confirmed. Fluorescence quenching in PS IIα was adequately described by the matrix model, except for a decrease in the energy transfer between units during photoreduction of Q_α, possibly due to the formation of ‘islets’ of closed centers. PS II reaction centers in which Q is reduced do not significantly quench fluorescence. The ratio of variable to maximum fluorescence, 0.77 in PS IIα and 0.92 in PS IIβ, multiplied by the fraction of Q remaining in the reduced state after one saturating flash, 0.88 in PS IIα and greater than 0.95 in PS IIβ, leads to a net quantum efficiency of Q reduction in the presence of DCMU and NH₂OH of 0.68 in PS IIα and about 0.90 in PS IIβ. These values are in good agreement with the measured overall quantum efficiency of Q reduction.     

Electron transfer in the water-oxidizing complex of Photosystem II

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, –3 during the so-called S₀ S₁ S₂ S₃ S₀ redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S₂ and the lowest in state S₁.     

Photosystem II cycle and alternative electron flow in leaves

Sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.) were grown in the laboratory and leaves were taken from field-grown birch trees (Betula pendula Roth). Chlorophyll fluorescence, CO2 uptake and O2 evolution were measured and electron transport rates were calculated, J(C) from the CO2 uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, J(O) from the O2 evolution rate, and J(F) from Chl fluorescence parameters. Mesophyll diffusion resistance, r(md), used for the calculation of J(C), was determined such that the in vivo Rubisco kinetic curve with respect to the carboxylation site CO2 concentration became a rectangular hyperbola with Km(CO2) of 10 microM at 22.5 degrees C. In sunflower, in the absence of external O2, J(O) = 1.07 J(C) when absorbed photon flux density (PAD) was varied, showing that the O2-independent components of the alternative electron flow to acceptors other than CO2 made up 7% of J(C). Under saturating light, J(F), however, was 20-30% faster than J(C), and J(F)-J(C) depended little on CO2 and O2 concentrations. The inter-relationship between J(F)-J(C) and non-photochemical quenching (NPQ) was variable, dependent on the CO2 concentration. We conclude that the relatively fast electron flow J(F)-J(C) appearing at light saturation of photosynthesis contains a minor component coupled with proton translocation, serving for nitrite, oxaloacetate and oxygen reduction, and a major component that is mostly cyclic electron transport around PSII. The rate of the PSII cycle is sufficient to release the excess excitation pressure on PSII significantly. Although the O2-dependent Mehler-type alternative electron flow appeared to be under the detection threshold, its importance is discussed considering the documented enhancement of photosynthesis by oxygen.