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A new EPR signal attributed to the primary plastoquinone acceptor in Photosystem II
Abstract
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q−AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin− signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin− signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.
... The most important reaction in the current study is the primary charge separation reaction. In PS II centres with Q A H 2 (double reduced and protonated Q A ) or samples lacking Q A , the primary charge separation can be followed by photo-accumulation of the Pheo − radical under reducing conditions [30][31][32] . This reaction is also known to be functional using far-red illumination 22 . ...
... Figure 2 shows the EPR signal from the Pheo − radical in PS II enriched membranes where Q A was doubly reduced. The radical EPR spectrum formed by either visible or far-red (732 nm) light was 13G wide with g = 2.0035 [30][31][32] . Both parameters are indicative that the signal originates from the Pheo − radical. ...
... For the measurements of the Pheo − radical, doubly reduced PS II membranes (6.0 mg Chl/ml) were subjected to white or far-red (732 nm) continuous wave light illumination as described earlier [30][31][32] , at 20 °C, for 6 and 10 minutes respectively at room temperature. Thereafter, the samples were frozen within 1 sec in a 200 K dry ice/ethanol bath and subsequently transferred into N 2 (l) before the measurements. ...
Charge separation is a key component of the reactions cascade of photosynthesis, by which solar energy is converted to chemical energy. From this photochemical reaction, two radicals of opposite charge are formed, a highly reducing anion and a highly oxidising cation. We have previously proposed that the cation after far-red light excitation is located on a component different from PD1, which is the location of the primary electron hole after visible light excitation. Here, we attempt to provide further insight into the location of the primary charge separation upon far-red light excitation of PS II, using the EPR signal of the spin polarized 3P680 as a probe. We demonstrate that, under far-red light illumination, the spin polarized 3P680 is not formed, despite the primary charge separation still occurring at these conditions. We propose that this is because under far-red light excitation, the primary electron hole is localized on ChlD1, rather than on PD1. The fact that identical samples have demonstrated charge separation upon both far-red and visible light excitation supports our hypothesis that two pathways for primary charge separation exist in parallel in PS II reaction centres. These pathways are excited and activated dependent of the wavelength applied.
... In order to obtain a sample with doubly reduce Q A [6,14,17], the film was pre-illuminated in the cryostat (λ N 600 nm;~15 mW/cm 2 ) for 5 min at 285 K and then frozen to 95 K after subsequent 1.5 h dark adaptation at 285 K. Light-induced triplet-minus-singlet difference spectra were calculated as lightminus-relaxation spectra, i.e. as a difference between the FTIR spectra (64 scans; accumulation time of 21 s) measured under and after continuous actinic illumination of a sample (λ N 600 nm;~15 mW/ cm 2 ). This approach made it possible to separate reversible IR absorbance changes associated with formation of 3 Chl from those due to stable photoaccumulation of Pheo D1 − [17,45]. Cycles of illumination were repeated to improve the signal-to-noise ratio. ...
... Previously, the FTIR difference spectra [13,[53][54][55] and the difference resonance Raman spectrum [56] for photoaccumulation of Pheo D1 − , as well as the difference resonance Raman spectrum associated with the chemical exchange of Pheo D1 with 13 1 -deoxo-13 1 -hydroxypheophytin a [57] have shown that the stretching frequency of the hydrogen-bonded 13 1 -keto C=O group of Pheo D1 in PSII particles is at 1677-1681 cm −1 . It is unlikely, however, that the negative peak at 1680 cm −1 in the light-minus-relaxation FTIR difference spectrum of core complexes (Fig. 3B) could be ascribed to photoaccumulation of Pheo D1 − because this radical anion is stable [17,45] and did not relax in the dark on the time scale of our measurements. In addition, photoaccumulation of Pheo D1 − would be accompanied by an appearance of the Pheo D1 − marker band at 1586-1588 cm −1 [13,[54][55][56][57], but such a band is not observed in Fig. 3B. ...
Phosphorescence measurements at 77 K and light-induced FTIR difference spectroscopy at 95 K were applied to study of the triplet state of chlorophyll α ((3)Chl) in photosystem II (PSII) core complexes isolated from spinach. Using both methods, (3)Chl was observed in the core preparations with doubly reduced primary quinone acceptor QA. The spectral parameters of Chl phosphorescence resemble those in the isolated PSII reaction centers (RCs). The main spectral maximum and the lifetime of the phosphorescence corresponded to 955±1nm and of 1.65±0.05ms respectively; in the excitation spectrum, the absorption maxima of all core complex pigments (Chl, pheophytin α (Pheo), and β-carotene) were observed. The differential signal at 1667(-)/1628(+)cm(-1) reflecting a downshift of the stretching frequency of the 13(1)-keto CO group of Chl was found to dominate in the triplet-minus-singlet FTIR difference spectrum of core complexes. Based on FTIR results and literature data, it is proposed that (3)Chl is mostly localized on the accessory chlorophyll that is in triplet equilibrium with P680. Analysis of the data suggests that the Chl triplet state responsible for the phosphorescence and the FTIR difference spectrum is mainly generated due to charge recombination in the reaction center radical pair P680(+)PheoD1(-), and the energy and temporal parameters of this triplet state as well as the molecular environment and interactions of the triplet-bearing Chl molecule are similar in the PSII core complexes and isolated PSII RCs.
... Firstly, in both WT*1-PSII and PsbJ-43H/PSII, the addition of DCMU resulted in the formation of QA -Fe 2+ /DCMU to the detriment of Fe 2+ QB - (Velthuys 1981). In both samples, QA -Fe 2+ /DCMU exhibited either the g = 1.9 signal or the g = 1.82 signal (Rutherford et al. 1983, Rutherford andZimmermann 1984), see below for a better description of the signals in Figure 6. Secondly, in WT*1-PSII, the addition of DCMU also modified the non-heme iron signal as previously observed (Diner and Petrouleas 1987). ...
Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, and Tsl0063. The presence of Psb27 has been proposed (Zabret et al. in Nat Plants 7:524–538, 2021; Huang et al. Proc Natl Acad Sci USA 118:e2018053118, 2021; Xiao et al. in Nat Plants 7:1132–1142, 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28, and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ∆PsbJ–PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, thermoluminescence, EPR spectroscopy, and UV–visible time-resolved spectroscopy. In the purified ∆PsbJ–PSII, an active Mn4CaO5 cluster is present in 60–70% of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB⁻ couple increases by ≥ 120 mV , thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA⁻ and QB/QB⁻ could contribute in a protection against the charge recombination between the donor side and QB⁻, identified at the origin of photoinhibition under low light (Keren et al. in Proc Natl Acad Sci USA 94:1579–1584, 1997), and possibly during the slow photoactivation process.
... Firstly, in both WT*1-PSII and PsbJ-43H/PSII, the addition of DCMU resulted in the formation of QA -Fe 2+ /DCMU to the detriment of Fe 2+ QB -(Velthuys 1981). In both samples, QA -Fe 2+ /DCMU exhibited either the g = 1.9 signal or the g = 1.82 signal (Rutherford et al. 1983, Rutherford andZimmermann 1984), see below for a better description of the signals in Figure 6. Secondly, in WT*1-PSII, the addition of DCMU also modified the non-heme iron signal as previously observed (Diner and Petrouleas 1987). ...
Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, Tsl0063. The presence of Psb27 has been proposed (Zabret et al. 2021; Huang et al. 2021; Xiao et al. 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28 and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ΔPsbJ-PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, Thermoluminescence, EPR spectroscopy and UV-visible time-resolved spectroscopy. In the purified ΔPsbJ-PSII, an active Mn4CaO5 cluster is present in 60-70 % of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB- couple increases by ≈120 mV thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA- and QB/QB- could contribute in a protection against the charge recombination between the donor side and QB-, identified at the origin of photoinhibition under low light (Keren et al. 1997), and possibly during the slow photoactivation process.
... And second, a similar shift is caused by replacing bicarbonate as a ligand of the non-heme iron with format, with a reported + 70 mV increase in QA/QAredox potential [13]. Interestingly, this substitution shifts the characteristic EPR signal of the QA -Fe 2+ /QB -Fe 2+ complexes from g = 1.9 [40] to g = 1.84, a value characteristic for the bacterial reaction centre [41]. In PSII-I the D1 Glu241 residue (which was not certified by peer review) is the author/funder. ...
Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.
... As mentioned above, FTIR data suggest that it is indeed bicarbonate ( Hienerwadel and Berthomieu 1995). In apparent contradiction to this conclusion, simulations of the characteristic EPR signal of the Q À A Fe II complex ( Rutherford and Zimmermann 1984;Nugent et al. 1992) were interpreted as indicating a carbonate ligand ( Cox et al. 2009). However, the calculations by Saito et al. (2013) again favor the bicarbonate variant. ...
Photosystem II (PSII), the light-driven water:plastoquinone (PQ) oxidoreductase of oxygenic photosynthesis, contains a nonheme iron (NHI) at its electron acceptor side. The NHI is situated between the two PQs QA and QB that serve as one-electron transmitter and substrate of the reductase part of PSII, respectively. Among the ligands of the NHI is a (bi)carbonate originating from CO2, the substrate of the dark reactions of oxygenic photosynthesis. Based on recent advances in the crystallography of PSII, we review the structure of the NHI in PSII and discuss ideas concerning its function and the role of bicarbonate along with a comparison to the reaction center of purple bacteria and other enzymes containing a mononuclear NHI site.
... The latter is a specialized plastoquinone molecule termed QA (124,176,188), and it can be photore duced to its plastosemiquinone anion form QA' When excitation energy from the light-harvesting pigment arrives at the reaction center, an endergonic charge separation occurs between P680 and pheophytin, followed by electron transfer to QA according to the reaction mechanism: Closely associated with the primary quinone acceptor QA of PSII is one atom of Fe(II), which does not participate directly as an intermediate in the electron-transport process. The Fe(II) is known to be magnetically coupled to the semiquinone anion form of QA(QA) (58, 159) and is probably situated about 7 A. away from QA and from the secondary quinone QB (47). The oxidation of Fe(Il) to Fe(lII) by exogeneously added ferricyanide (150) coincides with the oxidation of a previously unidentified high-potential elec tron acceptor of PSII (Q400; Ref. 87). ...
... Carbonate. Recently, the characteristic g-value of ∼1.9 for the semiquinone and nonheme Fe complex in EPR spectroscopy (40) was reinvestigated in theoretical simulations. On the basis of the simulations, it was proposed that the native ligand to the nonheme Fe was carbonate (CO 3 2-) rather than bicarbonate (HCO 3 -) (41). ...
Photosystem II uses light to drive water oxidation and plastoquinone (PQ) reduction. PQ reduction involves two PQ cofactors, Q(A) and Q(B), working in series. Q(A) is a one-electron carrier, whereas Q(B) undergoes sequential reduction and protonation to form Q(B)H(2). Q(B)H(2) exchanges with PQ from the pool in the membrane. Based on the atomic coordinates of the Photosystem II crystal structure, we analyzed the proton transfer (PT) energetics adopting a quantum mechanical/molecular mechanical approach. The potential-energy profile suggests that the initial PT to Q(B)(•-) occurs from the protonated, D1-His252 to Q(B)(•)(-) via D1-Ser264. The second PT is likely to occur from D1-His215 to Q(B)H(-) via an H-bond with an energy profile with a single well, resulting in the formation of Q(B)H(2) and the D1-His215 anion. The pathway for reprotonation of D1-His215(-) may involve bicarbonate, D1-Tyr246 and water in the Q(B) site. Formate ligation to Fe(2+) did not significantly affect the protonation of reduced Q(B), suggesting that formate inhibits Q(B)H(2) release rather than its formation. The presence of carbonate rather than bicarbonate seems unlikely because the calculations showed that this greatly perturbed the potential of the nonheme iron, stabilizing the Fe(3+) state in the presence of Q(B)(•-), a situation not encountered experimentally. H-bonding from D1-Tyr246 and D2-Tyr244 to the bicarbonate ligand of the nonheme iron contributes to the stability of the semiquinones. A detailed mechanistic model for Q(B) reduction is presented.
Plastoquinones (PQs) act as electron and proton mediators in photosystem II (PSII) for solar-to-chemical energy conversion. It is known that the redox potential of PQ varies in a wide range spanning hundreds of millivolts, however, its structural origin is not known yet. Here by developing a pump-probe ultraviolet resonance Raman technique, we measured the vibrational structures of PQs including QA and QB in cyanobacterial PSII directly. The conversion of QA to QA•- in the Mn-depleted PSII is verified by direct observation of the distinct QA•- vibrational bands. A frequency upshift of the ring C=O/C=C stretch band at 1565 cm-1 for QA•- was observed, which suggests a π-π interaction between the quinone ring and Trp253. In contrast, proton-coupled reduction of QA to QAH upon light-driven electron transfer is demonstrated in PSII without QB bound. The H-bond between QA and His214 is likely the proton origin of this proton-coupled electron transfer.
Over the last few years it has become increasingly clear that considerable homology exists between the acceptor side components of photosystem II (PS II) and those of reaction centers from purple photosynthetic bacteria (Parson and Ke 1982, Crofts and Wraight 1983, Dutton, Chap. 5, Michel and Deisenhofer, Chap. 8.4, both this Vol.). While one might hardly have expected to see familiar faces among the high potential donors of PS II, recent evidence suggests that some of the same old dogs may have learned new tricks.
The main electron transfer events in PS2 are:
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Multifrequency EPR and 55Mn ENDOR spectroscopy were used to characterize the S2′ state of the Ca2+- depleted oxygen-evolving complex. The 55Mn ENDOR spectrum of the S2′ state is broader than that of the native S2 state. Simulations of the data were performed using the spin Hamiltonian formalism. It was observed that the magnitudes of the four 55Mn hyperfine tensors (A1,iso≈ 300 MHz; A2, iso, A3, iso, A4, iso≈200MHz) are approximately the same as in the native S2 state. In addition, the geometries of the anisotropic hyperfine tensors are not changed. Thus, the same oxidation states are assigned to the Mn ions both in S2 and S2′ (MnA, MnB, MnC: IV, MnD: III). The isotropic hyperfine values of the individual Mn ions, especially A2, iso and A4 iso, do change upon Ca2+ depletion, indicating that, nonetheless, the electronic spin coupling scheme of the cluster is affected by Ca2+ removal.
This chapter discusses the primary reactions of photosystems I and II of algae and higher plants. In photosynthetic organisms, the “primary reactions” fulfil the objective of converting the energy of light into a primary form of chemical energy which lasts for a time compatible with ordinary biochemical processes—that is, milliseconds. In these reactions, a rather large fraction, approximately 40%, of the photon energy is stored as chemical free energy. The primary reactions can be viewed from two major perspectives. Firstly, from a photochemical point of view: pigment molecules are excited to their lowest excited singlet state, which reacts in an electron transfer reaction, the first step of a process of charge separation. Secondly, from a biochemical point of view the reactions take place in highly organized complexes, the reaction centres, which are made up of several classes of molecules that cooperate in fulfilling complementary roles, such as: architectural support, light absorption, energy transfer and electron transfer. All oxygenic organisms, ranging from cyanobacteria to algae and higher plants, contain photosystem I (PS I) and PS II reaction centres, with only minor variations in spite of their large taxonomic and ecological diversity.
Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a non-heme Fe2+ located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA•- and QB•- are magnetically couple to the Fe2+ giving weak and complex EPR signals. The distorted octahedral Fe2+ has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB•- Fe2+ EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA•- Fe2+ in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA•- Fe2+ and QB•- Fe2+ EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD•). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB•-Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB•-/QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA•-Fe2+ state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII.On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.
Publisher Summary This chapter discusses electron paramagnetic resonance (EPR) in photosynthesis. Practically, all aspects of EPR spectroscopy come to the fore, individually or in combination, in the various photosynthetic systems of plants and bacteria, in intact cells or in isolated subcellular particles or purified reaction center proteins. EPR has been instrumental in the demonstration that the primary electron donor, P, in bacterial reaction centers (RC) is a bacteriochlorophyll dimer. In normal photosynthesis, in all photosystems the charge on the photoreduced intermediary acceptor is quickly transported to the next primary acceptor. When this acceptor is (photo) chemically pre-reduced or removed by extraction, however, this negative charge cannot be further transported, and recombines with the positive charge on the primary donor. The recombination product is either the singlet ground or excited state, or the triplet excited state of P. The primary donor of photosystem II (P-680) is much more difficult to observe with EPR than that of photosystem PS I, because in normally functioning PS II the photo-oxidized donor is very rapidly (within at most a few hundred ns) reduced by an electron donor called Z. The reduced intermediary acceptor I (BPh) is normally too short-lived to be observable by EPR. However, it can be photoaccumulated at cryogenic temperatures in isolated RCs of, for example, Rb. Sphaeroides, when reduced Cyt c is added, because of slow, irreversible electron donation to P + .
Photosynthesis involves the capture of the Sun's energy into biochemical energy that sustains all life on the Earth. It is a massive process that has radically changed the composition of the atmosphere. Its scale can be judged from the fact that man's entire fossil fuel use from 1860 to 1988 was equivalent to only 2 years’ global photosynthesis. Although photosynthesis occurs in a wide range of organisms (bacteria, cyanobacteria, algae, and plants), and about a third of it occurs in the oceans, this discussion concentrates on plants.
Ca2+-depleted and Ca2+-reconstituted spinach photosystem II was studied using polarized X-ray absorption spectroscopy of oriented PS II preparations to investigate the structural and functional role of the Ca2+ ion in the Mn4O5Ca cluster of the oxygen-evolving complex (OEC). Samples were prepared by low pH/citrate treatment as one-dimensionally ordered membrane layers, and poised in the Ca2+-depleted S1 (S1'), S2 (S2') and S2'YZ• states, at which point the catalytic cycle of water oxidation is inhibited, and the Ca2+-reconstituted S1 state. Polarized Mn K-edge XANES and EXAFS spectra exhibit pronounced dichroism. Polarized EXAFS data of all states of Ca2+-depleted PS II investigated show only minor changes in distances and orientations of the Mn-Mn vectors compared to the Ca2+-containing OEC, which may be attributed to some loss of rigidity of the core structure. Thus, removal of the Ca2+ ion does not lead to fundamental distortion or rearrangement of the tetranuclear Mn cluster, which indicates that the Ca2+ ion in the OEC is not critical for structural maintenance of the cluster at least in the S1 and S2 states, but fulfills a crucial catalytic function in the mechanism of the water oxidation reaction. On the basis of this structural information, reasons for the inhibitory effect of Ca2+ removal are discussed, attributing to the Ca2+ ion a fundamental role in organizing the surrounding (substrate) water framework and in proton-coupled electron transfer to YZ• (D1-Tyr161).
Quantum theory is used to rationalize the results of recent high-precision X-ray diffraction studies of photosystem II. It is proposed that a single molecule transistor regulates the flow of electrons through this remarkable system. At the core of the device, electrons flow through an iron(II) d-orbital by a process of superexchange, at a rate which is gated by the ambient ligand field. The transistor operates in the negative feedback mode, and its existence suggests that man-made molecular logic gates are technologically feasible. We believe this is the first recorded example of a single molecule electronic transistor in a living system.
The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.
Inhibition of electron flow from H2O to methylviologen by 3-(3′4′ 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.
In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385-398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q400 component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.
A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.
It has been found that plastoquinone (PQ) and α-tocopherol quinone (α-TQ) form quinhydrone-type charge-transfer (CT) complexes on PQH2 and α-TQH2, respectively, in oleic, linoleic and linolenic acid throughout the whole range of temperatures examined, i.e., from −30°C to 60°C. The CT spectra of the PQ-PQH2 complex at room temperature showed one peak at 371.8–383 nm and a shoulder at 454–494 nm, depending on the fatty acid used. The corresponding CT spectra for the α-TQ—α-TQH2 complex were similar in shape with one peak at 371.4–378.1 nm and a shoulder at 439–469 nm. The temperature dependences of CT spectra were not large above the melting point of fatty acids. On the other hand, the changes in CT spectra in the frozen state were striking. A long-wavelength shift of both CT bands (up to 90 nm) and an increase in intensities of the bands, especially of the long-wavelength band, was observed, and as a result the intensity ratio of both bands was reversed with the decrease in temperature. The observed effects were explained in terms of different orientational isomers of the complex, the relative quantities of which varied with temperature changes.
It is well known that two photosystems, I and II, are needed to transfer electrons from H2O to NADP+ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS IIα and PS IIβ units, (b) RC complex containing electron acceptor Q1 or Q2; and (c) electron acceptor complex: QA (having two different redox potentials QL and QH) and QB (QB-type; Q'B type; and non-QB type); additional components such as iron (Q-400), U (Em,7=−450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.
The authors report the detection of a new electron paramagnetic resonance (EPR) signal that demonstrates the presence of a paramagnetic intermediate in the resting (S{sub 1}) state of the photosynthetic oxygen-evolving complex. The signal was detected using the method of parallel polarization EPR, which is sensitive to {Delta}m = 0 transitions in high spin systems. The properties of the parallel polarization EPR signal in the S{sub 1} state are consistent with an S=1 spin state of and exchange-coupled manganese center that corresponds to the reduced form of the species giving rise to the multiline EPR signal in the light-induced S{sub 2} state. The implications for the electronic structure of the oxygen-evolving complex are discussed. 36 refs., 2 fig., 1 tab.
A Fe2+ ion on the acceptor side of plant photosystem II has been substituted by Zn2+ and an anion radical of the primary acceptor quinone, Q−A, has been studied by electron spin echo method. The electron spin echo modulation shows the interaction of the unpaired electron of Q−A with nitrogen nuclei of the histidine and, probably, alanine residues situated nearby. The comparison of the modulation spectra of Q−A with those of the anion radical of plastoquinone-9 stabilized in protonated and deuterated isopropanol matrices allows one to distinguish between the spectrum lines due to the quinone protons and due to the protons of other molecules that form hydrogen bonds with the oxygen atoms of the quinones.
Light that exceeds the photosynthetic capacity of a plant can impair the ability of photosystem II to oxidize water. The light-induced inhibition is initiated by inopportune electron transport reactions that create damaging redox states. There is evidence that secondary electron transport pathways within the photosystem II reaction center can protect against potentially damaging redox states. Experiments using thylakoid membranes poised at different ambient redox potentials demonstrate that light-induced damage to photosystem II can be controlled by a redox component within the reaction center. The rate of photoinhibition is slow when the redox component is oxidized, but increases by more than 10-fold when the redox. component is reduced. Here, using spinach thylakoid membranes, we provide evidence that the redox component is cytochrome bâââ, an intrinsic heme protein of the photosystem II reaction center. The results support a model in which the low-potential (LP) form of cytochrome bâââ protects photosystem II by deactivating a rarely formed, but hazardous redox state of photosystem II, namely, P680/Pheoâ»/Q{sub A}â». Cytochrome bâââLP is proposed to deactivate this potentially lethal redox state by accepting electrons from reduced pheophytin.
Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones Q(A) and Q(B) in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to these of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents, Q-band EPR yielded the principal values of the g-tensor for PQ-9(.-) in 2-propanol and Q(A)(.-) in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9(.-) in vitro and on QA(.-) in the iron-depleted PS II samples, For the former a complete set of isotropic H-1 hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. H-1-ENDOR spectra of Q(A)(.-) in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of Q(A) and specific protein residues, The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.
The nonheme iron of the photosystem II reaction center was converted to its low-spin state (S = 0) by treatment with CN-. This allowed the study of the plastoquinone, Q(A)(-) anion radical by electron spin-echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of the ESEEM data of Q(A)(-) in N-14- and N-15-labeled PSII demonstrates the existence of a protein nitrogen nucleus coupled to the Q(A)(-). The N-14 coupling is characterized by a quadrupolar coupling constant e(2)qQ/4h = 0.82 MHz, an asymmetry parameter eta = 0.45, and hyperfine coupling constant A similar to 2.1 MHz. The N-15 hyperfine coupling is characterized by T = 0.41 MHz and alpha(iso) similar to 3.3 MHz. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acids thought to be close to the Q(A)(-) in PSII. Based on a comparison of the N-14 ESEEM with N-14-NQR and N-14-ESEEM data from the literature, the most likely candidate is the amide nitrogen of the peptide backbone of Ala261 of the polypeptide D2, although the indole nitrogen of Trp254 and the imino nitrogen of His215 of D2 also remain candidates.
The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca2+-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g almost-equal-to 2.004; DELTAH(pp) = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca2+-depleted PS II membranes obtained from N-14- and N-15-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca2+-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and N-14 and N-15 samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.
The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity to a site that structurally interacts with the primary electron acceptor FeQ/sub A//sup -/. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQ/sub A/) to g = 2.1 on the first turnover. These results indicate that the binding site for NH/sub 2/OH overlaps with or interacts with the binding site for Q/sub B/. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH/sub 2/OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH/sub 2/OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, whichmore » exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. The authors also find that the high-affinity binding of NH/sub 2/OH within the WOC produces no observable EPR-active products in the dark. They conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b/sub 559/ or signal II (/sup 160/Tyr x D/sub 1/ protein).« less
Electron paramagnetic resonance (EPR) spectroscopy has been applied in an investigation on the mechanism for photoinhibition of the electron transport in Photosystem II. The experiments were performed in vitro in thylakoid membranes and preparations of Photosystem-II-enriched membranes. Photoinhibition resulted in inhibition of the oxygen evolution and EPR measurements of the S2 state multiline EPR signal show that its induction by illumination at 198 K was decreased with the same kinetics as the oxygen evolution. Further EPR measurements show that the reduction of QA was inhibited with the same kinetics as the oxygen evolution. The amount of photoreducible pheophytin was estimated from photoaccumulation experiments under reducing conditions and the results show that the primary charge separation reaction was inhibited much slower than the oxygen evolution or the reduction of QA. These results indicate that photoinhibition inhibits the electron transfer between pheophytin and QA probably by impairment of the function of QA. In the inhibited centers the primary charge separation reaction is still operational. It is suggested that the event leading to photoinhibition of the electron transport is the double reduction of QA which then leaves its site. Photoinhibition also results in rapid oxidation of cytochrome b-559 and a change of cytochrome b-559 from its high potential form to its low potential form. The reaction is quantitative and proceeds with the same kinetics as the inhibition of oxygen evolution. The potential shift of cytochrome b-559 suggests that photoinhibition induces early conformational changes in Photosystem II.
Replacement of H2O by 2H2O in oxygen-evolving Photosystem II preparations caused an increased resolution of the fine structure of the S2 state EPR spectrum. In both 2H2O and H2O samples, comparison of the S2 spectra generated by illumination at 200 and 283 K (10°C) showed a difference in the fine structure on the hyperfine lines. A reduction in the spacing of the outer hyperfine lines was also observed when samples illuminated at 283 K were compared to those where S2 was formed by 200 K illumination. The observations are interpreted as due to proton binding, perhaps as water, at or near the manganese complex giving rise to the S2 signal.
It has been found that the solubility of PQH2 and α-TQH2 in hexane increased with the increase in PQ and α-TQ concentrations, respectively, that is connected with the formation of quinhydrone-type charge-transfer complexes. Measurements of the solubility of both prenylquinones and their reduced forms in hexane and acetone, at − 30°C and room temperature, showed a much higher affinity of the quinol forms for acetone than for hexane. In the case of quinones, the difference in affinity was not significant. The possibility of charge-transfer complex formation by PQ and α-TQ in thylakoid membranes and the influence of such complexes on the diffusion of PQH2 and α-TQH2 molecules have been considered.
We report spectral hole-burning as well as spontaneous and photo-induced hole-filling in Photosystem II (PSII) core complexes. A comparison is made between measurements on initially dark-adapted PSII cores in which the plastoquinone QA is either neutral or photo-reduced to QA- by illumination at 1.7K, with samples where QA is reduced by either 260K illumination or chemical treatment. The latter preparations have the advantage of exhibiting minimal change in QA- with either time or illumination at 1.7K. This stability allowed us to investigate the association of rapid hole-filling reported for PSII core complexes with the spontaneous re-oxidation of QA-, which occurs in dark-adapted samples after 1.7K illumination. We find that spontaneous hole-filling also occurs in PSII with stable QA- configurations. In either class of sample, spontaneous hole-filling occurs at significantly greater rates than for ‘normal’ two level system hole-burning processes in chlorophyll-protein antenna complexes. It is suggested that the rapid spontaneous hole-filling is associated with protein relaxation within the energy landscape of the PSII core proteins. This landscape may also be involved in influencing other processes such as low-temperature QA reduction and re-oxidation. Shallow holes burnt in QA samples exhibit a narrow (∼2cm-1) photoproduct on the low energy side of the hole. Subsequent illumination of holes burnt at 690nm with 705nm light induces strong hole-filling. These results are discussed with respect to charge and excitation energy transfer processes in PSII cores.
We have examined the effects of a number of carboxylate anions on the iron-quinone complex of Photosystem II (PS II). Typical effects are the following. In the state Q−AFe2+ oxalate enhances significantly the g = 1.84 EPR resonance while, for example, glycolate and glyoxylate suppress it. The anions have variable effects on the iron midpoint potential. Formate and oxalate raise significantly the Em of the iron. Glycolate lowers the Em significantly and the Em shows a weak pH dependence. In the presence of glycolate the native plastosemiquinone () can oxidise the iron. Glyoxylate also lowers the Em, but the Em shows a greater pH dependence than with glycolate but still weaker than the −60 mV/pH unit of the untreated iron. The Fe3+ EPR spectra are characterised by small but distinct shifts, while in addition an unusual resonance at close to g = 4.3 is observed. These as well as the temperature dependence of the spectra are analysed by a spin-Hamiltonian model. Comparison with competition studies in the companion paper indicates that the anions bind as iron ligands displacing bicarbonate.
pH-dependent regulation of Photosystem (PS) II (observed as ‘high-energy quenching’) has been characterized by chlorophyll fluorescence and thermoluminescence measurements in PS II particles, thylakoid membranes, alga cells and leaf tissue. Steady state redox titration of fluorescence yield performed at pH 6.5 revealed that the midpoint redox potential of the primary quinone acceptor, QA, is shifted towards positive direction from Em = −80 mV to Em = +40 mV (the absolute values for Em were varying by about 40 mV between different preparations) after incubation of PS II particles at pH 4.2 for 15 min in the presence of the Ca2+ chelator, PPi. The original midpoint potential was restored after the addition of 300 μM CaCl2. Low-pH treatment (pH 4.6) of PS II particles also resulted in a decrease of the Q band of thermoluminescence (appearing between 10–14°C after DCMU addition) with a concomitant appearance or intensification of a high temperature band between 42–50°C (C band). In accordance with the results of the redox titration of fluorescence yield the C band is attributed to a low-pH-induced high potential form of QA. The interconversion of Q band into the C band was more pronounced in the presence of the Ca2+ chelator, EGTA. Addition of CaCl2 to the low-pH-treated particles diminished the C band and restored the Q band. Light-induced acidification of the thylakoid lumen (ΔpH formation under illumination conditions of ‘high-energy quenching’) was also accompanied by a transformation of the Q band to the C band in isolated thylakoids, in the green alga, Chlorella vulgaris and in pea leaves. The phenomenon was completely reversed by abolishing the pH gradient with 10 mM NH4Cl. Addition of the Ca2+-channel inhibitor verapamil to the thylakoid suspension before the formation of a ΔpH suppressed the transformation of Q band into the C band. In contrast, when a ΔpH was first established and then verapamil was added, the ΔpH-induced change in the glow curve was irreversible and conversion of C band back to the Q band was prevented. It is suggested that the appearance of the C band is associated with Ca2+-dependent reversible inactivation of the water-splitting system and with a shift in the redox potential of QA. We propose that pH-dependent Ca2+-release is a physiological process which controls the electron transport of PS II in vivo.
We have investigated the EPR characteristics of native QB and QB analogues in higher plant PS II. We show that, as in cyanobacteria, an interaction between QA and QB iron-semiquinones (QA−-Fe2+-QB−) is observed which gives an EPR signal near g=1.6. Bicarbonate binding close to the non-haem iron is required to observe this interaction. The EPR signal of QB semiquinone is weak and difficult to distinguish from that of QA. The appearance of the g=1.6 signal from QA−-Fe2+-QB− after 77 K illumination is a better marker for the presence of QB semiquinone. The yield of QB semiquinone in plant PS II is lower than found in cyanobacteria. The brominated quinones DBMIB, TBTQ and bromanil were used as QB analogues to increase the yield of QA−-Fe2+-QB−. These analogues act by forming a stable semiquinone at the QB site and not by covalent binding.
We have studied the EPR signal in PS II from Phormidium laminosum with a g-value of 1.66, which we assign to an interaction between the semiquinones of Qa and Qb and the non-heme iron. 77 K illumination of samples from dark-poised redox titrations show the rise of the signal has a midpoint potential (Em) of about +60 mV, and it is lost with an Em of about −10 mV. Under the same conditions, the rise of the g = 1.9 signal from Q·−a-Fe2+ in the dark was found to be about +10-mV. The g = 1.66 signal can also be formed with a high yield by first illuminating dark-adapted PS II particles at 293 K, followed by a short period of darkness at 273 K and subsequent illumination at 77 K. We have measured the effect on signal yield of varying the period of darkness following 293 K illumination. Over 60% of the maximum signal size is seen after 1 min darkness, and increases further over 2 h. In these samples a signal attributed to Q·−b-Fe2+ is seen prior to 77 K illumination. Confirmation of the presence of Q·t-b was obtained by reductant-linked oxidation of the non-haem iron using phenyl-para-benzoquinone (PPBQ). Samples treated with the Qb-analogue tribromotoluquinone (TBTQ) give a modified EPR signal. We propose (i) that Qb is preserved in PS II preparations from P. laminosum; (ii) that Qb-semiquinone can be readily formed and trapped by freezing; and (iii) the g = 1.66 signal arises from a coupling between the primary and secondary plastosemiquinones and the non-haem iron.
Mild trypsinization of PS II particles at pH 6.0, which almost completely blocks reoxidation of the primary plastoquinone acceptor, QA−·Fe2+, by endogenous plastoquinone (PQ) as well as by exogenous (p-BQ) quinones, does not affect the shape or the amplitude of the EPR signal attributed to QA−·Fe2+. The effect of DCMU on the EPR signal [(1984) FEBS Lett. 105, 156-162] is completely eliminated in mildly trypsinized PS II particles. The lack of effect of mild trypsin treatment on the QA−·Fe2+ microenvironment is briefly discussed in relation to the functional and structural organization of the PS II acceptor side.Photosystem IISemiquinone-iron complexTrypsinHerbicidePhotosythesis
The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl- site(s) in PSII, and some even support activity. NO2- has been reported to be an activator in Cl--depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO2- with PSII. NO2- is shown to be inhibitory to PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O2-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O2 yield measurements. Enzyme kinetics analysis shows multiple sites of NO2- inhibition in PSII with significant inhibition of oxygen evolution at concentrations of NO2- below 5 mM. By EPR spectroscopy, the yield of the S2 state remains unchanged up to a concentration of 15 mM NO2-. However, the S2 state g = 4.1 signal is favored over the g = 2 multiline signal with increasing NO2- concentration. This could indicate competition of NO2- for the Cl- site at higher concentrations of NO2-. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO2-. The g = 1.9 Fe(II)-QA-• signal is replaced by a broad g = 1.6 signal in the presence of NO2-. Additionally, a g = 1.8 Fe(II)-Q-• signal is present in the dark, indicating the formation of a NO2--bound Fe(II)-QB-• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO2- on the activity of PSII is largely due to the donor side chemistry of NO2-. UV-visible spectroscopy and flash-induced polarographic O2 yield measurements indicate that NO2- is oxidized by the oxygen-evolving complex (OEC) in the higher S states, contributing to the donor-side inhibition by NO2-.
In photosynthetic oxygen evolution, redox active tyrosine Z (YZ) plays an essential role in proton-coupled electron transfer (PCET) reactions. Four sequential photooxidation reactions are necessary to produce oxygen at a Mn 4CaO5 cluster. The sequentially oxidized states of this oxygen-evolving cluster (OEC) are called the Sn states, where n refers to the number of oxidizing equivalents stored. The neutral radical, YZ•, is generated and then acts as an electron transfer intermediate during each S state transition. In the X-ray structure, YZ, Tyr161 of the D1 subunit, is involved in an extensive hydrogen bonding network, which includes calcium-bound water. In electron paramagnetic resonance experiments, we measured the YZ• recombination rate, in the presence of an intact Mn 4CaO5 cluster. We compared the S0 and S 2 states, which differ in Mn oxidation state, and found a significant difference in the YZ• decay rate (t1/2 = 3.3 ± 0.3 s in S0; t1/2 = 2.1 ± 0.3 s in S2) and in the solvent isotope effect (SIE) on the reaction (1.3 ± 0.3 in S 0; 2.1 ± 0.3 in S2). Although the YZ site is known to be solvent accessible, the recombination rate and SIE were pH independent in both S states. To define the origin of these effects, we measured the YZ• recombination rate in the presence of ammonia, which inhibits oxygen evolution and disrupts the hydrogen bond network. We report that ammonia dramatically slowed the YZ• recombination rate in the S2 state but had a smaller effect in the S0 state. In contrast, ammonia had no significant effect on YD•, the stable tyrosyl radical. Therefore, the alterations in YZ• decay, observed with S state advancement, are attributed to alterations in OEC hydrogen bonding and consequent differences in the YZ midpoint potential/pKa. These changes may be caused by activation of metal-bound water molecules, which hydrogen bond to YZ. These observations document the importance of redox control in proton-coupled electron transfer reactions.
The electron spin echo envelope modulation spectra of the reduced primary acceptor quinone, QA, in two preparations of plant photosystem II, have been studied. In one of these preparations the Fe2+ ion in the quinone–iron complex has been substituted by diamagnetic Zn2+. In the other preparation this iron ion has been converted into the diamagnetic state using a potassium cyanide treatment. A comparative analysis of two-dimensional three-pulse electron spin echo envelope modulation spectra has shown similar structure of the binding site of QA in both preparations. Two nitrogen nuclei have been found to contribute to the spectra in both preparations. One of these nitrogens is, most probably, an amino nitrogen in the imidazole ring of histidine 215 of the D2 protein. The other nitrogen has been assigned to the peptide group of alanine 261 of the D2 protein. The numerical simulations of the electron spin echo envelope modulation spectra have shown that both nitrogens are simultaneously bound to QA. © 1998 American Institute of Physics.
Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential E(h) = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo([unk]) (g = 2.0033, DeltaH approximately 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with beta-carotene) although not with vitamin K(1). TSF-II and TSF-IIa are found to contain approximately 2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO(4) plus 2.5 mM o-phenanthroline (but not with 0.55 M LiClO(4) alone) decreases this value to approximately 0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe(2+), but not with either Mn(2+) or Mg(2+). The Fe removal results in the development of a photoinduced EPR signal (g = 2.0044 +/- 0.0003, DeltaH = 9.2 +/- 0.5 G) at E(h) = 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable "primary" electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo([unk]), is responsible for the EPR doublet.
Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (Em ⋍ −50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (Em ⋍ −275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the Em-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.
Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q−AFe. o-Phenanthroline slightly broadens the Q−AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q−AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q−AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin− signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b559 to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.
Photosystem 2 preparations with very high rates of oxygen evolution from the thermophilic cyanobacterium Phormidium laminosum have been studied by EPR spectrometry. In the presence of DCMU the g = 1.82 signal of the iron—quinone electron acceptor (Q) can be observed. It is proposed that DCMU is necessary to disrupt a magnetic interaction, between the semiquinone forms of Q and the secondary acceptor B, which otherwise prevents detection of the Q−Fe signal. A doublet EPR signal arising from magnetic interaction between Q−Fe and the reduced intermediary electron acceptor pheophytin (I−), and a spin-polarized triplet signal assumed to arise from the back reaction between I− and P680+ can also be seen. Preliminary redox titrations of Q reduction have been carried out, indicating Em ⋍ 0 mV.
1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K.2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are are equivalent with respect to the light induced (BChl)2⨥ At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity.3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow () oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinoneiron. The state (oxidized ) relaxes in the dark at 200 K in approx. 20 min but below 77 K it is trapped on a days time scale.4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I⨪ is consistent with the idea that the reduced intermediary carrier is not simply BPh⨪ but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state. As such, the single electron would be shared in some way, and it is probable that one of these centers will be very close to the paramagnetism of the iron-quinone. Alternatively, it is possible that the electron only occupies BPh⨪ (the optical changes associated with the 800 nm bacteriochlorophyll occurring on a secondary basis) and that some of the BPh⨪ population of the trapped state is not close enough to interact with the quinone-iron.5. Light-induced triplet state formation is dramatically diminished in material in which I as well as the quinone-iron is reduced before illumination. This supports the idea that with quinone-iron alone reduced before illumination, triplet formation requires light activated electron transfer from the bacteriochlorophyll dimer to I (not possible if I is already reduced) and that the triplet is formed by the return of the electron from I⨪ to (BChl)2⨥.6. Results indicate that although the two cytochrome c553 hemes may be equivalent at the point of activation, once one has become oxidized the other becomes less competent for oxidation by the (BChl)2⨥.
Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y4) is lower compared to Y3 than at lower flash frequency. At 4 Hz, the calculated S0 concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent ratio increases to about 0.2. This is explained by a relatively fast donation () of one electron by an electron donor to S2 and S3 in 15–25% of the Photosystem II reaction chains. The one-electron donor to S2 and S3 appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal IIs. (ii) The probability for the fast one-electron donation to S2 and S3 has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S2 decay becomes 10-fold faster () in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S2 decay was extremely slow. The S3 decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S2 is reduced mainly by Q−A, whereas S3 is not. (iii) In the absence of CO2/HCO−A and in the presence of formate, the fast one-electron donation to S2 and S3 does not occur. Addition of HCO−3 restores the fast decay of part of S2 and S3 to almost the same extent as in control thylakoids. The slow phase of S2 and S3 decay is not influenced significantly by CO2/HCO−3. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q−A oxidation without interference of QB, were 2.3-fold slower in the absence of CO2/HCO−3 than in its presence. (iv) An almost 3-fold decrease in decay rate of S2 is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q−A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q−B is responsible for this shift of equilibrium Q−A concentration, these data would suggest that the pKa value for Q−B protonation is somewhat higher than 7.6, assuming that the protonated form of Q−B cannot reduce QA.
The light-induced EPR multiline signal is studied in O2-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S2 state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q−AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S2. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S2 signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.
Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).
The photoreductive trapping of the transient, intermediate acceptor, I⁻, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I⁻ was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 440, 587–599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301–304). The optical changes of I⁻ showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I⁻ were observed: one was a narrow singlet at g = 2.0035, ΔH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I⁻ on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI⁻Q⁻Fe²⁺ and the singlet EPR signal is due to DI⁻Q²⁻Fe²⁺. where D is the primary donor (BChl)⁺2.
The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.
Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.
ESR studies on light induced reactions in Chromatium D at liquid helium temperatures reveals that the primary electron acceptor of reaction center bacteriochlorophyll has a signal at approximately g 1.82. Hence, the primary electron acceptor is probably an iron-sulphur protein. Using the same approach with spinach chloroplasts, a signal which is typical of a reduced iron-sulphur protein of plants is formed after a brief period of illumination.
ESR studies at liquid helium temperatures have been conducted on chromatophore and subchromatophore preparations from Chromatium D. If the primary electron acceptor of reaction center bacteriochlorophyll is chemically in a reduced state before illumination, the light activated exited state bacteriochlorophyll is prevented from undergoing oxidation. This is evidenced under these conditions by the absence of the familiar g ≅ 2 signal. Instead, a new ESR spectrum is generated in the light. This is comprised of both absorption and emission bands. The oxidation-reduction potential dependence and kinetics of the ESR changes, activated by laser pulses, suggest the signals represent bacteriochlorophyll in the triplet state. This state could be a primary intermediate in the early light activated transitions of photosynthesis.
Photochemically active reaction centers from Rhodopseudomonas spheroides R-26 were prepared in which the electron donor is P(865) and the electron acceptor is ubiquinone. The latter was identified by comparing the EPR characteristics of the light-induced signal with those obtained from a ubiquinone radical.
A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.