EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor QA

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron-quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D| = 0.0286 cm⁻¹, |E| = 0.0044 cm⁻¹ was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T = 100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron-quinone primary acceptor complex.     

On the mechanism of photoinduced electron transfer in photosystem II reaction centers

The EPR spectrum of the triplet state of photosystem II reaction centers has been studied in the case of the singly reduced primary acceptor complex QAFe2+. It was demonstrated that the shape of the spectrum does not change much when the relaxation of the primary acceptor is accelerated and when magnetic interaction between the reduced quinone molecule QA and the non-heme iron Fe2+ is disrupted. This observation confirms the earlier conclusion that the anomalous shape of the EPR spectrum is due mainly to the anisotropy of the quatum yield of the triplet state. A scheme of primary events in photosystem II is discussed, which is consistent with the observed properties of the EPR spectrum of the triplet state.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T₀ mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex Q_AFe²⁺ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule Q_A and the nonheme iron ion Fe²⁺ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.     

Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model.

A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.     

The primary charge separation,cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations.Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex.The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840⁺ was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.     

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm^–1 and |E|=0.0039 cm^–1 (chlorobactene), and |D|=0.0355 cm^–1 and |E|=0.0039 cm^–1 (isorenieratene). In the intermediate frequency range 300–1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm^–1 and |E|=0.0050 cm^–1.Abbreviations BChl - bacteriochlorophyll - BPh - bacteriopheophytin - Chl. - Chlorobium - F(A)(O)DMR - fluorescence (absorption) (optical) detected magnetic resonance - FF - fluorescence fading - ISC - intramolecular intersystem crossing - RC - reaction center - ZFS - zero field splitting     

Electron transport and triplet formation in membranes of the photosynthetic bacterium Heliobacterium chlorum

The spectroscopic and thermodynamic properties of the electron-transport components of the photosynthetic bacterium Heliobacterium chlorum were studied by means of absorbance-difference spectroscopy. Upon flash illumination of membranes of H. chlorum photooxidation of the primary electron donor, P-798, was observed. In about 15% of the reaction centers P-798⁺ was reduced by cytochrome c-553, while in the remaining reaction centers P-798⁺ reduction occurred via a back reaction with a reduced electron acceptor. Titration experiments indicated a midpoint potential of −440 mV for the electron acceptor. At low redox potentials the formation of the triplet of P-798 was observed after a flash. The triplet was formed in about 30 ns by a back reaction with a reduced electron acceptor and decayed with a time constant of 35 μs. The yield of triplet formed in a flash was 30%. Upon continuous illumination at low redox potentials the accumulation in the reduced state of an electron acceptor was observed. The difference spectrum of this acceptor indicates that it is an iron-sulfur center. The yield of triplet formation was independent of the redox state of the iron-sulfur center, which indicates that the center is not located in the main electron-transport chain. A scheme with three acceptors in the main electron-transport chain is presented to accomodate our results and those of others.     

Role of bicarbonate in photosystem II, the water-plastoquinone oxido-reductase of plant photosynthesis

Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.     

EPR characterization of the CP47-D1-D2-cytochrome b-559 complex of photosystem II

A photosystem II complex consisting of a 47-kDa chlorophyll-binding protein (CP47), the reaction center proteins D1 and D2, and cytochrome b-559 was characterized. Trace amounts of plastoquinone were found, indicating that the primary acceptor quinone QA has been extracted during purification. However, in the presence of ferricyanide, an EPR signal with the characteristic line shape and g value of the tyrosine radicals associated with photosystem II could be photoaccumulated in the majority of the reaction centers; in the absence of ferricyanide, or under low-temperature illumination conditions, a 9.5-11-G wide signal with a Gaussian line shape was observed at g = 2.003. Neither signal is observed in D1-D2-b-559 complexes, indicating that retention of CP47 produces a more native, but quinone-depleted photosystem II reaction center. The tyrosine radical photogenerated at room temperature can be trapped at cryogenic temperatures; results are presented showing that this radical can arise from tyrosine YZ, from tyrosine YD, or from both species. Low-temperature EPR spectroscopy also revealed a pronounced split signal with contributions at g = 6.05 and g = 5.75, which is attributed to high-spin, non-heme Fe3+ with axial ligation symmetry which is probably the non-heme iron associated with the acceptor side of photosystem II.     

Electrogenic reactions and dielectric properties of photosystem II

This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P(680) and primary quinone acceptor Q(A); (ii) P(680)(+) reduction by the redox-active tyrosine Y(Z) of polypeptide D1; (iii) oxidation of Mn cluster by Y(Z)(ox) followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor Q(B). The electrogenicity related to (i) proton-coupled electron transfer between Q(A)(-) and preoxidized non-heme iron (Fe(3+)) in native and (ii) electron transfer between protein-water boundary and Y(Z)(ox) in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.     

Chlorophyll triplet states associated with photosystem II of thylakoids

The analysis of FDMR thylakoid spectra, determined at multiple emission wavelengths, by a global decomposition technique, has revealed the presence of three previously undescribed triplet populations at emission wavelengths characteristic of Photosystem II chlorophyll/protein complexes. Their zero-field splitting parameters have been determined in order to compare them with the well-studied PSII recombination triplet state. None of these triplets have the zero-field splitting parameters characteristic of the recombination triplet and are therefore probably not generated directly in the reaction center. On the basis of their microwave-induced emission spectra, it is suggested that two are probably generated in the core complex(es) while the third may be generated in the external antenna. These triplets are formed under nonreducing redox conditions, when the recombination triplet is undetectable. It is suggested that they may be involved in the photoinhibitory damage of Photosystem II. The triplet-minus-singlet spectrum associated with the recombination triplet state has been determined for thylakoids after reduction of the secondary acceptors. Its main peak is at 685 nm, slightly red shifted with respect to earlier reports, with a weak signal, of opposite sign at approximately 675 nm. The 685 nm peak indicates that at cryogenic temperatures, the triplet is located on the long-wavelength chlorophyll state present in the reaction center complex of Photosystem II (D1.D2.Cytb(559) complex). From the absence of a clear structure in the 680 nm absorption region, this long-wavelength absorbing state does not appear to be strongly coupled to P(680), though it must be associated with one of the "inner core" pigments recently identified in the photosystem II crystallographic structure [Zouni et al. (2001) Nature 408, 739-743].     

Spectral properties of stabilized D1/D2/cytochrome b-559 photosystem II reaction center complex Effects of Triton X-100, the redox state of pheophytin, and β-carotene

Absorption, fluorescence, and CD spectral properties of the isolated D1/D2/cytochrome b-559 photosystem II reaction center complex were examined in stabilized reaction center material at 77 K. Spectral properties were dependent on the presence or absence of 0.05% Triton X-100 in the RC suspension medium, on the redox state of pheophytin, and on the state of inactivation of the complex. The specific spectral properties of the PS II RC complex in the red suggest that the primary donor is not a bacterial-type special pair and could be a monomer. Furthermore, the spectral properties in the PS II RC may be the result of excitonic interactions among all the porphyrin molecules in the complex. Interactions between β-carotene and porphyrins indicate a significant role for β-carotene in the PS II RC.     

High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction centers. Protein interactions modify the properties of quinones such that similar quinone species can perform diverse functions in reaction centers. Both type I and type II (oxygenic and nonoxygenic, respectively) reaction centers contain quinone cofactors that serve very different functions as the redox potential of similar quinones can operate at up to 800 mV lower reduction potential when present in type I reaction centers. However, the factors that determine quinone function in energy transduction remain unclear. It is thought that the location of the quinone cofactor, the geometry of its binding site, and the "smart" matrix effects from the surrounding protein environment greatly influence the functional properties of quinones. Photosystem II offers a unique system for the investigation of the factors that influence quinone function in energy transduction. It contains identical plastoquinones in the primary and secondary quinone acceptor sites, Q(A) and Q(B), which exhibit very different functional properties. This study is focused on elucidating the tuning and control of the primary semiquinone state, Q(A)(-), of photosystem II. We utilize high-resolution two-dimensional hyperfine sublevel correlation spectroscopy to directly probe the strength and orientation of the hydrogen bonds of the Q(A)(-) state with the surrounding protein environment of photosystem II. We observe two asymmetric hydrogen bonding interactions of reduced Q(A)(-) in which the strength of each hydrogen bond is affected by the relative nonplanarity of the bond. This study confirms the importance of hydrogen bonds in the redox tuning of the primary semiquinone state of photosystem II.     

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by beta-carotene, alpha-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress.     

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.     

Primary events in the photosynthetic reaction centre from Rhodopseudomonas spheroides strain R26: Triplet and oxidized states of bacteriochlorophyll and the identification of the primary electron acceptor

ESR studies at approximately 10 °K on the reaction centre complex of the photosynthetic bacterium Rhodopseudomonas spheroides (strain R26), have revealed bacteriochlorophyll triplet states and a component which has an ESR absorption centred at g = 1.82. The triplet-state bacteriochlorophyll is induced only in the light and is only detectable when the reaction-centre bacteriochlorophyll and its primary electron acceptor are reduced; the ESR triplet state signals are composed of both ESR absorption and ESR emission bands. The oxidation-reduction properties of the g = 1.82 component and its flash-induced kinetic behavior in relation to that of P870 are those expected for the primary electron acceptor in bacterial photosynthesis.