Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.     

ESE relaxation measurements in photosystem II. The influence of the reaction center non-heme iron on the spin-lattice relaxation of Tyr D.

The spin-lattice relaxation of the tyrosine radical D. in Photosystem II particles was studied at 4.2 K in samples in which flash-induced oscillations of the oxidation state of the Mn-cluster of the oxygen evolving system were abolished by addition of ANT2P, leaving Fe2+/Fe3+ oscillations intact. Samples subjected to 0, 1 or 2 light-flashes all showed the same relaxation kinetics. No period-2 oscillation in the spin-lattice relaxation corresponding to the Fe2+/Fe(3+)-oscillation was observed. Thus the T1-oscillations of D. as a function of flash number in untreated samples are solely caused by the charge-oscillations of the Mn-cluster (1989, Biochim, Biophys. Acta 973, 428-442).     

Photoelectric response generated under non-heme iron reduction on the photosystem II acceptor side

Proteoliposomes containing oxygen-evolving particles of Photosystem II and associated with a planar phospholipid membrane generate a transmembrane electric potential difference (DeltaPsi) induced by a laser flash. With direct electrometrical technique, it was shown that the direction of the electrical field ("minus" inside the proteoliposome) corresponds to acceptor side of the Photosystem II complex facing inside and donor side facing outside of the liposomes. In addition to the fast phase (tau < 0.1 microsec) of the DeltaPsi generation due to electron transfer between YZ of the water-oxidizing complex and the primary plastoquinone QA, a phase with tau approximately 120 microsec and maximum amplitude approximately 30% of the amplitude of the fast phase was observed under the first flash in proteoliposomes containing potassium ferricyanide, which is known as an oxidant of the non-heme iron (Fenh) on the acceptor side of Photosystem II. This additional phase was absent under the second laser flash but was completely restored after 5 min dark adaptation. The phase of the photoelectric response with tau approximately 120 microsec is probably due to electron transfer from QA to Fenh(III) and likely includes a component related to H+ transfer.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO₂ limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO₂ with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the "classical" scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO(2) limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO(2) with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: studies of the mechanism.

The effects of photosystem II (PSII) exogenous electron donors and acceptors on the kinetics of weak light photoinhibition of NH2OH/EDTA-extracted spinach PSII membranes were examined. Under aerobic conditions, Mn2+ (approximately 1 Mn/reaction center; Km approximately 400 nM) inhibited photoinactivation and approximately 1 Mn/reaction center plus 100 microM NH2NH2 gave almost complete protection. In the absence of electron donors, strict anaerobiosis greatly inhibited photoinactivation even in the presence of an electron acceptor. Under aerobic conditions, the addition of electron acceptors (FeCN, DCIP), oxyradical scavengers, or superoxide dismutase strongly suppressed rates of photodamages. Increase in the concentrations of superoxide above those produced by illuminated NH2OH/EDTA-photosystem II membranes increased the rates of damage in the light but gave no damage in the dark. Scavengers of hydroxyl radicals and singlet oxygen did not suppress the rates of aerobic photoinhibition. These findings, along with others, lead us to conclude that photodamage of the secondary donors of the PSII reaction center occurs by two mechanisms: (1) a rapid superoxide and tyrosine YZ+ dependent process and (2) a slower process in which P680+/Chl+ catalyze the damages.     

Electrostatic role of the non-heme iron complex in bacterial photosynthetic reaction center

To elucidate the role of the non-heme iron complex (Fe-complex) in the electron transfer (ET) events of bacterial photosynthetic reaction centers (bRC), we calculated redox potentials of primary/secondary quinones Q(A/B) (E(m)(Q(A/B))) in the Fe-depleted bRC. Removing the Fe-complex, the calculated E(m)(Q(A/B)) are downshifted by approximately 220 mV/ approximately 80 mV explaining both the 15-fold decrease in ET rate from bacteriopheophytin (H(A)(-)) to Q(A) and triplet state occurrence in Fe-depleted bRC. The larger downshift in E(m)(Q(A)) relative to E(m)(Q(B)) increases the driving-energy for ET from Q(A) to Q(B) by 140 meV, in agreement with approximately 100 meV increase derived from kinetic studies.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B(A)), bacteriopheopytin (H(A)), the primary quinone (Q(A)) to the secondary quinone (Q(B)). Although the non-heme iron complex (Fe complex) is located between Q(A) and Q(B), it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q(A) and Q(B) in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q(A/B) (E(m)(Q(A/B))) and the Fe complex (E(m)(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E(m)(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E(m)(Fe) downshift by 230 mV upon formation of Q(A)(-) (but not Q(B)(-)) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B_A), bacteriopheopytin (H_A), the primary quinone (Q_A) to the secondary quinone (Q_B). Although the non-heme iron complex (Fe complex) is located between Q_A and Q_B, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q_A and Q_B in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q_A/B (E_m(Q_A/B)) and the Fe complex (E_m(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E_m(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E_m(Fe) downshift by 230 mV upon formation of Q_A⁻ (but not Q_B⁻) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Photoinhibition of photosystem II in vitro: Spectral and kinetic analyses

Two different preparations of photosystem II (PSII) (BBY-type membrane fragments and PSII core complexes) were isolated from 14-day-old pea seedlings (Pisum sativum L.) and used for spectral and kinetic study of photobleaching of chlorophyll (Chl) and amino acids under photoinhibitory conditions. A short-term (2–4 min) illumination of PSII preparations with high-intensity red light (λ > 610 nm, 800 W/m²) resulted in irreversible photobleaching of Chl at 672 and 682 nm under conditions of both acceptor- and donor-side photoinhibition. At longer illumination exposures (> 10 min) the photobleaching maximum at 682 nm was predominant. The calculated kinetic constants for Chl photobleaching in both absorption bands at temperatures of 20 and 4°C had similar values under different photoinhibitory conditions. The shape of action spectrum for Chl photooxidation indicates that photoinhibition of PSII was sensitized by two spectral forms of Chl with absorption maxima at 670 and 680 nm. The photobleaching of amino acids in PSII membrane fragments was only observed during acceptor-side photoinhibition and displayed the photobleaching peaks at 220 and 274 nm. The photogeneration of superoxide anion radical during donor-side photoinhibition was 4–6 times larger than during acceptor-side photoinhibition. Nevertheless, the kinetics of Chl and amino acid photobleaching in PSII preparations showed no appreciable differences. The activation energies for Chl photooxidation were estimated around 3.5 and 9 kcal/mol during acceptor- and donor-side photoinhibition, respectively, providing evidence for the involvement of biochemical stages in PSII photoinhibition. Based on the data obtained, it is proposed that the antenna Chl, rather than Chl of the reaction center, is the sensitizer for both acceptor- and donor-side photoinhibition of PSII in vitro.     

The nonheme iron in photosystem II

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 Q_A and Q_B 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.     

Photoinhibition of manganese enzymes: insights into the mechanism of photosystem II photoinhibition

Evidence has recently been presented that photoinhibition of photosystem II (PSII) is triggered by absorption of light by the oxygen-evolving manganese cluster. To get insight into the effects of light on enzymes containing manganese or other transition metal cofactors, the photosensitivities of Mn catalase, Mn superoxide dismutase, the haem (Fe)-containing bovine liver catalase, and CuZn superoxide dismutase were investigated. Glucose oxidase was studied as an example of an enzyme that does not have a metal cofactor. Sensitivities of these five enzymes to UVC, UVA, and visible light were compared in anaerobic conditions. The Mn(III)-oxo-Mn(III)-containing Mn catalase was found to be more sensitive to both visible and UV light than bovine liver catalase. Furthermore, the action spectrum of photoinhibition of Mn catalase was found to be fairly similar to that of photoinhibition of PSII. The Mn(II)-containing Mn superoxide dismutase was sensitive to UVC light and somewhat sensitive to UVA light, while only UVC light caused some inhibition of CuZn superoxide dismutase. Glucose oxidase was the least photosensitive of the enzymes studied. The photosensitivity of Mn enzymes supports the hypothesis that the oxygen-evolving manganese complex of PSII can be damaged by UV and visible light absorbed by its Mn(III) or Mn(IV) ions.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Redox potentials of chlorophylls in the photosystem II reaction center

Water oxidation generating atmospheric oxygen occurs in photosystem II (PSII), a large protein-pigment complex located in the thylakoid membrane. The recent crystal structures at 3.2 and 3.5 A resolutions provide novel details on amino acid side chains, especially in the D1/D2 subunits. We calculated the redox potentials for one-electron oxidation of the chlorophyll a (Chla) molecules in PSII, considering the protein environment in atomic detail. The calculated redox potentials for the dimer Chla (P(D1/D2)) and accessory Chla (Chl(D1/D2)) were 1.11-1.30 V relative to the normal hydrogen electrode at pH 7, which is high enough for water oxidation. The D1/D2 proteins and their cofactors contribute approximately 390 mV to the enormous upshift of 470 mV compared to the redox potential of monomeric Chla in dimethylformamide. The other subunits are responsible for the remaining 80 mV. The high redox potentials of the two accessory Chla Chl(D1/D2) suggests that they also participate in the charge separation process.     

Photoinhibition of photosynthesis without net loss of photosystem II components inPopulus deltoides

The photoinhibition of photosynthesis was investigated on intact attached leaves and isolated thylakoid membranes of Populus deltoides.Our studies demonstrate that in intact leaves photoinhibition takes place under high irradiance which is more pronounced at higher temperatures. No net loss of Dl and other proteins associated with photosystem II (PSII) were observed even after 64 % photoinhibition suggesting that the degradation of polypeptides associated with PSII is not the only key step responsible for photoinhibition as observed by other workers. Electron transport studies in isolated thylakoid membranes suggested water oxidation complex as one of the damaged site during high light exposure. The possible mechanisms of photoinhibition without net loss of D1 protein are discussed.