PSII-enriched Membranes Research Articles

PsbS-зависимые изменения в содержании липидов и фотосинтетических пигментов в растениях риса

Plants acclimate to fluctuations in light conditions by adjusting their photosynthetic apparatus. When the light intensity exceeds, an unbalanced excitation of the two photosystems occurs. It results in reduced photosynthetic efficiency. Photosystem II (PSII) is the most susceptible and dynamically regulated part of the light reactions in the thylakoid membrane. Non-photochemical quenching of chlorophyll fluorescence (NPQ) is one of the short-term photoprotective mechanisms, which consist of the number of components. The strongest NPQ component — qE is localized in the PSII antenna and induced in plants by lumen acidification, the activation of the pH sensor PsbS, and the conversion of the violaxanthin to zeaxanthin within the xanthophyll cycle. Here, I present data that characterizes the role of the PsbS protein in organization of PSII structural components in isolated PSII-enriched membranes. The preparations were isolated from wild-type (WT) and PsbS-less (PsbS-KO) mutant rice plant. Based on the obtained results, the PSII-enriched membranes from WT and PsbS-KO differ as in the level of lipids, also in carotenoids. I conclude that the PsbS-dependent changes in membrane fluidity in PsbS-KO mutant plants compensated with increased lipid level in mutant plants.

The Main Structural and Functional Characteristics of Photosystem-II-Enriched Membranes Isolated from Wild Type and cia3 Mutant Chlamydomonas reinhardtii

Photosystem II (PSII)-enriched membranes retain the original PSII architecture in contrast to PSII cores or PSII supercomplexes, which are usually isolated from Chlamydomonas reinhardtii. Here, we present data that fully characterize the structural and functional properties of PSII complexes in isolated PSII-enriched membranes from C. reinhardtii. The preparations were isolated from wild-type (WT) and CAH3-deficient mutant cia3 as the influence of CAH3 on the PSII function was previously proposed. Based on the equal chlorophyll content, the PSII-enriched membranes from WT and cia3 have the same amount of reaction centers (RCs), cytochrome b559, subunits of the water-oxidizing complex, Mn ions, and carotenes. They differ in the ratio of other carotenoids, the parts of low/intermediate redox forms of cytochrome b559, and the composition of outer light-harvesting complexes. The preparations had 40% more chlorophyll molecules per RC compared to higher plants. Functionally, PSII-enriched membranes from WT and cia3 show the same photosynthetic activity at optimal pH 6.5. However, the preparations from cia3 contained more closed RCs even at pH 6.5 and showed more pronounced suppression of PSII photosynthetic activity at shift pH up to 7.0, established in the lumen of dark-adapted cells. Nevertheless, the PSII photosynthetic capacities remained the same.

Determining the Activation Energy of Photoassembly of the Oxygen‐evolving Complex Using an Alternative Redox Mediator

The oxygen‐evolving complex (OEC) is a well‐researched Mn4CaO5 cluster with a cubane‐like structure that is responsible for oxidizing water to fuel photosynthetic electron transfer. It is a cofactor of photosystem II (PSII), which catalyzes light‐induced charge separation and oxidation of the OEC. PSII is comprised of pigment molecules and cofactors that transport electrons to a mobile plastoquinone that is located at the QB site. These reducing equivalents can eventually be used to reduce CO2 into simple sugars. In nature, the OEC is constantly degraded as a result of PSII photodamage and must be reassembled through a light‐dependent process after the affected PSII is repaired. OEC assembly occurs in the presence of light in vivo, but if conducted in situ is significantly enhanced by including a dark incubation after the first light dependent step. This dark‐dependent step is the rate‐limiting step of OEC assembly. Determining the activation energy will provide insight into the mechanism of OEC formation and provide clues on how artificial systems could be engineered.To conduct these studies, subcellular thylakoid membranes were isolated and enriched in PSII and the Mn4CaO5 cluster was removed via an alkaline incubation to generate apo‐PSII. The apo‐PSII enriched membranes were then used to measure the efficiency of OEC assembly in situ at temperatures ranging from 5 – 45 °C using Clark type oximetry. However, when OEC assembly occurs in apo‐PSII‐enriched membranes the rate‐determining step is electron shuttling from the QB site to the terminal electron acceptor {K3[Fe(CN)6]} via the native plastoquinones, thus preventing an accurate determination of the activation energy. This issue was remedied by supplementing with an alternative redox mediator know as phenyl‐p‐benzoquinone (PPBQ), which has been shown to increase the rate of electron shuttling from the QB site to a terminal electron acceptor. OEC photoassembly was successful in the presence of PPBQ and the oxygen production at varying temperatures was used to produce an Arrhenius plot to calculate the activation energy of OEC assembly.Currently, the activation energies have been determined for photoassembled PSII‐enriched membranes in the presence of PPBQ (18 kJ/mol) and native plastoquinones (49 kJ/mol). From these results, we speculate that OEC photoassembly in apo‐PSII membranes with PPBQ allows us to measure the true rate‐limiting step.Support or Funding InformationE.N. was supported by a fellowship from the SURE Program funded through the WV Research Challenge Fund, and administered by the WV HEPC, Division of Science and Research, Award Number DSR.17.09. E.N. had additional support from the WV NASA Space Grant Consortium’s NASA WVSG Undergraduate Fellowship Program and from the DOW Undergraduate Research Fellowship.D.K. and A.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952. Amanda Smythers was also funded by the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant #NNX15AI01H.J.B. had additional support from the WV NASA Space Grant Consortium’s NASA WVSG Undergraduate Fellowship Program and from the DOW Undergraduate Research Fellowship.

Conjugated Polymer Enhanced Photoelectric Response of Self-Circulating Photosynthetic Bioelectrochemical Cell.

A water-oxygen-water photosynthetic bioelectrochemical cell (PBEC) comprising hybrid poly(fluorene-alt-phenylene) (PFP)/PSII-enriched membranes (BBY) photoanode and bilirubin oxidase (BOD) biocathode has been designed and fabricated. In the PBEC, water is split into oxygen, protons, and electrons through light-dependent reaction of PSII at the photoanode, and oxygen is converted into water catalyzed by BOD at the biocathode, forming the electronic circuit and generating current. At the photoanode, PFP can simultaneously accelerate the photosynthetic water oxidation and the electron transfer between BBY and electrode. Interestingly, the photocurrent density produced by PBEC after the introduction of PFP reaches 1.05 ± 0.01 μA/cm2, which is 2.5 times more than that of the BBY electrode, indicating that conjugated polymer can enhance the photoelectric response of PBEC.

Mechanism of Photodamage of the Oxygen Evolving Mn Cluster of Photosystem II by Excessive Light Energy

Photodamage to Photosystem II (PSII) has been attributed either to excessive excitation of photosynthetic pigments or by direct of light absorption by Mn4CaO5 cluster. Here we investigated the time course of PSII photodamage and release of Mn in PSII-enriched membranes under high light illumination at 460 nm and 660 nm. We found that the loss of PSII activity, assayed by chlorophyll fluorescence, is faster than release of Mn from the Mn4CaO5 cluster, assayed by EPR. Loss of PSII activity and Mn release was slower during illumination in the presence of exogenous electron acceptors. Recovery of PSII activity was observed, after 30 min of addition of electron donor post illumination. The same behavior was observed under 460 and 660 nm illumination, suggesting stronger correlation between excessive excitation and photodamage compared to direct light absorption by the cluster. A unified model of PSII photodamage that takes into account present and previous literature reports is presented.

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.

A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ∼300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.

Isolation of photosystem II-enriched membranes and the oxygen-evolving complex subunit proteins from higher plants.

We describe methods to isolate highly active oxygen-evolving photosystem II (PSII) membranes and core complexes from higher plants, and to purify subunits of the oxygen-evolving complex (OEC). The membrane samples used as the material for various in vitro studies of PSII are prepared by solubilizing thylakoid membranes with the nonionic detergent Triton X-100, and the core complexes are prepared by further solubilization of the PSII membranes with n-dodecyl-β-D-maltoside (β-DDM). The OEC subunit proteins are dissociated from the PSII-enriched membranes by alkaline or salt treatment, and are then purified by ion-exchange chromatography using an automated high performance liquid chromatography system.

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn(4) cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S(2)-, S(3)-, and S(0)-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.

Two-Electron Reactions S2QB →S0QB and S3QB →S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S0–S4), and dark incubation leads to 25% S0 and 75% S1. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor QB−. We measured flash-induced oxygen evolution to understand how S3 and S2 are converted to lower S states when the electron required to reduce the manganese cluster does not come from QB−. Thylakoid samples preconditioned to make the concentration of the S1 state 100% and to oxidize tyrosine YD were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S2 and S3 are converted to lower S states via recombination from S2QB− and S3QB− and by a slow change of the state of oxygen-evolving complex from S3 and S2 to S1 and S0 in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S2QB →S0QB, and S3QB →S1QB. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S3QB →S2QB →S1QB in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H2O2 acts as an electron donor for the reaction S2QB →S0QB but added H2O2 did not enhance this reaction.

Probing tyrosine Z oxidation in photosystem II core complex isolated from spinach by EPR at liquid helium temperatures

Tyrosine Z (Tyr(Z)) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr(Z) in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr(Z) oxidation in the S(1) and S(2) states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S(1) state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S(1)Tyr(Z)(*) EPR signal. Furthermore, EPR signals of the S(2) state of the Mn-cluster, the oxidation of the non-heme iron, and the S(1)Tyr(Z)(*) in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.

Detection of the D0→D1 transition of β-carotene radical cation photoinduced in photosystem II

The D0→D1 absorption band of a β-carotene radical cation in the near-infrared (NIR) region was detected for the first time in photosystem II (PSII). PSII-enriched membranes and isolated reaction center (RC) complexes (D1/D2/Cytb559) from spinach were illuminated at 80 and 150 K, respectively, in the presence of electron acceptors. In both preparations, UV-Vis-NIR difference spectra upon illumination exhibited a medium-intensity band at ∼1450 nm along with a strong band at ∼990 nm. The latter band has been assigned to the D0→D2 transition of the radical cation of the β-carotene in the secondary electron transfer pathway in PSII. These NIR bands disappeared at 210 K in the PSII membranes, and diminished their intensities in the RC complexes partially depleted of carotenoid. The absence or diminish of the β-carotene cation with little change in the formation of chlorophyll cations under these conditions were also confirmed by detecting light-induced FTIR difference spectra in the mid-IR region. From these results, it was concluded that the NIR band observed at ∼1450 nm arose from the D0→D1 transition of the β-carotene radical cation. It was shown that the observed band in the RC complexes was a mixture of the band of one β-carotene cation (Car507+) at 1464 nm and that of the other cation (Car489+) at a wavelength shorter than 1434 nm, indicating that the D0→D1 transition is sensitive to the protein environment. It is proposed that the position and the relative intensity of the D0→D1 band together with the well-known D0→D2 band can be useful monitors to investigate the properties of the radical cation and the molecular interaction of β-carotene in the PSII proteins.

Water Molecules Coupled to the Redox-Active Tyrosine YD in Photosystem II as Detected by FTIR Spectroscopy

The redox-active tyrosine YD (D2-Tyr160) in photosystem II (PSII) serves as a side-path electron donor to P680. When YD is oxidized, a proton is released from phenolic OH, and a neutral radical YD* is formed. A hydrogen bond network around YD must be deeply involved in the mechanism of the YD reaction. In this study, we have detected water molecules structurally coupled to YD by means of Fourier transform infrared (FTIR) spectroscopy. Light-induced YD*/YD FTIR difference spectrum of a hydrated film of the PSII core complexes from Thermosynechococcus elongatus showed major signals at 3636(-)/3617(+) and 3594(+)/3585(-) cm-1 in the weakly hydrogen bonded OH stretching region. These peaks downshifted by 11-12 cm-1 upon H218O substitution and almost disappeared upon H/D exchange, and hence, they were definitely assigned to the water OH vibrations. Small intramolecular couplings of 3-6 cm-1 estimated from the OH frequencies of residual HOD species in a deuterated film indicate that these OH signals arise from two different water molecules that have significantly asymmetric hydrogen bond structures. Similar OH signals were observed in PSII-enriched membranes from spinach, suggesting that two water molecules commonly exist near YD irrespective of biological species. These water molecules are coupled to YD most probably through a hydrogen bond network or one of them possibly interacts directly with YD, and thus, they may play crucial roles in the YD reaction by forming a proton-transfer pathway and tuning the redox potential of YD.

Differential response of chloride binding sites to elevated temperature: a comparative study in spinach thylakoids and PSII-enriched membranes

A study of heat effects was performed in thylakoids and photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl(-)-induced activation of PSII catalyzed oxygen evolution and the retention of Cl(-) in the PSII complex. For this, Cl(-)-sufficient membranes and low-Cl(-) membranes were used. The presence of Cl(-) in the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40 degrees C in PSII membranes and up to 42.5 degrees C in thylakoids. Heat resistance of Cl(-)-induced activation of oxygen evolution was found to be independent of the presence of 'bound Cl(-)' in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Cl(-) in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/Y (D) (+) showed that there was little loss of Mn(2+) up to 42 degrees C in our preparations, although the PSII activity was significantly lowered. These observations together with data from steady state chlorophyll a fluorescence imply that the site of action of Cl(-) causing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The high-affinity (tightly bound, slow exchanging) site of chloride is affected earlier ( approximately 37 degrees C) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (42.5 degrees C in thylakoids and 40 degrees C in the case of PSII-enriched membranes).

Structural Perturbation of the Carboxylate Ligands to the Manganese Cluster upon Ca2+/Sr2+ Exchange in the S-State Cycle of Photosynthetic Oxygen Evolution As Studied by Flash-Induced FTIR Difference Spectroscopy

A Ca(2+) ion is an indispensable element in the oxygen-evolving Mn cluster in photosystem II (PSII). To investigate the structural relevance of Ca(2+) to the Mn cluster, the effects of Sr(2+) substitution for Ca(2+) on the structures and reactions of ligands to the Mn cluster during the S-state cycle were investigated using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra representing the four S-state transitions, S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1), were recorded by applying four consecutive flashes either to PSII core complexes from Thermosynechococcus elongatus or to PSII-enriched membranes from spinach. The spectra were also recorded using biosynthetically Sr(2+)-substituted PSII core complexes from T. elongatus and biochemically Sr(2+)-substituted PSII membranes from spinach. Several common spectral changes upon Sr(2+) substitution were observed in the COO(-) stretching region of the flash-induced spectra for both preparations, which were best expressed in Ca(2+)-minus-Sr(2+) double difference spectra. The significant intensity changes in the symmetric COO(-) peaks at approximately 1364 and approximately 1418 cm(-)(1) at the first flash were reversed as opposite intensity changes at the third flash, and the slight shift of the approximately 1446 cm(-)(1) peak at the second flash corresponded to the similar but opposite shift at the fourth flash. Analyses of these changes suggest that there are at least three carboxylate ligands whose structures are significantly perturbed by Ca(2+)/Sr(2+) exchange. They are (1) the carboxylate ligand having a bridging or unidentate structure in the S(2) and S(3) states and perturbed in the S(1) --> S(2) and S(3) --> S(0) transitions, (2) that with a chelating or bridging structure in the S(1) and S(0) states and perturbed also in the S(1) --> S(2) and S(3) --> S(0) transitions, and (3) that with a chelating structure in the S(3) and S(0) states and changes in the S(2) --> S(3) and S(0) --> S(1) transitions. Taking into account the recent FTIR studies using site-directed mutagenesis and/or isotope substitution [Chu et al. (2004) Biochemistry 43, 3152-3116; Kimura et al. (2005) J. Biol. Chem. 280, 2078-2083; Strickler et al. (2006) Biochemistry 45, 8801-8811], it was concluded that these carboxylate groups do not originate from either D1-Ala344 (C-terminus) or D1-Glu189, which are located near the Ca(2+) ion in the X-ray crystallographic model of the Mn cluster. It was thus proposed that if the X-ray model is correct, the above carboxylate groups sensitive to Sr(2+) substitution are ligands to the Mn ions strongly coupled to the Ca(2+) ion rather than direct ligands to Ca(2+).

Enhancement of Y D• spin relaxation by the CaMn 4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y D • of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn 4 cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y D • EPR intensity upon the application of a single laser flash to S 1 state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y D • by the OEC in the S 2 state, as a consequence of the single laser flash turnover. The post-flash decay reflected S 2 → S 1 back-turnover, as confirmed by their correlations with independent measurements of S 2 multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y D • as a probe.

Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was applied for the first time to detect the structural changes upon photoreactions of redox cofactors in photosystem II (PSII). The PSII-enriched membranes from spinach were adsorbed on the surface of a silicon prism, and FTIR measurements of various redox cofactors were performed for the same sample but under different conditions by exchanging buffers in a flow cell. Light-induced FTIR difference spectra upon redox reactions of the oxygen-evolving Mn cluster, the primary quinone electron acceptor Q A, the redox-active tyrosine Y D, the primary electron acceptor pheophytin, and the primary electron donor chlorophyll P680 were successively recorded in buffers including different redox reagents and inhibitors. All of these cofactors remained active in the PSII membranes on the silicon surface, and the resultant spectra were basically identical to those previously recorded by the conventional transmission method. These ATR-FTIR measurements enable accurate comparison between reactions of different active sites in a single PSII sample. The present results demonstrated that the ATR-FTIR spectroscopy is a useful technique for investigation of the reaction mechanism of PSII.

Rapid Loss of Structural Motifs in the Manganese Complex of Oxygenic Photosynthesis by X-ray Irradiation at 10–300 K

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-mu-oxo bridge between manganese atoms, previously separated by approximately 2.7 A, to a mono-mu-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at approximately 200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final Mn(II)4 state the complete loss of inter-manganese distances <3 A was observed, even at 10 K, because of the destruction of mu-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.