Quality control of photosystem II: impact of light and heat stresses

Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress

Moderate heat stress (40 degrees C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40 degrees C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved.     

Photosynthetic control and photophosphorylation in photosystem II of isolated spinach chloroplasts

Two cycles of photosynthetic control have been observed in isolated spinach chloroplasts in the presence of lipophilic class III electron acceptors, which may accept electrons at PS II. $\text{ADP}\text{O}$ ratios of 0.8 to 0.9 were recorded;rates of oxygen evolution were stimulated by phosphorylating reagents and uncouplers. Addition of the plastoquinone antagonist DBMIB decreased photosynthetic control, oxygen evolution and photophosphorylation. We believe that there is a coupling site associated with PSII which can be rate limiting. Comparison of the $\text{P}\text{2e}$ ratios observed with class I and class III electron acceptors leads us to propose that more than 0.6 and possibly approaching one molecule of ATP can be formed for every pair of electrons transported from water to PSII acceptors.     

Inactive photosystem II centers: A resolution of discrepencies in photosystem II quantitation

The abundance of photosystem II in chloroplast thylakoid membranes has been a contentious issue because different techniques give quite different estimates of photosystem II titer. This discrepancy led in turn to disagreements regarding the stoichiometry of photosystem II to photosystem I in these membranes. We believe that the discrepancy in photosystem II quantitation is resolved by evidence which shows that a large population of photosystem II centers with negligible turnover rates are present in isolated thylakoid membranes as well as in normally developed leaves of healthy plants.     

Subunit proteins of photosystem II

Recipient of the Society Award for Young Scientists 1991.     

Multiple functions of photosystem II

The most important function of photosystem II (PSII) is its action as a water-plastoquinone oxido-reductase. At the expense of light energy, water is split, and oxygen and plastoquinol are formed. In addition to this most important activity, PSII has additional functions, especially in the regulation of (light) energy distribution. The downregulation of PSII during photoinhibition is a protection measure. PSII is an anthropogenic target for many herbicides. There is a unique action of bicarbonate on PSII. Decrease in the activity of PSII is the first effect in a plant under stress; this decreased activity can be most easily measured with fluorescence. PSII is a sensor for stress, and induces regulatory processes with different time scales: photochemical quenching, formation of a proton gradient, state transitions, downregulation by photoinhibition and gene expression.     

Water oxidation in photosystem II

Photosynthesis Research - Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for...     

Carotenoid photooxidation in photosystem II

Carotenoids are known to function as light-harvesting pigments and they play important roles in photoprotection in both plant and bacterial photosynthesis. These functions are also important for carotenoids in photosystem II. In addition, beta-carotene recently has been found to function as a redox intermediate in an alternate pathway of electron transfer within photosystem II. This redox role of a carotenoid in photosystem II is unique among photosynthetic reaction centers and stems from the very highly oxidizing intermediates that form in the process of water oxidation. In this minireview, an overview of the electron-transfer reactions in photosystem II is presented, with an emphasis on those involving carotenoids. The carotenoid composition of photosystem II and the physical methods used to study the structure of the redox-active carotenoid are reviewed. Possible roles of carotenoid cations in photoprotection of photosystem II are discussed.     

Electron transfer in photosystem II

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.     

Heterogeneity in chloroplast photosystem II

Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.Abbreviations and symbols ATP Adenosine triphosphate - Chl Chlorophyll - C₅₅₀ Absorbance bandshift at 550 nm; proportional to [Q_A ^-] - D, D Components involved ir electron donation to P680 - pH Transthylakoid proton gradient - Transthylakoid electrical gradient - DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea referred to as diuron - E _h Oxidation-reduction potential - E _m Cxidation-reduction midpoint potential - EPR Electron paramagnetic resonance - Fm Fluorescence yield when all traps are closed - Fo Fluorescence yield when all traps are open - Fv Variable fluorescence equal to Fm-Fo - Fi Initial fluorescence yield, (usually equivalent to Fo in dark-adapted thylakoids) - Hepes 2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - LHCP Light-harvesting chlorophyll a/b binding protein - PQ Plastoquinone - PSII Photosystem II - P680 Reaction centre chlorophyll of PSII - P₅₁₈ Absorbance bandshift at 518 nm, reflects asymmetric charge distribution across the thylakoid membrane - Q_A, _QH , Q₁ Primary stable plastoquinone electron acceptor of PSII; a quencher of fluorescence - Q _B , B, R Plastoquinone associated with the Q _B -protein, the two-electron gate - Q _D , Q₂, X _a Non-quinone electron acceptor of PSII - X₃₂₀ Absorbance bandshift at 320 nm; semiquinone anion indicator     

Tyrosyl radicals in photosystem II

In PSII, there are two redox-active tyrosines, D and Z, with different midpoint potentials and different reduction kinetics. The factors responsible for these functional differences have not yet been elucidated. Recent model compound studies of tyrosinate and of tyrosine-containing dipeptides have demonstrated that perturbations of the amino and amide/imide group occur when the tyrosyl aromatic ring is oxidized [J. Am. Chem. Soc. 124 (2002) 5496]. Accompanying density functional calculations suggested that this perturbation is due to spin density delocalization from the aromatic ring onto the amino nitrogen. The implication of this finding is that spin density delocalization may occur in redox-active, tyrosine-containing enzymes, like Photosystem II. In this paper, we review the supporting evidence for the hypothesis that tyrosyl radical spin density delocalizes into the peptide bond in a conformationally sensitive, sequence-dependent manner. Our experimental measurements on tyrosyl radicals in dipeptides have suggested that the magnitude of the putative spin migration may be sequence-dependent. Vibrational spectroscopic studies on the tyrosyl radicals in Photosystem II, which are consistent with spin migration, are reviewed. Migration of the unpaired spin may provide a mechanism for control of the direction and possibly the rate of electron transfer.     

Light harvesting in photosystem II

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some “extra” Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.     

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.     

Water oxidation chemistry of photosystem II

Photosystem II (PSII) uses light energy to split water into protons, electrons and O2. In this reaction, nature has solved the difficult chemical problem of efficient four-electron oxidation of water to yield O2 without significant amounts of reactive intermediate species such as superoxide, hydrogen peroxide and hydroxyl radicals. In order to use nature's solution for the design of artificial catalysts that split water, it is important to understand the mechanism of the reaction. The recently published X-ray crystal structures of cyanobacterial PSII complexes provide information on the structure of the Mn and Ca ions, the redox-active tyrosine called YZ and the surrounding amino acids that comprise the O2-evolving complex (OEC). The emerging structure of the OEC provides constraints on the different hypothesized mechanisms for O2 evolution. The water oxidation mechanism of PSII is discussed in the light of biophysical and computational studies, inorganic chemistry and X-ray crystallographic information.     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.     

Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes.

We present an extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII-LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry 38, 2233-2239], the following results were obtained. The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about -20 degrees and -50 degrees, respectively. The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions. A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips. Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.     

Quality control of photosystem II. Cleavage of reaction center D1 protein in spinach thylakoids by FtsH protease under moderate heat stress

When spinach thylakoids were subjected to moderate heat stress (40 degrees C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mM ZnCl2 and 1 mM ATP and was completely inhibited by 1 mM EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the Q(B) site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 M KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70-80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70-80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70-80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.