Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

• Klíčová slova
• EPR, mass spectrometry, photosystem II, reactive oxygen species, tocopherol,
• MeSH
• alfa-tokoferol chemie   metabolismus   MeSH
• aminokyseliny chemie   metabolismus   MeSH
• Arabidopsis enzymologie   genetika   účinky záření   MeSH
• fotosyntéza fyziologie   účinky záření   MeSH
• fotosystém II (proteinový komplex) chemie   genetika   metabolismus   MeSH
• hydroxylový radikál chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• intramolekulární transferasy chemie   genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• kyslík chemie   metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• oxidace-redukce MeSH
• superoxidy chemie   metabolismus   MeSH
• světlo MeSH
• termodynamika MeSH
• Thermosynechococcus enzymologie   genetika   účinky záření   MeSH
• tylakoidy enzymologie   genetika   účinky záření   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• železo chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alfa-tokoferol MeSH
• aminokyseliny MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxylový radikál MeSH
• intramolekulární transferasy MeSH
• kyslík MeSH
• superoxidy MeSH
• tocopherol cyclase MeSH Prohlížeč
• železo MeSH

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.

• Klíčová slova
• aggregate, fragment, hydroxyl radical, photosystem II, protein, protein radical, reactive oxygen species, singlet oxygen,
• Publikační typ
• časopisecké články MeSH

The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O2•- and HO• are involved in the photoinhibitory process. Using tandem mass spectroscopy, we have identified a number of oxidatively modified D1 and D2 residues. Our analysis indicates that these oxidative modifications are associated with formation of HO• at both the Mn4O5Ca cluster and the nonheme iron. Additionally, O2•- appears to be formed by the reduction of O2 at either PheoD1 or QA Early oxidation of D1:332H, which is coordinated with the Mn1 of the Mn4O5Ca cluster, appears to initiate a cascade of oxidative events that lead to the oxidative modification of numerous residues in the C termini of the D1 and D2 proteins on the donor side of the photosystem. Oxidation of D2:244Y, which is a bicarbonate ligand for the nonheme iron, induces the propagation of oxidative reactions in residues of the D-de loop of the D2 protein on the electron acceptor side of the photosystem. Finally, D1:130E and D2:246M are oxidatively modified by O2•- formed by the reduction of O2 either by PheoD1•- or QA•- The identification of specific amino acid residues oxidized by reactive oxygen species provides insights into the mechanism of damage to the D1 and D2 proteins under light stress.

• Klíčová slova
• Photosystem II, mass spectrometry, photo inhibition, photosynthesis, reactive oxygen species,
• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• antioxidancia metabolismus   MeSH
• chloridy metabolismus   MeSH
• elektronová paramagnetická rezonance MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• hydroxylový radikál metabolismus   MeSH
• kyslík metabolismus   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• oxidace-redukce * MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• aminokyseliny MeSH
• antioxidancia MeSH
• chloridy MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxylový radikál MeSH
• kyslík MeSH
• reaktivní formy kyslíku MeSH

The effect of various abiotic stresses on photosynthetic apparatus is inevitably associated with formation of harmful reactive oxygen species (ROS). In this review, recent progress on ROS production by photosystem II (PSII) as a response to high light and high temperature is overviewed. Under high light, ROS production is unavoidably associated with energy transfer and electron transport in PSII. Singlet oxygen is produced by the energy transfer form triplet chlorophyll to molecular oxygen formed by the intersystem crossing from singlet chlorophyll in the PSII antennae complex or the recombination of the charge separated radical pair in the PSII reaction center. Apart to triplet chlorophyll, triplet carbonyl formed by lipid peroxidation transfers energy to molecular oxygen forming singlet oxygen. On the PSII electron acceptor side, electron leakage to molecular oxygen forms superoxide anion radical which dismutes to hydrogen peroxide which is reduced by the non-heme iron to hydroxyl radical. On the PSII electron donor side, incomplete water oxidation forms hydrogen peroxide which is reduced by manganese to hydroxyl radical. Under high temperature, dark production of singlet oxygen results from lipid peroxidation initiated by lipoxygenase, whereas incomplete water oxidation forms hydrogen peroxide which is reduced by manganese to hydroxyl radical. The understanding of molecular basis for ROS production by PSII provides new insight into how plants survive under adverse environmental conditions.

• Klíčová slova
• free oxygen radicals, heat inactivation, lipid peroxidation, photoinhibition, singlet oxygen,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Native gel electrophoresis revealed that Psb27 was present mainly in monomeric PSII core complexes but also in smaller amounts in dimeric PSII core complexes, in large PSII supercomplexes, and in the unassembled protein fraction. We conclude from analysis of assembly mutants and isolated histidine-tagged PSII subcomplexes that Psb27 associates with the "unassembled" CP43 complex, as well as with larger complexes containing CP43, possibly in the vicinity of the large lumenal loop connecting transmembrane helices 5 and 6 of CP43. A functional role for Psb27 in the biogenesis of CP43 is supported by the decreased accumulation and enhanced fragmentation of unassembled CP43 after inactivation of the psb27 gene in a mutant lacking CP47. Unexpectedly, in strains unable to assemble PSII, a small amount of Psb27 comigrated with monomeric and trimeric PSI complexes upon native gel electrophoresis, and Psb27 could be copurified with histidine-tagged PSI isolated from the wild type. Yeast two-hybrid assays suggested an interaction of Psb27 with the PsaB protein of PSI. Pull-down experiments also supported an interaction between CP43 and PSI. Deletion of psb27 did not have drastic effects on PSII assembly and repair but did compromise short-term acclimation to high light. The tentative interaction of Psb27 and CP43 with PSI raises the possibility that PSI might play a previously unrecognized role in the biogenesis/repair of PSII.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• fotosystém II (proteinový komplex) genetika   metabolismus   MeSH
• multiproteinové komplexy metabolismus   MeSH
• mutace MeSH
• stabilita proteinů MeSH
• Synechocystis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém I (proteinový komplex) MeSH
• fotosystém II (proteinový komplex) MeSH
• multiproteinové komplexy MeSH
• photosystem II, chlorophyll binding protein, CP-43 MeSH Prohlížeč

The selective replacement of photodamaged D1 protein within the multisubunit photosystem II (PSII) complex is an important photoprotective mechanism in chloroplasts and cyanobacteria. FtsH proteases are involved at an early stage of D1 degradation, but it remains unclear how the damaged D1 subunit is recognized, degraded, and replaced. To test the role of the N-terminal region of D1 in PSII biogenesis and repair, we have constructed mutants of the cyanobacterium Synechocystis sp PCC 6803 that are truncated at the exposed N terminus. Removal of 5 or 10 residues blocked D1 synthesis, as assessed in radiolabeling experiments, whereas removal of 20 residues restored the ability to assemble oxygen-evolving dimeric PSII complexes but inhibited PSII repair at the level of D1 degradation. Overall, our results identify an important physiological role for the exposed N-terminal tail of D1 at an early step in selective D1 degradation. This finding has important implications for the recognition of damaged D1 and its synchronized replacement by a newly synthesized subunit.

• MeSH
• autotrofní procesy účinky léků   účinky záření   MeSH
• biologické modely MeSH
• dimerizace MeSH
• fluorescenční spektrometrie MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• linkomycin farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• posttranslační úpravy proteinů * účinky léků   účinky záření   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• světlo MeSH
• Synechocystis cytologie   účinky léků   metabolismus   účinky záření   MeSH
• tylakoidy účinky léků   metabolismus   účinky záření   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• linkomycin MeSH
• mutantní proteiny MeSH
• podjednotky proteinů MeSH