In this study, electron paramagnetic resonance spin-trapping spectroscopy was used to study the light-induced production of superoxide anion (O2 (•-)) and carbon-centered (R(•)) radicals by Photosystem II (PSII). It is evidenced here that exposure of PSII membranes to high light (2,000 μmol photons m(-2) s(-1)) or heat (47 °C) treatments prior to the illumination suppressed O2 (•-) production, while R(•) was formed. Formation of R(•) in the both high light- and heat-treated PSII membranes was enhanced by DCMU. Removal of molecular oxygen by glucose/glucose oxidase/catalase system and O2 (•-) scavenging by exogenous superoxide dismutase completely suppressed carbon-centered radical formation. It is proposed here that the oxidation of polyunsaturated fatty acids and amino acids by O2 (•-) on the electron acceptor side of PSII results in the formation of R(•), known to initiate a cascade reaction leading to the lipid peroxidation and protein degradation, respectively.

• MeSH
• elektronová paramagnetická rezonance MeSH
• fotochemické procesy MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• kyslík chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• spin trapping metody   MeSH
• Spinacia oleracea MeSH
• superoxidy chemie   metabolismus   MeSH
• světlo MeSH
• volné radikály chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• kyslík MeSH
• superoxidy MeSH
• volné radikály 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

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

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

• Klíčová slova
• HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
• MeSH
• acetofenony farmakologie   MeSH
• barvení a značení MeSH
• buněčná diferenciace * účinky léků   MeSH
• elektronová paramagnetická rezonance MeSH
• HL-60 buňky MeSH
• hydroxylový radikál MeSH
• lidé MeSH
• monocyty cytologie   účinky léků   metabolismus   MeSH
• NADP metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny metabolismus   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• tvar buňky účinky léků   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• volné radikály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• hydroxylový radikál MeSH
• NADP MeSH
• proteiny MeSH
• tetradekanoylforbolacetát MeSH
• volné radikály MeSH

Photosystem II (PSII) is a multisubunit protein complex in cyanobacteria, algae and plants that use light energy for oxidation of water and reduction of plastoquinone. The conversion of excitation energy absorbed by chlorophylls into the energy of separated charges and subsequent water-plastoquinone oxidoreductase activity are inadvertently coupled with the formation of reactive oxygen species (ROS). Singlet oxygen is generated by the excitation energy transfer from triplet chlorophyll formed by the intersystem crossing from singlet chlorophyll and the charge recombination of separated charges in the PSII antenna complex and reaction center of PSII, respectively. Apart to the energy transfer, the electron transport associated with the reduction of plastoquinone and the oxidation of water is linked to the formation of superoxide anion radical, hydrogen peroxide and hydroxyl radical. To protect PSII pigments, proteins and lipids against the oxidative damage, PSII evolved a highly efficient antioxidant defense system comprising either a non-enzymatic (prenyllipids such as carotenoids and prenylquinols) or an enzymatic (superoxide dismutase and catalase) scavengers. It is pointed out here that both the formation and the scavenging of ROS are controlled by the energy level and the redox potential of the excitation energy transfer and the electron transport carries, respectively. The review is focused on the mechanistic aspects of ROS production and scavenging by PSII. This article is part of a Special Issue entitled: Photosystem II.

• MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• přenos energie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• scavengery volných radikálů metabolismus   MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• reaktivní formy kyslíku MeSH
• scavengery volných radikálů MeSH

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.

• Klíčová slova
• antioxidants, bioactive compounds, macrophage, malondialdehyde, monocyte, nutraceuticals, phorbol 12-myristate 13-acetate, protein modification, reactive oxygen species, redox reactions,
• MeSH
• lidé MeSH
• malondialdehyd MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• reaktivní formy kyslíku farmakologie   MeSH
• volné radikály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malondialdehyd MeSH
• reaktivní formy kyslíku MeSH
• volné radikály MeSH

Metal ions play a crucial role in enzymatic reactions in all photosynthetic organisms such as cyanobacteria, algae and plants. It well known that metal ions maintain the binding of substrate in the active site of the metalloenzymes and control the redox activity of the metalloenzyme in the enzymatic reaction. A large pigment-protein complex, PSII, known to serve as a water-plastoquinone oxidoreductase, contains three metal centers comprising non-heme iron, heme iron of Cyt b559 and the water-splitting manganese complex. Metal ions bound to PSII proteins maintain the electron transport from water to plastoquinone and regulate the pro-oxidant and antioxidant activity in PSII. In this review, attention is focused on the role of PSII metal centers in (i) the formation of superoxide anion and hydroxyl radicals by sequential one-electron reduction of molecular oxygen and the formation of hydrogen peroxide by incomplete two-electron oxidation of water; and (ii) the elimination of superoxide anion radical by one-electron oxidation and reduction (superoxide dismutase activity) and of hydrogen peroxide by two-electron oxidation and reduction (catalase activity). The balance between the formation and elimination of reactive oxygen species by PSII metal centers is discussed as an important aspect in the prevention of photo-oxidative damage of PSII proteins and lipids.

• Klíčová slova
• Cytochrome b559, Non-heme iron, Photo-oxidative damage, Photosystem II, Reactive oxygen species, Water-splitting manganese complex,
• MeSH
• cytochromy typu b chemie   metabolismus   MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• katalytická doména MeSH
• kovy metabolismus   MeSH
• kyslík metabolismus   MeSH
• mangan metabolismus   MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostliny enzymologie   MeSH
• scavengery volných radikálů metabolismus   MeSH
• sinice enzymologie   MeSH
• voda metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cytochrome b559 MeSH Prohlížeč
• cytochromy typu b MeSH
• fotosystém II (proteinový komplex) MeSH
• kovy MeSH
• kyslík MeSH
• mangan MeSH
• reaktivní formy kyslíku MeSH
• scavengery volných radikálů MeSH
• voda MeSH
• železo MeSH

When photosystem II (PSII) is exposed to excess light, singlet oxygen ((1)O(2)) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair (3)[P680(•+)Pheo(•-)] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of (1)O(2) in the donor side photoinhibition of PSII. Light-induced (1)O(2) production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R(•)) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R(•) by propyl gallate significantly suppressed (1)O(2). Based on our results, it is concluded that (1)O(2) formation correlates with R(•) formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680(•+)/TyrZ(•). It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R(•) with molecular oxygen is a plausible mechanism for (1)O(2) formation in the donor side photoinhibition of PSII.

• MeSH
• chemické modely MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotochemie metody   MeSH
• fotosystém II (proteinový komplex) fyziologie   MeSH
• koncentrace vodíkových iontů MeSH
• kyslík chemie   MeSH
• propylgalan chemie   MeSH
• singletový kyslík * MeSH
• spin trapping metody   MeSH
• Spinacia oleracea MeSH
• světlo MeSH
• Synechococcus metabolismus   MeSH
• uhlík chemie   MeSH
• volné radikály MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• kyslík MeSH
• propylgalan MeSH
• singletový kyslík * MeSH
• uhlík MeSH
• volné radikály MeSH

During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic Sediminicoccus sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium Sediminicoccus sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.

• Klíčová slova
• AAP, Proteobacteria, Sediminicoccus, gene expression, light adaptation, photosynthesis,
• MeSH
• Bacteria metabolismus   MeSH
• bakteriochlorofyly * metabolismus   MeSH
• fotosyntetická reakční centra (proteinové komplexy) * genetika   MeSH
• fotosyntéza genetika   MeSH
• kyslík metabolismus   MeSH
• reaktivní formy kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Island MeSH
• Názvy látek
• bakteriochlorofyly * MeSH
• fotosyntetická reakční centra (proteinové komplexy) * MeSH
• kyslík MeSH
• reaktivní formy kyslíku MeSH