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Neutrophils mediate the early innate immune response through extracellular traps comprising intracellular protein and DNA. These traps play a pivotal role in both immunity against invading pathogens and the development of immunopathological reactions through the production of reactive oxygen species (ROS). Proteins serve as the main target for ROS, resulting in the formation of protein adducts. Herein, we report that the superoxide anion radical (O2Ë-) plays a vital role in neutrophil function through sequential events involving 5-lipoxygenase (5-LOX) and NADPH oxidase (NOX). More specifically, differences in NOX homologs expression were observed post-stimulation with PMA and LPS. Differentiation conditions and O2Ë- generation were confirmed using flow cytometry. Immunoblotting analysis confirmed the time-dependent expression of NOX underlying its requirement and 5-LOX-mediated lipid peroxidation events in neutrophil function. Protein-malondialdehyde (MDA) adducts formed were detected using immunoblotting, and quercetin was evaluated for its ability to scavenge free radicals through electron paramagnetic resonance (EPR) spin-trapping spectroscopy and results were confirmed with blotting analysis. Free radical-mediated protein oxidation events influence neutrophil function and protein adducts formed serve as markers of neutrophil activation upon infection and inflammation. The study warrants further corroboration and the study of specific proteins involved in neutrophil activation and their role in inflammation.
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Extracellular vesicles (EVs) are a type of cytoplasmic vesicles secreted by a variety of cells. EVs originating from cells have been known to participate in cell communication, antigen presentation, immune cell activation, tolerance induction, etc. These EVs can also carry the active form of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Hydrogen (NADPH) oxidase, which is very essential for the production of reactive oxygen species (ROS) and that can then modulate processes such as cell regeneration. The aim of this study is to characterize the EVs isolated from U-937 and THP-1 cells, identify the NADPH oxidase (NOX) isoforms, and to determine whether EVs can modulate NOX4 and NOX2 in monocytes and macrophages. In our study, isolated EVs of U-937 were characterized using dynamic light scattering (DLS) spectroscopy and immunoblotting. The results showed that the exogenous addition of differentiation agents (either phorbol 12-myristate 13-acetate (PMA) or ascorbic acid) or the supplementation of EVs used in the study did not cause any stress leading to alterations in cell proliferation and viability. In cells co-cultured with EVs for 72 h, strong suppression of NOX4 and NOX2 is evident when monocytes transform into macrophagic cells. We also observed lower levels of oxidative stress measured using immunoblotting and electron paramagnetic resonance spectroscopy under the EVs co-cultured condition, which also indicates that EVs might contribute significantly by acting as an antioxidant source, which agrees with previous studies that hypothesized the role of EVs in therapeutics. Therefore, our results provide evidence for NOX regulation by EVs in addition to its role as an antioxidant cargo.
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The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O2 â¢-) that functions as a precursor for antimicrobial hydrogen peroxide (H2O2) production, and H2O2 is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. H2O2 modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.
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Peróxido de Hidrógeno , Superóxidos , Oxidación-Reducción , Estrés Oxidativo , Ácido Hipocloroso , Inmunidad InnataRESUMEN
In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.
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Human skin is exposed to various physical and chemical stress factors, which commonly cause the oxidation of lipids and proteins. In this study, azo initiator AAPH [2,2' -azobis(2-methylpropionamidine) dihydrochloride] was employed to initiate lipid peroxidation in porcine skin as an ex vivo model for human skin. We demonstrate that malondialdehyde (MDA), a secondary product of lipid peroxidation, is covalently bound to collagen in the dermis, forming MDA-collagen adducts. The binding of MDA to collagen results in an unfolding of the collagen triple helix, formation of the dimer of α-chains of collagen, and fragmentation of the collagen α-chain. It is proposed here that the MDA is bound to the lysine residues of α-chain collagen, which are involved in electrostatic interaction and hydrogen bonding with the glutamate and aspartate of other α-chains of the triple helix. Our data provide crucial information about the MDA binding topology in the skin, which is necessary to understand better the various types of skin-related diseases and the aging process in the skin under stress.
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Colágeno , Estrés Oxidativo , Humanos , Porcinos , Malondialdehído/metabolismo , Oxidación-Reducción , Colágeno/metabolismo , Peroxidación de Lípido , AnimalesRESUMEN
Acetaldehyde can be found in human cells as a byproduct of various metabolic pathways, including oxidative processes such as lipid peroxidation. This secondary product of lipid peroxidation plays a role in various pathological processes, leading to various types of civilization diseases. In this study, the formation of free acetaldehyde induced by oxygen-centred radicals was studied in monocyte-like cell line U937. Exposure of U937 cells to peroxyl/alkoxyl radicals induced by azocompound resulted in the formation of free acetaldehyde. Acetaldehyde is formed by the cleavage of fatty acids, which represents the breakdown of fatty acids into smaller fragments initiated by the cyclization of lipid peroxyl radical and ß-scission of lipid alkoxyl radical. The cleavage of fatty acids alters the integrity of the plasma and nuclear membrane, leading to the loss of cell viability. Understanding the pathological processes of acetaldehyde formation is an active area of research with potential implications for preventing and treating various diseases associated with oxidative stress.
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Acetaldehído , Monocitos , Humanos , Peroxidación de Lípido , Radicales Libres/metabolismo , Células U937 , Monocitos/metabolismo , Ácidos Grasos/metabolismo , Especies Reactivas de OxígenoRESUMEN
Under environmental conditions, plants are exposed to various abiotic and biotic stress factors, which commonly cause the oxidation of lipids and proteins. Lipid peroxidation constantly produces malondialdehyde (MDA), a secondary product of lipid peroxidation, which is covalently bound to proteins forming MDA-protein adducts. The spatial distribution of MDA-protein adducts in Arabidopsis leaves shows that MDA-protein adducts are located in the chloroplasts, uniformly spread out over the thylakoid membrane. At the lumenal side of thylakoid membrane, MDA interacts with PsbP, an extrinsic subunit of the photosystem II (PSII), which is in electrostatic interaction with the PSII core proteins. Under heat stress, when MDA is moderately enhanced, the electrostatic interaction between PsbP and PSII core proteins is weakened, and PsbP with bound MDA is released in the lumen. It is proposed here that the electrophilic MDA is bound to the nucleophilic lysine residues of PsbP, which are involved in electrostatic interactions with the negatively charged glutamate of the PSII core protein. Our data provide crucial information about the MDA binding topology in the higher plant PSII complex, which is necessary to understand better the physiological functions of MDA for plant survival under stress.
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Arabidopsis , Malondialdehído , Ácido Glutámico , Lisina , Respuesta al Choque TérmicoRESUMEN
Oxidative processes in all types of organisms cause the chemical formation of electronically excited species, with subsequent ultraweak photon emission termed biological auto(chemi)luminescence (BAL). Imaging this luminescence phenomenon using ultrasensitive devices could potentially enable monitoring of oxidative stress in optically accessible areas of the human body, such as skin. Although oxidative stress induced by UV light has been explored, for chemically induced stress, there is no in vivo-quantified imaging of oxidative processes in human skin using BAL under the controlled extent of oxidative stress conditions. Furthermore, the mechanisms and dynamics of BAL from the skin have not been fully explored. Here, we demonstrate that different degrees of chemically induced oxidative stress on the skin can be spatially resolved quantitatively through noninvasive label-free BAL imaging. Additionally, to gain insight into the underlying mechanisms, a minimal chemical model of skin based on a mixture of lipid, melanin, and water was developed and used to show that it can be used to reproduce essential features of the response of real skin to oxidative stress. Our results contribute to novel, noninvasive photonic label-free methods for quantitative sensing of oxidative processes and oxidative stress.
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Luminiscencia , Piel , Humanos , Piel/metabolismo , Estrés Oxidativo , Rayos Ultravioleta , FotonesRESUMEN
The heart of oxygenic photosynthesis is the water-splitting photosystem II (PSII), which forms supercomplexes with a variable amount of peripheral trimeric light-harvesting complexes (LHCII). Our knowledge of the structure of green plant PSII supercomplex is based on findings obtained from several representatives of green algae and flowering plants; however, data from a non-flowering plant are currently missing. Here we report a cryo-electron microscopy structure of PSII supercomplex from spruce, a representative of non-flowering land plants, at 2.8 Å resolution. Compared with flowering plants, PSII supercomplex in spruce contains an additional Ycf12 subunit, Lhcb4 protein is replaced by Lhcb8, and trimeric LHCII is present as a homotrimer of Lhcb1. Unexpectedly, we have found α-tocopherol (α-Toc)/α-tocopherolquinone (α-TQ) at the boundary between the LHCII trimer and the inner antenna CP43. The molecule of α-Toc/α-TQ is located close to chlorophyll a614 of one of the Lhcb1 proteins and its chromanol/quinone head is exposed to the thylakoid lumen. The position of α-Toc in PSII supercomplex makes it an ideal candidate for the sensor of excessive light, as α-Toc can be oxidized to α-TQ by high-light-induced singlet oxygen at low lumenal pH. The molecule of α-TQ appears to shift slightly into the PSII supercomplex, which could trigger important structure-functional modifications in PSII supercomplex. Inspection of the previously reported cryo-electron microscopy maps of PSII supercomplexes indicates that α-Toc/α-TQ can be present at the same site also in PSII supercomplexes from flowering plants, but its identification in the previous studies has been hindered by insufficient resolution.
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Complejo de Proteína del Fotosistema II , alfa-Tocoferol , Complejo de Proteína del Fotosistema II/metabolismo , Microscopía por Crioelectrón , alfa-Tocoferol/análisis , alfa-Tocoferol/metabolismo , Tilacoides/metabolismo , Fotosíntesis , Plantas/metabolismoRESUMEN
Geothermal energy installations are becoming increasingly common in new city developments and renovations. With a broad range of technological applications and improvements in this field, the demand for suitable monitoring technologies and control processes for geothermal energy installations is also growing. This article identifies opportunities for the future development and deployment of IoT sensors applied to geothermal energy installations. The first part of the survey describes the technologies and applications of various sensor types. Sensors that monitor temperature, flow rate and other mechanical parameters are presented with a technological background and their potential applications. The second part of the article surveys Internet-of-Things (IoT), communication technology and cloud solutions applicable to geothermal energy monitoring, with a focus on IoT node designs, data transmission technologies and cloud services. Energy harvesting technologies and edge computing methods are also reviewed. The survey concludes with a discussion of research challenges and an outline of new areas of application for monitoring geothermal installations and innovating technologies to produce IoT sensor solutions.
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Energía Geotérmica , Internet de las Cosas , Nube Computacional , Tecnología de la Información , TecnologíaRESUMEN
Skin plays an important role in protection, metabolism, thermoregulation, sensation, and excretion whilst being consistently exposed to environmental aggression, including biotic and abiotic stresses. During the generation of oxidative stress in the skin, the epidermal and dermal cells are generally regarded as the most affected regions. The participation of reactive oxygen species (ROS) as a result of environmental fluctuations has been experimentally proven by several researchers and is well known to contribute to ultra-weak photon emission via the oxidation of biomolecules (lipids, proteins, and nucleic acids). More recently, ultra-weak photon emission detection techniques have been introduced to investigate the conditions of oxidative stress in various living systems in in vivo, ex vivo and in vitro studies. Research into two-dimensional photon imaging is drawing growing attention because of its application as a non-invasive tool. We monitored spontaneous and stress-induced ultra-weak photon emission under the exogenous application of a Fenton reagent. The results showed a marked difference in the ultra-weak photon emission. Overall, these results suggest that triplet carbonyl (3C=O∗) and singlet oxygen (1O2) are the final emitters. Furthermore, the formation of oxidatively modified protein adducts and protein carbonyl formation upon treatment with hydrogen peroxide (H2O2) were observed using an immunoblotting assay. The results from this study broaden our understanding of the mechanism of the generation of ROS in skin layers and the formation/contribution of various excited species can be used as tools to determine the physiological state of the organism.
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Peróxido de Hidrógeno , Piel , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Piel/metabolismo , Estrés Oxidativo , Oxidación-Reducción , Proteínas/metabolismoRESUMEN
Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.
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Macrófagos , Proteínas , Especies Reactivas de Oxígeno/metabolismo , Radicales Libres/análisis , Radicales Libres/metabolismo , Detección de Spin/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Macrófagos/metabolismo , Proteínas/químicaRESUMEN
The acclimation of higher plants to different light intensities is associated with a reorganization of the photosynthetic apparatus. These modifications, namely, changes in the amount of peripheral antenna (LHCII) of photosystem (PS) II and changes in PSII/PSI stoichiometry, typically lead to an altered chlorophyll (Chl) a/b ratio. However, our previous studies show that in spruce, this ratio is not affected by changes in growth light intensity. The evolutionary loss of PSII antenna proteins LHCB3 and LHCB6 in the Pinaceae family is another indication that the light acclimation strategy in spruce could be different. Here we show that, unlike Arabidopsis, spruce does not modify its PSII/PSI ratio and PSII antenna size to maximize its photosynthetic performance during light acclimation. Its large PSII antenna consists of many weakly bound LHCIIs, which form effective quenching centers, even at relatively low light. This, together with sensitive photosynthetic control on the level of cytochrome b6f complex (protecting PSI), is the crucial photoprotective mechanism in spruce. High-light acclimation of spruce involves the disruption of PSII macro-organization, reduction of the amount of both PSII and PSI core complexes, synthesis of stress proteins that bind released Chls, and formation of "locked-in" quenching centers from uncoupled LHCIIs. Such response has been previously observed in the evergreen angiosperm Monstera deliciosa exposed to high light. We suggest that, in contrast to annuals, shade-tolerant evergreen land plants have their own strategy to cope with light intensity changes and the hallmark of this strategy is a stable Chl a/b ratio.
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Arabidopsis , Picea , Aclimatación , Arabidopsis/metabolismo , Clorofila/metabolismo , Clorofila A/metabolismo , Complejo de Citocromo b6f/metabolismo , Citocromos b/metabolismo , Proteínas de Choque Térmico/metabolismo , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Picea/metabolismoRESUMEN
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.
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Estrés Oxidativo , Radicales Libres/metabolismo , Humanos , Malondialdehído , Oxidación-Reducción , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
Reactive oxygen species (ROS) are formed in photosystem II (PSII) under various types of abiotic and biotic stresses. It is considered that ROS play a role in chloroplast-to-nucleus retrograde signaling, which changes the nuclear gene expression. However, as ROS lifetime and diffusion are restricted due to the high reactivity towards biomolecules (lipids, pigments, and proteins) and the spatial specificity of signal transduction is low, it is not entirely clear how ROS might transduce signal from the chloroplasts to the nucleus. Biomolecule oxidation was formerly connected solely with damage; nevertheless, the evidence appears that oxidatively modified lipids and pigments are be involved in chloroplast-to-nucleus retrograde signaling due to their long diffusion distance. Moreover, oxidatively modified proteins show high spatial specificity; however, their role in signal transduction from chloroplasts to the nucleus has not been proven yet. The review attempts to summarize and evaluate the evidence for the involvement of ROS in oxidative signaling in PSII.
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Cloroplastos , Complejo de Proteína del Fotosistema II , Cloroplastos/metabolismo , Lípidos , Estrés Oxidativo , Complejo de Proteína del Fotosistema II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The largest stable photosystem II (PSII) supercomplex in land plants (C2S2M2) consists of a core complex dimer (C2), two strongly (S2) and two moderately (M2) bound light-harvesting protein (LHCB) trimers attached to C2 via monomeric antenna proteins LHCB4-6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C2S2M2 supercomplex. Here, we performed structure-function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the "spruce-type" PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The "spruce-type" PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C2S2 semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C2S2 arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión a Clorofila/genética , Complejo de Proteína del Fotosistema II/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Picea/metabolismoRESUMEN
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 (HOâ) 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.
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Diferenciación Celular , Radicales Libres/metabolismo , Monocitos/citología , Monocitos/metabolismo , Proteínas/metabolismo , Acetofenonas/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón , Células HL-60 , Humanos , Radical Hidroxilo , Monocitos/efectos de los fármacos , NADP/metabolismo , Coloración y Etiquetado , Acetato de Tetradecanoilforbol/farmacología , Células U937RESUMEN
Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4ß2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4ß2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood-brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.
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Alcaloides , Receptores Nicotínicos , Alcaloides/farmacología , Animales , Ansiedad/tratamiento farmacológico , Nicotina , Pez CebraRESUMEN
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 (O2â¢-) and the hydroxyl (HOâ¢) 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 O2â¢- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2â¢- and HOâ¢, 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.