Role of Hydroperoxyl Radicals in Heterogeneous Oxidation of Oxygenated Organic Aerosols.

Heterogeneous oxidative aging of organic aerosols (OA) occurs ubiquitously in the atmosphere, initiated by oxidants, such as the hydroxyl radicals (•OH). Hydroperoxyl radicals (HO2•) are also an important oxidant in the troposphere, and its gas-phase chemistry has been well studied. However, the role of HO2• in heterogeneous OA oxidation remains elusive. Here, we carry out •OH-initiated heterogeneous oxidation of several OA model systems under different HO2• conditions in a flow tube reactor and characterize the molecular oxidation products using a suite of mass spectrometry instrumentation. By using hydrogen-deuterium exchange (HDX) with thermal desorption iodide-adduct chemical ionization mass spectrometry, we provide direct observation of organic hydroperoxide (ROOH) formation from heterogeneous HO2• and peroxy radicals (RO2•) reactions for the first time. The ROOH may contribute substantially to the oxidation products, varied with the parent OA chemical structure. Furthermore, by regulating RO2• reaction pathways, HO2• also greatly influence the overall composition of the oxidized OA. Last, we suggest that the RO2• + HO2• reactions readily occur at the OA particle interface rather than in the particle bulk. These findings provide new mechanistic insights into the heterogeneous OA oxidation chemistry and help fill the critical knowledge gap in understanding atmospheric OA oxidative aging.

Unexpectedly Efficient Aging of Organic Aerosols Mediated by Autoxidation.

Multiphase oxidative aging is a ubiquitous process for atmospheric organic aerosols (OA). But its kinetics was often found to be slow in previous laboratory studies where high hydroxyl radical concentrations ([•OH]) were used. In this study, we performed heterogeneous oxidation experiments of several model OA systems under varied aging timescales and gas-phase [•OH]. Our results suggest that OA heterogeneous oxidation may be 2-3 orders of magnitude faster when [•OH] is decreased from typical laboratory flow tube conditions to atmospheric levels. Direct laboratory mass spectrometry measurements coupled with kinetic simulations suggest that an intermolecular autoxidation mechanism mediated by particle-phase peroxy radicals greatly accelerates OA oxidation, with enhanced formation of organic hydroperoxides, alcohols, and fragmentation products. With autoxidation, we estimate that the OA oxidation timescale in the atmosphere may be from less than a day to several days. Thus, OA oxidative aging can have greater atmospheric impacts than previously expected. Furthermore, our findings reveal the nature of heterogeneous aerosol oxidation chemistry in the atmosphere and help improve the understanding and prediction of atmospheric OA aging and composition evolution.

Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1's specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.

Isoprene photochemistry over the Amazon rainforest.

Isoprene photooxidation is a major driver of atmospheric chemistry over forested regions. Isoprene reacts with hydroxyl radicals (OH) and molecular oxygen to produce isoprene peroxy radicals (ISOPOO). These radicals can react with hydroperoxyl radicals (HO2) to dominantly produce hydroxyhydroperoxides (ISOPOOH). They can also react with nitric oxide (NO) to largely produce methyl vinyl ketone (MVK) and methacrolein (MACR). Unimolecular isomerization and bimolecular reactions with organic peroxy radicals are also possible. There is uncertainty about the relative importance of each of these pathways in the atmosphere and possible changes because of anthropogenic pollution. Herein, measurements of ISOPOOH and MVK + MACR concentrations are reported over the central region of the Amazon basin during the wet season. The research site, downwind of an urban region, intercepted both background and polluted air masses during the GoAmazon2014/5 Experiment. Under background conditions, the confidence interval for the ratio of the ISOPOOH concentration to that of MVK + MACR spanned 0.4-0.6. This result implies a ratio of the reaction rate of ISOPOO with HO2 to that with NO of approximately unity. A value of unity is significantly smaller than simulated at present by global chemical transport models for this important, nominally low-NO, forested region of Earth. Under polluted conditions, when the concentrations of reactive nitrogen compounds were high (>1 ppb), ISOPOOH concentrations dropped below the instrumental detection limit (<60 ppt). This abrupt shift in isoprene photooxidation, sparked by human activities, speaks to ongoing and possible future changes in the photochemistry active over the Amazon rainforest.

Global Transcriptional Response to Organic Hydroperoxide and the Role of OhrR in the Control of Virulence Traits in Chromobacterium violaceum.

A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides.

BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.

Two pathways of photoproduction of organic hydroperoxides on the donor side of photosystem 2 in subchloroplast membrane fragments.

Earlier the catalase-insensitive formation of organic hydroperoxides (via the interaction of organic radicals produced due to redox activity of P680+· (or TyrZ·) with molecular oxygen) has been found in Mn-depleted PS2 preparations (apo-WOC-PS2) by Khorobrykh et al. (Biochemistry 50:10658-10665, 2011). The present work describes a second pathway of the photoproduction of organic peroxides on the donor side of PS2. It was shown that illumination of CaCl2-treated PS2 membranes (deprived of the PS2 extrinsic proteins without removal of the Mn-containing water-oxidizing complex) (CaCl2-PS2) led to the photoproduction of highly lipophilic organic hydroperoxides (LP-OOH) (in amount corresponding to 1.5 LP-OOH per one reaction center of PS2) which significantly increased upon the addition of exogenous electron acceptor potassium ferricyanide (to 4.2 LP-OOH per one reaction center). Addition of catalase (200 U/ml) before illumination inhibited ferricyanide-induced photoproduction of hydroperoxides while no effect was obtained by adding catalase after illumination or by adding inactivated catalase before illumination. The hydroperoxide photoproduction was inhibited by the addition of exogenous electron donor for PS2, diphenylcarbazide or diuron (inhibitor of the electron transfer in PS2). The addition of exogenous hydrogen peroxide to the CaCl2-PS2 led to the production of highly lipophilic organic hydroperoxides in the dark (3.2 LP-OOH per one reaction center). We suggest that the photoproduction of highly lipophilic organic hydroperoxides in CaCl2-PS2 preparations occurs via redox activity of H2O2 produced on the donor side of PS2.

Carotenoid-dependent singlet oxygen photogeneration in light-harvesting complex 2 of Ectothiorhodospira haloalkaliphila leads to the formation of organic hydroperoxides and damage to both pigments and protein matrix.

Earlier, it was suggested that carotenoids in light-harvesting complexes 2 (LH2) can generate singlet oxygen, further oxidizing bacteriochlorophyll to 3-acetyl-chlorophyll. In the present work, it was found that illumination of isolated LH2 preparations of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with light in the carotenoid absorption region leads to the photoconsumption of molecular oxygen, which is accompanied by the formation of hydroperoxides of organic molecules in the complexes. Photoformation of two types of organic hydroperoxides were revealed: highly lipophilic (12 molecules per one LH2) and relatively hydrophobic (68 per one LH2). It has been shown that illumination leads to damage to light-harvesting complexes. On the one hand, photobleaching of bacteriochlorophyll and a decrease in its fluorescence intensity are observed. On the other hand, the photoinduced increase in the hydrodynamic radius of the complexes, the reduction in their thermal stability, and the change in fluorescence intensity indicate conformational changes occurring in the protein molecules of the LH2 preparations. Inhibition of the processes described above upon the addition of singlet oxygen quenchers (L-histidine, Trolox, sodium L-ascorbate) may support the hypothesis that carotenoids in LH2 preparations are capable of generating singlet oxygen, which, in turn, damage to protein molecules.

Ohr - OhrR, a neglected and highly efficient antioxidant system: Structure, catalysis, phylogeny, regulation, and physiological roles.

Ohrs (organic hydroperoxide resistance proteins) are antioxidant enzymes that play central roles in the response of microorganisms to organic peroxides. Here, we describe recent advances in the structure, catalysis, phylogeny, regulation, and physiological roles of Ohr proteins and of its transcriptional regulator, OhrR, highlighting their unique features. Ohr is extremely efficient in reducing fatty acid peroxides and peroxynitrite, two oxidants relevant in host-pathogen interactions. The highly reactive Cys residue of Ohr, named peroxidatic Cys (Cp), composes together with an arginine and a glutamate the catalytic triad. The catalytic cycle of Ohrs involves a condensation between a sulfenic acid (Cp-SOH) and the thiol of the second conserved Cys, leading to the formation of an intra-subunit disulfide bond, which is then reduced by dihydrolipoamide or lipoylated proteins. A structural switch takes place during catalysis, with the opening and closure of the active site by the so-called Arg-loop. Ohr is part of the Ohr/OsmC super-family that also comprises OsmC and Ohr-like proteins. Members of the Ohr, OsmC and Ohr-like subgroups present low sequence similarities among themselves, but share a high structural conservation, presenting two Cys residues in their active site. The pattern of gene expression is also distinct among members of the Ohr/OsmC subfamilies. The expression of ohr genes increases upon organic hydroperoxides treatment, whereas the signals for the upregulation of osmC are entry into the stationary phase and/or osmotic stress. For many ohr genes, the upregulation by organic hydroperoxides is mediated by OhrR, a Cys-based transcriptional regulator that only binds to its target DNAs in its reduced state. Since Ohrs and OhrRs are involved in virulence of some microorganisms and are absent in vertebrate and vascular plants, they may represent targets for novel therapeutic approaches based on the disruption of this key bacterial organic peroxide defense system.

Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity.

Typical 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous Cys-based peroxidases, which are stable as decamers in the reduced state, and may dissociate into dimers upon disulfide bond formation. A peroxidatic Cys (CP) takes part of a catalytic triad, together with a Thr/Ser and an Arg. Previously, we described that the presence of Ser (instead of Thr) in the active site stabilizes yeast 2-Cys Prx as decamers. Here, we compared the hyperoxidation susceptibilities of yeast 2-Cys Prx. Notably, 2-Cys Prx containing Ser (named here Ser-Prx) were more resistant to hyperoxidation than enzymes containing Thr (Thr-Prx). In silico analysis revealed that Thr-Prx are more frequent in all domains of life, while Ser-Prx are more abundant in bacteria. As yeast 2-Cys Prx, bacterial Ser-Prx are more stable as decamers than Thr-Prx. However, bacterial Ser-Prx were only slightly more resistant to hyperoxidation than Thr-Prx. Furthermore, in all cases, organic hydroperoxide inhibited more the peroxidase activities of 2-Cys Prx than hydrogen peroxide. Moreover, bacterial Ser-Prx displayed increased thermal resistance and chaperone activity, which may be related with its enhanced stability as decamers compared to Thr-Prx. Therefore, the single substitution of Thr by Ser in the catalytic triad results in profound biochemical and structural differences in 2-Cys Prx.

An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses.

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.

OsmC proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis protect against organic hydroperoxide stress.

Bacterial antioxidants play a critical role in the detoxification of endogenously and host derived oxidative radicals during host-pathogen interactions. Recently, the osmotically induced bacterial protein C (OsmC) is included in the antioxidant category of enzymes as it shows structural and functional relationships with organic hydroperoxide reductase (Ohr) enzyme. A copy of the gene encoding OsmC is conserved across mycobacterial species, including Mycobacterium tuberculosis (Rv2923c) and Mycobacterium smegmatis (MSMEG2421), but its role in protecting these species against oxidative stress is unknown. To determine the role of OsmC in mycobacterial oxidative stress, we overexpressed and purified OsmCs of M. tuberculosis and M. smegmatis and assessed their ability to reduce peroxide substrates like hydrogen peroxide (H(2)O(2)), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP) in Ferrous Ion Oxidation in Xylenol (FOX) assay. This revealed that OsmCs from both species were capable of reducing both inorganic (H(2)O(2)) and organic (CHP and t-BHP) peroxides. Further, an M. smegmatis mutant (MS∆osmC) deficient in OsmC exhibited reduced reduction of CHP and t-BHP than the parental wild type strain, indicating that OsmC protein contributes significantly for the total peroxide reductase activity of mycobacteria. The MS∆osmC strain was also sensitive to organic hydroperoxides, which could be reversed by complementing with a plasmid borne osmC. Plasmid borne osmC also increased the resistance of M. smegmatis wild type strain to isoniazid (INH) but at a relatively lower level than ahpC, an organic hydroperoxide reductase. These results suggest that OsmC plays an important role in peroxide metabolism and protecting mycobacteria against oxidative stress.