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Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H2O2 interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar.

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this exidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as messured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death

Recent studies have revealed that dietary flavonoids are potent radical scavengers, acting in a manner similar to ascorbate and alpha-tocopherol. However, it is still not clear whether flavonoids have a similar antioxidative function in plants. We examined the possibility that flavonoids could function as stress protectants in plant cells by scavenging H2O2. Two major flavonoids, quercetin and kaempferol glycosides, were isolated from leaves of the tropical tree Schefflera arboricola Hayata. Both glycosides and aglycones of isolated flavonols were oxidized by H2O2 in the presence of horseradish peroxidase and/or in a soluble fraction of S. arboricola leaf extract. The rates of oxidation were in the order quercetin kaempferol quercetin glycoside kaempferol glycoside. Judging from the effects of inhibitors such as KCN, p-chloromercuribenzoate, and 3-amino-1H-1,2,4-triazole, we conclude that guaiacol peroxidase in the soluble fraction catalyzes H2O2-dependent oxidation of flavonols. In the flavonol-guaiacol peroxidase reaction, ascorbate had the potential to regenerate flavonols by reducing the oxidized product. These results provide further evidence that the flavonoid-peroxidase reaction can function as a mechanism for H2O2 scavenging in plants

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2 and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed

Exposure of Arabidopsis plants that were maintained under low light (200 micromoles of photons m-2 Sec-1) to excess light (2000 micromoles of photons m-2 sec-1) for 1 hr caused reversible photoinhibition of photosynthesis. Measurements of photosynthetic parameters and the use of electron transport inhibitors indicated that a novel signal transduction pathway was initiated at plastoquinone and regulated, at least in part, by the redox status of the plastoquinone pool. This signal, which preceded the photooxidative burst of hydrogen peroxide (H2O2) associated with photoinhibition of photosynthesis, resulted in a rapid increase (within 15 min) in mRNA levels of two cytosolic ascorbate peroxidase genes (APX1 and APX2). Treatment of leaves with exogenous reduced glutathione abolished this signal, suggesting that glutathione or the redox status of the glutathione pool has a regulatory impact on this signaling pathway. During recovery from photooxidative stress, transcripts for cytosolic glutathione reductase (GOR2) increased, emphasizing the role of glutathione in this stress

Measurements of the quantum efficiencies of photosynthetic electron transport through photosystem II (phi(PSII)) and CO2 assimilation (phi(CO2)) were made simultaneously on leaves of maize (Zea mays) crops in the United Kingdom during the early growing season, when chilling conditions were experienced. The activities of a range of enzymes involved with scavenging active O2 species and the levels of key antioxidants were also measured. When leaves were exposed to low temperatures during development, the ratio of phi(PSII)/phi(CO2) was elevated, indicating the operation of an alternative sink to CO2 for photosynthetic reducing equivalents. The activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and superoxide dismutase and the levels of ascorbate and alpha-tocopherol were also elevated during chilling periods. This supports the hypothesis that the relative flux of photosynthetic reducing equivalents to O2 via the Mehler reaction is higher when leaves develop under chilling conditions. Lipoxygenase activity and lipid peroxidation were also increased during low temperatures, suggesting that lipoxygenase-mediated peroxidation of membrane lipids contributes to the oxidative damage occurring in chill-stressed leaves

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment