Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Human erythrocytes exposure to juglone leads to an increase of superoxide anion production associated with cytochrome b5 reductase uncoupling

Cytochrome b₅ reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the cytochrome b₅ reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human cytochrome b₅ reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to cytochrome b₅ and ferricyanide. Upon complex formation with cytochrome b₅ reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that cytochrome b₅ reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by cytochrome b₅ reductase.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.     

Generation of superoxide anion and hydrogen peroxide at the surface of plant cells

In addition to well-known cell wall peroxidases, there is now evidence for the presence of this enzyme at the plasma membrane of the plant cells (surface peroxidase). Both are able to catalyze, through a chain of reactions involving the superoxide anion, the oxidation of NADH to generate hydrogen peroxide. The latter is oxidized by other wall-bound peroxidases to convert cinnamoyl alcohols into radical forms, which, then polymerize to generate lignin. However, there are other enzymes at the surface of plasma membranes capable of generating hydrogen peroxide (cell wall polyamine oxidase), superoxide anion (plasma membrane Turbo reductase), or both (plasma membrane flavoprotein?). These enzymes utilize NAD(P)H as a substrate. The Turbo reductase and the flavoprotein catalyze the univalent reduction of Fe³⁺ and then of O₂ to produce Fe²⁺ and \(O_2^{\bar \cdot } \) , respectively. The superoxide anion, in the acidic environment of the cell wall, may then dismutate to H₂O₂. These superoxide anion- and hydrogen peroxide-generating systems are discussed in relation to their possible involvement in physiological and pathological processes in the apoplast of plant cells.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pre-treatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Adaptation of the Phytopathogenic Fungus Fusarium decemcellulare to Oxidative Stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H₂O₂ and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H₂O₂ and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main enzymes involved in the defense against oxidative stress.     

Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Effect of Aluminium on Oxidative Stress Related Enzymes Activities in Barley Roots

The impact of aluminium stress on activities of enzymes of the oxidative metabolism: superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), NADH peroxidase (NADH-POD) and oxalate oxidase (OXO) was studied in barley (Hordeum vulgare L. cv. Alfor) root tips. SOD appeared to be involved in detoxification mechanisms at highly toxic Al doses and after long Al exposure. POD and APX, H₂O₂ consuming enzymes, were activated following similar patterns of expression and exhibiting significant correlation between their elevated activities and root growth inhibition. The signalling role of NADH-POD in oxidative stress seems to be more probable than that of OXO, which might be involved in Al toxicity mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.