Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

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.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

Polarographic Measurements of Spontaneous and Mitochondria-Promoted Oxidation of 5,6-and 5,7-Dihydroxytryptamine

Abstract: Spontaneous oxygen consumption by 5,6- and 5,7-DHT (dihydroxytryptamine), related indoleethylamines, and 6-hydroxydopamine and oxygen consumption by these compounds in the presence of rat liver mitochondria were measured by the polarographic oxygen electrode technique. 5,6- and 5,7-DHT react with oxygen at very different rates (2.7 nmol O₂/min and 33.4 nmol O₂/min, respectively) when incubated in buffer, pH 7.2, at a concentration of 1 mm and with different kínetic characteristics. While the oxidation of 5,7-DHT obeys a reaction of second-order type, the oxidation of 5,6-DHT is more complex and characterized by autocatalytic promotion. Coloured quinoidal oxidation products appeared during the degradation of both indoleamines. Glutathione, ascorbate, dithiothreitol, cysteine, albumin, and superoxide dismutase partially prevented 5,6- and 5,7-DHT from oxidative destruction. Catalase saved oxygen only in the case of 5,6-DHT by recycling of O₂ released from near-stoichiometrically formed H₂O₂ during oxidation of 5,6-DHT: 5,7-DHT did not generate H₂O₂ in measurable amounts. Oxygen consumption rates of 5,6- and 5,7-DHT were enhanced after addition of rat liver mitochondria to the incubation medium; this resulted in an accelerated formation of quinoidal products. This stimulatory effect on the oxidation rates of both 5,6- and 5,7-DHT was blocked by cyanide, but not rotenone, and was abolished by boiling of the mitochondria fraction. The observed increase in oxygen consumption in the presence of mitochondria was found not to be influenced by monoamine oxidase-dependent deamination of 5,6- and 5,7-DHT. It is postulated that 5,6- and 5,7-DHT are capable of participating in the electron transfer of the mitochondrial respiration chain beyond complex III. Results obtained in determinations of ADP:0 ratios in respiratory control experiments exclude a possible interference of 5,6-DHT, 5,7-DHT, and 6-OH-DA with phosphorylating sites. During the activated state of respiration, no signs of electron transfer inhibition by 5,6- and 5,7-DHT were detectable. A comparison and evaluation of the autoxidation rates of various hydroxylated indoleethylamines, of their affinity to the 5-HT transport sites, and their neurotoxic potency in vivo reveals that interaction of these compounds with oxygen at restricted reaction velocity is a prerequisite for efficient toxicity in monoaminergic neurons following active accumulation in these neurons via the high-affinity uptake systems.     

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver.

In isolated hepatic mitochondria, the oxidation of NAD⁺-dependent substrates was decreased after chronic consumption of ethanol or by the addition of acetaldehyde in vitro. Reversed electron transport from succinate to NAD^?, which requires transfer of electrons through the NADH dehydrogenase complex and energy transduction through coupling site 1, was depressed by ethanol feeding and by acetaldehyde in vitro, whereas NADH formation from glutamate, which is mediated directly by substrate oxidation and is not energy-dependent, was slightly increased. By contrast, reactions involving the terminal portion of the phosphorylation chain, e.g., ATP-³²P exchange or dinitrophenolstimulated ATPase activity, were not affected. Adenine nucleotide translocase activity was not altered by chronic consumption of ethanol or the addition of acetaldehyde in vitro. These data suggest that the NADH-ubiquinone oxidoreductase complex of the respiratory chain, a segment which contains several iron-sulfur centers which participate in electron transport and energy transduction, may be impaired by chronic consumption of ethanol and is especially sensitive to inhibition by acetaldehyde in vitro. Neither energy coupling sites II or III, nor the terminal reactions of oxidative phosphorylation share this sensitivity. CO₂ production from various labeled intermediates of the citric acid cycle was depressed after chronic consumption of ethanol and after the addition of acetaldehyde. Acetate had no effect on these reactions, indicating that the inhibition by acetaldehyde is not mediated via acetate. Impairment of the activities of the respiratory chain and the citric acid cycle, or both, may explain the decreases in oxygen uptake and CO₂ production from citric acid cycle intermediates and fatty acids, as well as the increase in ketone body production, found in mitochondria from ethanolfed rats.     

THE OXIDATION OF SODIUM LACTATE BY HYDROGEN PEROXIDE

By means of a modification of the technique of the Osterhout apparatus it is possible to follow the production of CO₂ from sodium lactate when acted upon by H₂O₂. The results of this process indicate that the reaction is not a simple one but is of an autocatalytic type. This conclusion is borne out by the fact that the determinations of H₂O₂ during the reaction show an increased amount of peroxide during the earlier stages of the reaction. This is considered to be due to the formation of a peroxide by the oxidation of the acetaldehyde (formed by the interaction of H₂O₂ and sodium lactate) with the oxygen of the air. When the reaction is carried out in an atmosphere of nitrogen no increase is observed. Further experiments in nitrogen tend to show that acetaldehyde is the end-product of the action of H₂O₂ alone. The effect of FeCl₃ upon the reaction depends upon the previous treatment of the iron salt. If the iron solution is added to the H₂O₂ before mixing with the lactate there is an increased amount of CO₂. If, however, the iron is added to the lactate before the addition of the peroxide, the action tends to inhibit the production of CO₂. The reaction of H₂O₂ with sodium lactate is comparable to the action of killed yeast and methylene blue as determined by Palladin and his coworkers.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

A kinetic study on iron stimulation of the xanthine oxidase dependent oxidation of ascorbate

Xanthine oxidase reduces molecular oxygen to H₂O₂ and superoxide radicals during its catalytic action on xanthine, hypoxanthine or acetaldehyde. Ascorbate is catalytically oxidized by the superoxide radicals generated, when present in the reaction solution (Nishikimi 1975). The present study shows that iron ions markedly stimulate the enzyme dependent ascorbate oxidation, by acting as a red/ox-cycling intermediate between the oxidase and ascorbate. An apparent K_m-value of 10.8 M characterized the iron stimulatory effect on the reaction at pH 6.0. Reduced transition-state metals can be oxidized by H₂O₂ through a Fenton-type reaction. Catalase was found to reduce the effect of iron on the enzyme dependent ascorbate oxidation, strongly suggesting that H₂O₂, produced during catalysis, is involved in the oxidation of ferrous ions.     

Hemin-mediated oxidation of dithiothreitol reduces oxygen to H2O

Summary Hemin catalyses the oxidation of dithiothreitol. One mole of oxygen is consumed for every 2 moles of dithiothreitol oxidized and the product is shown by spectral studies to be the intramolecular disulphide. The reaction shows a specificity for dithiol and for free heme moieties. Hemin molecules exhibit cooperativity in oxygen reduction. Oxygen radicals do not seem to be involved. H₂O₂ is not required for this oxidation of dithiothreitol and does not appear to be an intermediate in the reduction of O₂ to H₂O. However, an independent minor reaction involving a 2-electron transfer with the formation of H₂O₂ also occurs. These studies on the hemin-catalyzed oxidation of dithiothreitol provide a chemical model for a direct 4-electron reduction of O₂ to H₂O.Abbreviations HMGCoA 3-hydroxy-3-methylglutaryl coenzyme A - DTT dithiothreitol - Tris-HCl tris(hydroxymethyl)-aminomethane hydrochloride - HEPES N-2,hydroxylethypiperazine-N-2-ethane-sulphonic acid     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Alternatives to the ‘water oxidation pathway’ of biological ozone formation

Recent studies have shown that ozone (O₃) is endogenously generated in living tissues, where it makes both positive and negative physiological contributions. A pathway for the formation of both O₃ and hydrogen peroxide (H₂O₂) was previously proposed, beginning with the antibody or amino acid-catalyzed oxidation of water by singlet oxygen (¹O₂₎ to form hydrogen trioxide (H₂O₃) as a key intermediate. A key pillar of this hypothesis is that some of the H₂O₂ molecules incorporate water-derived oxygen atoms. However, H₂O₃ decomposes extremely readily in water to form ¹O₂ and water, rather than O₃ and H₂O₂. This article highlights key literature indicating that the oxidation of organic molecules such as the amino acids methionine, tryptophan, histidine, and cysteine by ¹O₂ is involved in ozone formation. Based on this, an alternative hypothesis for ozone formation is developed involving a further reaction of singlet oxygen with various oxidized organic intermediates. H₂O₂ having water-derived oxygen atoms is subsequently formed during ozone decomposition in water by known reactions.     

Microsomal metabolism of hydroxyl radical scavenging agents: Relationship to the microsomal oxidation of alcohols

Previous studies provided indirect evidence that hydroxyl radicals are involved in the oxidation of primary aliphatic alcohols by rat liver microsomes. In the current study, three ·OH scavengers were used as chemical probes to evaluate ·OH production by microsomes. The scavengers and their products were 3-thiomethylpropanal (methional) and 2-keto-4-thiomethylbutyric acid, which yield ethylene gas, and dimethylsulfoxide, which yields methane gas. We observed that microsomes actively generate the appropriate hydrocarbon gas from each scavenger when electron transport is initiated with NADPH. Hydrocarbon gas production is augmented by 0.5 mm azide, an agent which inhibits catalase and, thereby, permits H₂O₂ to accumulate. However, no metabolism of scavengers occurs when H₂O₂ is added in the absence of microsomes. These results are consistent with a presumed role for H₂O₂ as a precursor of hydroxyl radicals. In addition, no metabolism of scavengers occurs when azide and H₂O₂ are added either to boiled microsomes or to intact microsomes in the absence of electron transport (NADPH-generating system omitted). Therefore, both H₂O₂ and simultaneous electron transport are required. Ethanol inhibits the metabolism of the scavengers. Similarly, the scavengers inhibit the oxidation of ethanol to acetaldehyde; inhibition in the presence of azide is competitive. These latter results indicate a competition between the scavengers and ethanol for metabolically generated ·OH in microsomes. The specificity of this interaction is evident from the observation that the scavengers do not affect the activities of microsomal aminopyrine demethylase or aniline hydroxylase. Two model ·OH-generating systems (Fenton's reagent and iron-EDTA-ascorbate) were also studied and they produced acetaldehyde from ethanol and hydrocarbon gases from the scavengers. These results, as a whole, tend to verify a role for ·OH in the microsomal oxidation of alcohols.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Microsensor Measurements of Sulfate Reduction and Sulfide Oxidation in Compact Microbial Communities of Aerobic Biofilms

The microzonation of O₂ respiration, H₂S oxidation, and SO₄^2- reduction in aerobic trickling-filter biofilms was studied by measuring concentration profiles at high spatial resolution (25 to 100 μm) with microsensors for O₂, S^2-, and pH. Specific reaction rates were calculated from measured concentration profiles by using a simple one-dimensional diffusion reaction model. The importance of electron acceptor and electron donor availability for the microzonation of respiratory processes and their reaction rates was investigated. Oxygen respiration was found in the upper 0.2 to 0.4 mm of the biofilm, whereas sulfate reduction occurred in deeper, anoxic parts of the biofilm. Sulfate reduction accounted for up to 50% of the total mineralization of organic carbon in the biofilms. All H₂S produced from sulfate reduction was reoxidized by O₂ in a narrow reaction zone, and no H₂S escaped to the overlying water. Turnover times of H₂S and O₂ in the reaction zone were only a few seconds owing to rapid bacterial H₂S oxidation. Anaerobic H₂S oxidation with NO₃^- could be induced by addition of nitrate to the medium. Total sulfate reduction rates increased when the availability of SO₄^2- or organic substrate increased as a result of deepening of the sulfate reduction zone or an increase in the sulfate reduction intensity, respectively.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

Response properties of the genetically encoded optical H2O2 sensor HyPer

Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H₂O₂ reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H₂O₂ treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H₂O₂ was still present. Although HyPer should be H₂O₂ specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl⁻ exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H₂O₂—albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H₂O₂ increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H₂O₂ decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.