Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium.

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2 + Fe2+----HO. + HO- + Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH+HO.----R. + H2O; R. + O2----RO2.----products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g.1-1), cellobiose oxidase, O2 and Fe(III) (30 microM). An addition of catalase after 20-30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

Apical,but not basolateral,endotoxin preincubation protects alveolar epithelial cells against hydrogen peroxide-induced loss of barrier function: the role of nitric oxide synthesis

The influence of LPS preincubation on hydrogen peroxide (H(2)O(2))-induced loss of epithelial barrier function was investigated in rat alveolar epithelial type II cells (ATII). Both apical and basolateral H(2)O(2) administration caused a manyfold increase in transepithelial [(3)H]mannitol passage. Apical but not basolateral preincubation of ATII with LPS did not influence control barrier properties but fully abrogated the H(2)O(2)-induced leakage response. The effect of apical LPS was CD14 dependent and was accompanied by a strong up-regulation of NO synthase II mRNA and protein and NO release. Inhibition of NO by N(G)-monomethyl-L-arginine suppressed the LPS effect, whereas it was reproduced by exogenous application of gaseous NO or NO donor agents. Manipulation of the glutathione homeostasis (buthionine-(S,R)-sulfoximine) and the cGMP pathway (1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxaline-1-one; zaprinast) did not interfere with the protective effect of LPS. Superoxide (O*(-)(2)) generation by ATII cells was reduced by exogenous NO and LPS preincubation. O*(-)(2) scavenging with exogenous superoxide dismutase, the intracellular superoxide dismutase analog Mn(III)tetrakis(4-benzoic acid) porphyrin, and the superoxide scavenger nitroblue tetrazolium and, in particular, hydroxyl radical scavenging with hydroxyl radical scavenger 1,3-dimethyl-thiourea inhibited the H(2)O(2)-induced epithelial leakage response. In conclusion, apical but not basolateral LPS preincubation of ATII cells provides strong protection against H(2)O(2)-induced transepithelial leakage, attributable to an up-regulation of epithelial NO synthesis. It is suggested that the LPS-induced NO formation is effective via interaction with reactive oxygen species, including superoxide and hydroxyl radicals. The polarized epithelial response to LPS may be part of the lung innate immune system, activated by inhaled endotoxin or under conditions of pneumonia.     

Oxidative damage of DNA by the reaction of amino acid with methylglyoxal in the presence of Fe(III)

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. DNA cleavage induced by the reaction of MG with lysine in the presence of Fe3+ was investigated. When plasmid DNA was incubated with MG and lysine in the presence of Fe3+, DNA strand breakage was proportional to MG and lysine concentrations. The formation of superoxide anion was detected during this reaction, and catalase, hydroxyl radical scavengers and iron chelator, desferrioxamine inhibited DNA cleavage. Deoxyribose assays showed that hydroxyl radicals were generated during the MG/lysine/Fe3+ reaction. These results suggest that superoxide anion and H2O2 may be generated from the glycation reaction between lysine with MG, and that Fe3+ probably participates in a Fenton's type reaction to produce hydroxyl radicals, which may cause DNA cleavage. This mechanism, in part, may provide an explanation for the deterioration of organs under diabetic conditions.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction

Phenanthroline and bipyridine, strong chelators of iron, protect DNA from single-strand break formation by H2O2 in human fibroblasts. This fact strongly supports the concept that these DNA single-strand breaks are produced by hydroxyl radicals generated by a Fenton-like reaction between intracellular Fe2+ and H2O2: H2O2 + Fe2+----Fe3+ + OH- + OH: Corroborating this idea is the fact that thiourea, an effective OH radical scavenger, prevents the formation of DNA single-strand breaks by H2O2 in nuclei from human fibroblasts. The copper chelator diethyldithiocarbamate, a strong inhibitor of superoxide dismutase, greatly enhances the in vivo production of DNA single-strand breaks by H2O in fibroblasts. This supports the idea that Fe3+ is reduced to Fe2+ by superoxide ion: O divided by 2 + Fe3+----O2 + Fe2+; and therefore that the sum of this reaction and the Fenton reaction, namely the so-called Haber-Weiss reaction, H2O2 + O divided by 2----O2 + OH- + OH; represents the mode whereby OH radical is produced from H2O2 in the cell. EDTA completely protects DNA from single-strand break formation in nuclei. The chelator therefore removes iron from the chromatin, and although the Fe-EDTA complex formed is capable of reacting with H2O2, the OH radical generated under these conditions is not close enough to hit DNA. Therefore iron complexed to chromatin functions as catalyst for the Haber-Weiss reaction in vivo, similarly to the role played by Fe-chelates in vitro.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.     

Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket-fence-like' iron (III) complex with the superoxide oxygen couple

Solution properties of the iron-(III) 'picket-fence-like' porphyrin, Fe(III)-alpha,alpha,alpha, beta-tetra-ortho (N-methyl-isonicotinamidophenyl) porphyrin, (Fe(III)PFP) were investigated. These were acid/base properties of the aquo complex with pKa of 3.9 and its aggregation (formation of dimer with K = 1 X 10(-10) dm3 mol-1), complex formation with cyanide ions and 1-methyl imidazole (1-MeIm), spectral properties of the three iron complexes in their ferric and ferrous form and the one-electron reduction potential of these complexes. Knowing these properties, the reaction of the ferric complexes, aquo, dicyano and bis (1-MeIm), with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the iron (III) aquo complex which governs the catalytic efficiency of the metalloporphyrin upon the disproportionation of the superoxide radical was 7.6 X 10(7) dm3 mol-1 s-1, two orders of magnitude faster when compared to the reaction of each of the other complexes. The reduction by other radicals with all iron (III) complexes had similar second-order rate constants (10(9) to 10(10) dm3 mol-1 s-1). The reduction reaction in all cases produced Fe(II)PEP and no intermediate was found. The oxidation reaction of Fe(II)PEP by O2- was one order of magnitude faster when compared to the reduction of Fe(III)PFP by the same radical. Since the reactivity of O2- toward the three iron (III) porphyrin complexes follows their reduction potentials, it is suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The reactions of the Fe(II)PFP complexes with dioxygen were also studied. The aquo complex was found to be first order in O2 and second order in Fe(II)PFP, suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The intermediate formation was corroborated by evidence of the rapid CO binding reaction to the aquo complex of Fe(II)PFP. The two other complexes reacted very slowly with O2 as well as with CO.     

Superoxide dismutase enhanced the formation of hydroxyl radicals in a reaction mixture containing xanthone under UVA irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).     

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation

We have previously observed that both Fe(II) and Fe(III) are required for lipid peroxidation to occur, with maximal rates of lipid peroxidation observed when the ratio of Fe(II) to Fe(III) is approximately one (J. R. Bucher et al. (1983) Biochem. Biophys. Res. Commun. 111, 777-784; G. Minotti and S. D. Aust (1987) J. Biol. Chem. 262, 1098-1104). Consistent with the requirement for both Fe(II) and Fe(III), ascorbate, by reducing Fe(III) to Fe(II), stimulated iron-catalyzed lipid peroxidation but when the ascorbate concentration was sufficient to reduce all of the Fe(III) to Fe(II), ascorbate inhibited lipid peroxidation. The rates of lipid peroxidation were unaffected by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers. Exogenously added H2O2 also either stimulated or inhibited ascorbate-dependent, iron-catalyzed lipid peroxidation apparently by altering the ratio of Fe(II) to Fe(III). Thus, it appears that the prooxidant effect of ascorbate is related to the ability of ascorbate to promote the formation of a proposed Fe(II):Fe(III) complex and not due to oxygen radical production. The antioxidant effect of ascorbate on iron-catalyzed lipid peroxidation may be due to complete reduction of iron.     

Nonenzymatic reduction of Met-Hb under the action of NAD radicals generated in the presence of HAD.H and Fe(II) salts. Antioxidant properties of Met-Hb]

It has been found that superoxide radicals formed at autooxidation of Fe(II) ions or of their complexes with EDTA in the presence of NAD.H cause reduction of met-Hb into deoxy or oxy-form. Under the effect of hydroxyl radicals generated in the ultrasonic field in the presence of NADH reduction of met-Hb into the ferro-form (carboxy-Hb) was observed in Co atmosphere and not in O2 atmosphere. It was induced by a high oxidation rate of oxy-Hb by hydroxyl radicals into met-Hb as compared to carboxy-Hb. Reduction of met-Hb was shown to be accompanied with the formation of NAD+. The role of NAD. radicals in the reduction of met-Hb which acts as an antioxidant in respect to organic free radicals was discussed.     

DNA strand scission by iron complexes of meso-tetra(N-methylpyridyl)porphines

The DNA strand scission activities of three positional isomers of Fe(III) meso-tetra(N-methylpyridyl)porphine (Fe(III)TnMPyP, where n = 2, 3 or 4) have been investigated using PM2 DNA as a substrate. A significant degree of strand scission activity was noted in the presence of oxygen without the addition of a reducing agent. This activity was probably due to the presence of reducing agents in the agarose gels used to separate the DNA forms, as higher levels were recorded with reducing agents added to the strand scission mixture. The relative order of strand scission activity in the absence of added reducing agents was found to be Fe(III)T2MPyP greater than Fe(III)T4MPyP greater than Fe(III)T3MPyP. Comparative studies were also made with Fe(II)bleomycin. High concentrations of some reducing agents inhibited strand scission. Oxygen was required to produce optimal strand scission activity for all three porphyrins. It was also noted from spectroscopic measurements that the reduced porphyrins were degraded in the presence of oxygen. Studies with a series of potential strand scission inhibitors suggest that hydrogen peroxide and possibly peroxy radicals are intermediates in the reaction mechanism, while diffusible hydroxyl radicals appear to be excluded. However, superoxide radicals cannot be ruled out.     

Pathways for production of Fenton's reagent by wood-rotting fungi

Abstract: Many forms of Fe(II) react with H₂0₂ to generate hydroxyl radicals (Fenton reaction). There is evidence that hydroxyl radicals are important in brown-rot, while they can be formed by secondary reactions during lignin breakdown by white-rot fungi. Their involvement in cellulose breakdown creates a range of oxidized sugars. The two reactants of Fenton's reagent can be generated by Fe(II) autoxidation, or by superoxide in reaction with Fe(III). A rapid autoxidation is not possible for complexes with a high Fe(III)/Fe(II) redox potential. Turning to specific pathways for formation of Fenton's reagent, decomposition of Fe(III)-oxalate is probably solely a photochemical process. Lignin peroxidases can act indirectly as a source of superoxide, either by reactions that lead to a peroxyradical, or by 1-electron oxidation of an aliphatic compound creating a strong reductant. Cellobiose dehydrogenase can provide a direct enzymic source for Fenton's reagent (S.M. Kremer and P.M. Wood (1992) Eur. J. Biochem. 208, 807–814). In the experiments as published, hydroxyl radical production was limited by the slow interaction of cellobiose dehydrogenase with O₂. This limitation can be removed by the presence of an iron complex with an autoxidizable Fe(lI) state. The successful use of Fenton's reagent by a living organism requires a spatial separation between initiating enzyme(s) and the site of production of hydroxyl radicals. The mobility of the extra electron on Fe(II) by intermolecular transfer may be important for achieving this separation.