Mechanism of enzymatic dismutation of superoxide anion-radicals

The mechanism of interaction of the superoxide radical O2-. with lysine was studied by the methods of polarography and ab initio quantum chemical calculations. It was shown that O2-. interacts with lysine to form a thermodynamically stable complex with proton transfer and formation of the HO2. radical.     

Generation of the superoxide radical during autoxidation of oxymyoglobin.

Autoxidation of bovine oxymyoglobin to metmyoglobin induces co-oxidation of epinephrine to adrenochrome. This co-oxidation is markedly inhibited by superoxide dismutase [EC 1.15.1.1]. Electron transfer from oxymyoglobin to ferricytochrome c is partially inhibited by superoxide dismutase. These results indicate that autoxidation of oxymyoglobin results in generation of superoxide radicals. Autoxidation of oxymyoglobin is accelerated by superoxide dismutase and partially inhibited by catalase [EC 1.11.1.6].     

Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes

Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction.     

Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.     

ESR evidence of superoxide radical dismutation by human ceruloplasmin

The formation of the paramagnetic complex between human ceruloplasmin and radiation produced superoxide radicals was observed by the ESR method at low temperatures. The disappearance of the complex without changes in the oxidation state of copper give the direct evidence that ceruloplasmin, the major antioxidant in serum, is able to dismutate superoxide radicals.     

Antioxidant properties of fungal melanin pigments

Fungal melanin pigments were shown to display a high antioxidant activity. An increase in the number of methyl substituents in benzidine molecules of melanins obtained from micromycetes and macromycetes was accompanied by a decrease in the efficiency of inhibition of peroxidase-mediated oxidation. Melanins were found to have considerable gene-protecting properties. Pigments isolated from macromycetes and applied at a much lower concentration than those obtained from micromycetes prevented damage to bacteriophage-λ DNA induced by products of peroxidase-mediated degradation of aminobiphenyls.     

Antioxidant features of fungal melanin pigments

Fungal melanin pigments were shown to display a high antioxidant activity. An increase in the number of methyl substituents in benzidine molecules of melanins obtained from micromycetes and macromycetes was accompanied by a decrease in the efficiency of inhibition of peroxidase-catalyzed oxidation. Melanins were found to have considerable gene-protecting properties. Pigments isolated from macromycetes and applied at a much lower concentration than those obtained from micromycetes prevented damage to bacteriophage-lambda DNA induced by products of peroxidase-catalyzed degradation of aminobiphenyls.     

Water induced dismutation of superoxide anion generates singlet molecular oxygen

Direct spectroscopic measurement of 1268 nm singlet oxygen emission from KO2 suspensions at room temperature in three non-protonic solvents--CCl4, Cl2FCCClF2, and C6F14 by the action of water is reported. The results clearly show that the singlet oxygen generation is due to a water induced reaction, and suggest that one role of the enzyme superoxide dismutase may be the protection of biological structures, for example, lipid membranes, from degradation by singlet oxygen.     

Mechanism of autoxidation of oxyhaemoglobin

Oxyhaemoglobins from erythrocytes of different animals including fish, amphibians, reptiles, birds, mammals and human beings have been isolated by ion-exchange chromatography over phosphocellulose and the comparative rates of autoxidation of oxyhaemoglobin studied. The mechanism of autoxidationin vitro has been elucidated using toad as well as human oxyhaemoglobin. Autoxidation is markedly inhibited by carbon monoxide as well as by anion ligands, namely, potassium cyanide, sodium azide and potassium thiocyanate. The inhibition by anions is in the same order as their strength as nucleophiles, indicating that it is the oxyhaemoglobin and not the ligand-bound deoxy species which undergoes autoxidation. The structure of oxyhaemoglobin is considered to be mainly and determination of the rate of autoxidation with or without using superoxide dismutase and catalase indicates that the initial process of autoxidation takes place by dissociation of to methaemoglobin and superoxide to the extent of 24%. The superoxide thus produced reattacks oxyhaemoglobin to produce further methaemoglobin and hydrogen peroxide. H₂O₂ is a major oxidant of oxyhaemoglobin producing methaemoglobin to the extent of 53%. A tentative mechanism of autoxidation showing the sequence of reactions involving superoxide, H₂O₂ and OH has been presented.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

New water-soluble Mn-porphyrin with catalytic activity for superoxide dismutation and peroxynitrite decomposition

We have synthesized a new water-soluble cationic Mn-porphyrin with catalytic activity for both superoxide dismutation and peroxynitrite decomposition. The resulting Mn-porphyrin also showed higher stability for reactive oxygen species such as hydrogen peroxide and lower cytotoxicity, when compared with a control normal Mn-porphyrin. Furthermore, the new porphyrin recovered the viability of lipopolysaccharide-stimulated macrophage RAW 264.7 cells but the control Mn-porphyrin did not.     

Mechanism of copper-catalyzed autoxidation of cysteine

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant ^·OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.     

Effects of superoxide dismutase on the autoxidation of 1,4-hydroquinone

During autoxidation of 1,4-hydroquinone (H2Q, less than 1 mM) at pH 7.4 and 37 degrees C, stoichiometric amounts of 1,4-benzoquinone (Q) and hydrogen peroxide were formed during the initial reaction. The reaction kinetics showed a significant induction period which was abolished by minute amounts of Q. Hydrogen peroxide and catalase were without effect on the autoxidation process. Transition metals apparently were not involved, since chelators like EDTA, DETAPAC, and desferrioxamine or FeSO4 had no influence on the autoxidation kinetics. Superoxide dismutase (SOD) did not abolish the induction period but dramatically enhanced the autoxidation rate by more than two orders of magnitude. The stimulatory effect was first-order in SOD concentration but showed saturation kinetics. The dependence of Q and hydrogen peroxide formation rates on H2Q concentration shows a biphasic behaviour: dependence on the square at low H2Q, but on the square root at high H2Q concentration. As revealed by calculatory simulations the results can be adequately described by the known reaction rate constants. The reaction starts with the comproportionation of H2Q and Q to yield two semiquinone molecules which autoxidize to give two superoxide radicals and two molecules of Q which enter into a new cycle of comproportionation. Because of unfavourable equilibria the autocatalytic reaction soon comes to steady state, and the further reaction is governed by the rate of superoxide removal. At excess SOD, the comproportionation reaction is rate-limiting, thus explaining the saturation effects of SOD. The experiments do not allow a decision between the two functions of SOD; the conventional action as a superoxide:superoxide oxidoreductase or as a semiquinone:superoxide oxidoreductase. In the latter reaction SOD is thought to be reduced by semiquinone with Q formation. In the second step the reduced enzyme would be re-oxidized by a superoxide radical which is formed during autoxidation of the second semiquinone molecule generated in the comproportionation reaction. From thermodynamic considerations, the latter function of SOD appears to be plausible.     

The nature of melanin pigments from some micro- and macromycetes

New inhibitors of melanin formation by micromycetes Aspergillus carbonarius, Alternaria alternata, and Paecilomyces variotii and basidiomycetes Inonotus obliquus and Phellinus robustus were found. Precursors of melanin pigments were isolated and identified. p-Hydroxybenzoic acid was identified among the products of alkaline degradation of melanin formed by micromycetes, whereas in the case of macromycetes this was protocatechuic acid. Therefore, melanins of the former were found to belong to the dihydronaphthalene group, whereas those of the latter belong to catechols.     

Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone

The autoxidation of 1,4-naphthohydroquinone, in a phosphate, EDTA buffer at pH 7.4, exhibits an autocatalysis whose lag phase becomes more pronounced in the presence of either the Cu,Zn- or the Mn-containing superoxide dismutases. In contrast, the autoxidation of a second aliquot of the hydroquinone, added after complete oxidation of the first, is linear and is accelerated by superoxide dismutase. Catalase or inactive superoxide dismutase were without effect in either situation. These results are explicable in terms of a free radical chain reaction which is initially propagated by O2- and then, as the quinone accumulates, by univalent reduction of the quinone by the hydroquinone. Reduction of the quinone by O2- diminishes the overall rate of oxidation. It is not necessary to postulate catalysis by superoxide dismutase of the reduction of the semiquinone by O2-.