Superoxide radical production during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate.

1-Methyl-4-phenyl-2,3-dihydropyridinium perchlorate (MPDP+), an intermediate in the metabolism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected by their ability to reduce ferricytochrome c. Superoxide dismutase inhibited this reduction in a dose-dependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP+ but also on the pH of the system. Thus, the rate of autoxidation of MPDP+ and the sensitivity of this autoxidation to superoxide dismutase-inhibitable ferricytochrome c reduction were both augmented, as the pH was raised from 7.0 to 10.5. The rate constant (Kc) for the reaction of superoxide radical with ferricytochrome c to form ferricytochrome c was found to be 3.48 x 10(5) M-1 s-1. The rate constant (KMPDP+) for the reaction of MPDP+ with ferricytochrome3+ c was found to be only 4.86 M-1 s-1. These results, in conjunction with complexities in the kinetics, lead to the proposal that autoxidation of MPDP+ proceeds by at least two distinct pathways, one of which involves the production of superoxide radicals and hence is inhibitable by superoxide dismutase. It is possible that the free radicals so generated could induce oxidative injury which may be central to the MPTP/MPDP(+)-induced neuropathy.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.     

Superoxide anion production by the autoxidation of cytochrome P450cam

Chemiluminescence occurs on autoxidation of oxygenated ferrous cytochrome P450_cam and is abolished by reagents that scavenge free radicals, by superoxide dismutase and singlet oxygen quenchers. We attribute the chemiluminescence to the decay of an excited singlet oxygen which arises from a superoxide anion radical precursor.     

Superoxide radical inhibits catalase

Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these was rapidly established and could be as rapidly reversed by the addition of superoxide dismutase. The second developed slowly and was reversed by ethanol, but not by superoxide dismutase. The rapid inhibition was probably due to conversion of catalase to the ferrooxy state (compound III), while the slow inhibition was due to conversion to the ferryl state (compound II). Since neither compound III nor compound II occurs in the catalatic reaction pathway, they are inactive. This inhibition of catalase by O2- provides the basis for a synergism between superoxide dismutase and catalase. Such synergisms have been observed in vitro and may be significant in vivo.     

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].     

Superoxide dismutase and free radical pathology

Different defense systems against oxidative damage leading to pathological conditions are described. The superoxide radical plays a primary role in initiating and sustaining biological damage and is responsible for the production of other free radicals and lipoperoxides. Certain pathologies are associated with these events such as post-irradiation necrosis, or the Spanish Toxic Oil Syndrome. Superoxide dismutase has been used clinically with considerable success, particularly the liposomal form, to treat various diseases in which it has been shown that the superoxide radical plays an important role. Although the mechanism of the enzymic reaction catalysed by superoxide dismutase is now well defined, a complete explanation of the anti-inflammatory properties in vivo of the enzyme has not yet been established.     

Superoxide anion radical scavenging property of catecholamines

The direct effect of the four catecholamines (adrenaline, noradrenaline, dopamine and isoproterenol) on superoxide anion radicals () was investigated. The reaction between 18‐crown‐6‐ether and potassium superoxide in dimethylsulfoxide was used as a source of . The reactivity of catecholamines with was examined using chemiluminescence, reduction of nitroblue tetrazolium and electron paramagnetic resonance spin‐trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide was included as the spin trap. The results showed that the four catecholamines were effective and efficient in inhibiting chemiluminescence accompanying the potassium superoxide/18‐crown‐6‐ether system in a dose‐dependent manner over the range 0.05–2 mm in the following order: adrenaline > noradrenaline > dopamine > isoproterenol, with, IC₅₀ = 0.15 ± 0.02 mm 0.21 ± 0.03 mm , 0.27 ± 0.03 mm and 0.50 ± 0.04 mm , respectively. The catecholamines examined also exhibited a strong scavenging effect towards when evaluated this property by the inhibition of nitroblue tetrazolium reduction (56–73% at 1 m concentration). A very similar capacity of scavenging was monitored in the 5,5‐dimethyl‐1‐pyrroline‐N‐oxide spin‐trapping assay. The results suggest that catecholamines tested may involve a direct effect on scavenging radicals. Copyright © 2013 John Wiley & Sons, Ltd.     

The role of superoxide radical in the autoxidation of cytochrome c.

The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.     

Propionate-3-nitronate oxidase from Penicillium atrovenetum is a flavoprotein which initiates the autoxidation of its substrate by O2

On the fifth day following inoculation into an unstirred liquid surface culture, Penicillium atrovenetum abruptly, and reproducibly, secretes large quantities (2 g/liter) of the toxic antibiotic 3-nitropropionate into the medium. Concomitantly and with the same time course, crude extracts of the fungus acquire the ability to catalyze the oxidation of 3-nitropropionate by O2. We purified this activity some 300-fold to homogeneity and find it to be a soluble, dimeric (Mr = 73,000) flavoprotein oxidase having FMN as prosthetic group with lambda max = 363 and 433 nm. The preferred substrates are propionate-3-nitronate (3-NP-2) and O2 while the reaction products are malonate semialdehyde, NO2-, NO3-, O2-., and H2O2. Of 13 nitronates tested only butyrate-4-nitronate is more than 2% as reactive as 3-NP-2. 3-NP-2 (0.1 mM) rapidly reduces E-FMN anaerobically to E-FMNH., the flavin semiquinone (t1/2 less than 5 s), but reduces E-FMNH. to the fully reduced enzyme (E-FMNH2) very slowly (t1/2 approximately 900 s). The steady state turnover number with 0.1 mM 3-NP-2 and infinite O2 is 350 s-1. Therefore, the enzyme must oscillate almost exclusively between E-FMN and E-FMNH. during aerobic turnover. (Formula: see text). The complicated and non-integral reaction stoichiometry provides further support for this free radical mechanism. Each mole of 3-NP-. generated enzymatically initiates the nonenzymatic autoxidation of at least 2.2 mol of 3-NP-2 through a free radical chain reaction. An appropriate name for the newly characterized enzyme is propionate-3-nitronate oxidase.     

The effect of fixed valence metal ions on the free radical process of epinephrine autoxidation

The physiologically active metal ions with fixed valence Ca²⁺ and Mg²⁺ were shown to accelerate epinephrine autoxidation at an alkaline pH, which proceeds via the known quinoid pathway and is accompanied by the generation of reactive oxygen species. A higher efficiency was observed for Ca²⁺ ions compared with Mg²⁺ ions. The activation of epinephrine autoxidation was evident from a decrease in the time of the initiation of the chain reaction to begin (i.e., the reaction lag) and an increase in the rate of both oxygen uptake and the formation of adrenochrome. Based on the observed effects, Ca²⁺ and Mg²⁺ cations were assumed to have the potential to play a role in the free radical processes that are associated with redox reactions in the cell and can also modulate the effect of epinephrine in the organism its oxidation via the quinoid pathway.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Generation of superoxide radical, hydrogen peroxide and hydroxyl radical during the autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine

The autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at neutral pH has been shown to generate superoxide radical and hydrogen peroxide. The rate of formation of these species was increased in the presence of certain iron and copper compounds; in the presence of iron complexed with EDTA, hydroxyl radical was also produced. Hydrogen peroxide was detected in erythrocytes incubated with TMPD and these cells suffered oxidative damage as reflected by methaemoglobin formation and glutathione depletion; the one-electron oxidation product of TMPD, Wurster's Blue, was equally effective in producing such changes in erythrocytes. N-Methylated p-phenylenediamines are known to be mutagenic and myotoxic, and it is suggested that 'active oxygen' species may be involved in the initiation of these harmful effects.     

Superoxide radical as electron donor for oxidative phosphorylation of ADP

When isolated rat heart mitochondria are subject to xanthine/xanthine oxidase generated free radicals, nmol quantities of ADP are phosphorylated to ATP. This effect is proportional to xanthine oxidase concentration, and is relatively independent of ADP concentration. Exogenous superoxide dismutase partially suppresses the phosphorylation. Micromolar concentrations of iron salts completely eliminate the phosphorylation. Catalase has no effect. The likely electron source, then, is superoxide radicals. The reduced minus oxidised spectra of superoxide-bombarded mitochondria show that superoxide enters the electron transport chain by reducing cytochrome c and complex IV. Mitochondria retain their ability to phosphorylate ADP in more traditional ways under the experimental conditions described. Superoxide under physiological conditions in vivo may be a source of electrons for the oxidative phosphorylation of ADP.     

Steady-state kinetics of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical.

Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 +/- 0.13 x 10(4) M-1s-1 and 9.26 +/- 0.58 x 10(4) M-1s-1 at 25 degrees C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated.