Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^- ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^-  species initiate the formation of peroxynitrite (ONOO^- ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^-  decomposition and ONOO^- -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^-  is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

A membrane-associated Mn-superoxide dismutase protects the photosynthetic apparatus and nitrogenase from oxidative damage in the Cyanobacterium Anabaena sp. PCC 7120

We investigated the functions of a membrane-associated manganese superoxide dismutase (MnSOD) of the heterocystous cyanobacterium Anabaena sp. PCC 7120. The gene sodA encoding MnSOD was inactivated by interposon mutagenesis and it was confirmed by Southern hybridization and immunoblotting. The strain A17, lacking sodA, grew more slowly than the wild type, and the difference in growth rates between the two strains became larger with an increase in growth light intensity. More severe inhibition of growth of A17 was observed when the cells were grown in the absence of combined nitrogen. Complementation of A17 with a full copy of the sodA gene restored the wild-type phenotypes. Strain A17 produced more malondialdehyde than did the wild type, especially under high light intensity, indicating more lipid peroxidation in the absence of MnSOD. A17 was also more susceptible to photoinhibition by a high light, and it was shown that both PSII and PSI were more severely damaged by the photoinhibitory light in A17, suggesting that the MnSOD plays important roles in protection of both photosystems. Immunoblotting revealed that the MnSOD was present in vegetative cells and heterocysts. Light greatly stimulated nitrogenase activity in the wild type under both aerobic and anaerobic conditions, but stimulated nitrogenase activity in A17 only slightly in air. The results suggest that reactive oxygen species produced in heterocysts under aerobic conditions cause the inactivation of nitrogenase in the absence of MnSOD.     

The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations

Deinococcus radiodurans (Drad), a bacterium with an extraordinary capacity to tolerate high levels of ionizing radiation, produces only a manganese-containing superoxide dismutase (MnSOD). As MnSOD has been shown to remove superoxide radical with varying efficiency depending upon its cellular origin, a comparison of the Drad MnSOD efficiency with that of both human and Escherichia coli MnSODs was undertaken. Pulse radiolysis studies demonstrate that, under identical ratios of enzyme to superoxide radical, the dismutation efficiencies scaled as Drad MnSOD > E. coli MnSOD > human MnSOD. Further, Drad MnSOD is most effective at high superoxide fluxes found under conditions of high radioactivity. A mechanism is postulated to account for the differences in the activities of the MnSODs that considers the release of peroxide as not always an optimal process.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans.

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.     

Anaerobic induction of ProMn-superoxide dismutase in Escherichia coli

Escherichia coli growing anaerobically respond to NO3- plus PQ2+ with a 20-30-fold induction of an inactive form of the manganese-containing superoxide dismutase (MnSOD). Mutants lacking a functional nitrate reductase fail to show this response. This inactive enzyme can be activated by addition of Mn(II) salts to cell extracts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer. Direct addition of Mn(II) to cell extracts does not result in activation. However, addition of Mn(II) to purified apo-MnSOD results in partial activation. Inactive, reconstitutable MnSOD is induced 13-fold within 15 min of exposure to NO3- plus PQ2+. Western blot analysis revealed a 15-fold increase in immunoreactive MnSOD under these conditions, suggestive of de novo synthesis of this protein. A strain of E. coli bearing a multicopy plasmid carrying the MnSOD gene (sodA) overproduces inactive MnSOD 19-fold compared to the parent strain under anaerobic conditions. Strains of E. coli with an inactivating insertion in the sodA gene do not induce inactive, reconstitutable MnSOD in response to NO3- plus PQ2+ and lack the immunoreactive MnSOD band. These results, in toto, suggest that the inactive protein synthesized under anaerobic conditions in the presence of NO3- plus PQ2+, acting as an electron sink, is a product of the sodA gene and is devoid of activity due to occupation of the manganese site by another metal.     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.     

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.     

Mitochondrial superoxide decreases yeast survival in stationary phase

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.     

NMR method for superoxide dismutase assay in brain and liver homogenates.

A method for copper- and manganese-containing superoxide dismutase (Cu- and MnSOD) assay in tissue homogenates such as liver and brain, based on the measurement of the longitudinal nuclear relaxation time (T1) of F-, has been developed as a preliminary approach to in vivo measurement of these enzymes. The relaxation rate of F-, which increases linearly with the SOD concentration, also depends on the oxidation state of the metal ion present in the active site of the enzyme. The relaxivity values of the oxidized, reduced and turnovering CuSOD were found to be 9.6 x 10(6), much less than 1 x 10(2) and 5.2 x 10(6) M-1 s-1, respectively, while for MnSOD the corresponding values were 2.9 x 10(6), 4.2 x 10(6) and 3.6 x 10(6) M-1 s-1, respectively. These high relaxivity values allow the detection of SODs in brain and liver homogenates where, under aerobic conditions, these enzymes appear in the steady-state. The contribution of the two types of SOD to the F- relaxation rate in the homogenates was measured by addition of either diethyldithiocarbamate or cyanide, both of which selectively inhibit the CuSOD. The comparison between NMR and activity data confirmed the possibility of carrying out accurate and precise measurements of SODs in homogenates by NMR.     

The induction of manganese superoxide dismutase in response to stress in Nicotiana plumbaginifolia.

Superoxide dismutases (SODs) are metalloproteins that catalyse the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined, where it plays a major role in the defence against toxic reduced oxygen species which are generated in many biological oxidations. Here we report the complete primary structure of a plant manganese superoxide dismutase (MnSOD), deduced from a cDNA clone of Nicotiana plumbaginifolia. The plant protein is highly homologous to MnSODs from other organisms and also contains an N-terminal leader sequence resembling a transit peptide for mitochondrial targeting. The location of the mature protein within the mitochondria has been demonstrated by subcellular fractionation experiments. We have analysed the expression profile of this MnSOD and found that it is dramatically induced during stress conditions, most notably in tissue culture as a result of sugar metabolism and also as part of the pathogenesis response of the plant, being induced by ethylene, salicylic acid, and Pseudomonas syringae infection. This induction is always accompanied by an increase in cytochrome oxidase activity, which suggests a specific protective role for MnSOD during conditions of increased mitochondrial respiration.     

Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae.

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.