Induction of superoxide dismutases in Escherichia coli B by metal chelators

The effects of metal salts, chelating agents, and paraquat on the superoxide dismutases (SODs) of Escherichia coli B were explored. Mn(II) increased manganese-containing SOD (MnSOD), whereas Fe(II) increased iron-containing SOD (FeSOD). Chelating agents induced MnSOD but decreased FeSOD and markedly increased the degree of induction seen with Mn(II). Paraquat also exerted a synergistic effect with Mn(II). High levels of MnSOD were achieved in the combined presence of Mn(II), chelating agent, and paraquat. All of these effects were dependent on the presence of oxygen. MnSOD, not ordinarily present in anaerobically grown E. coli cells, was present when the cells were grown anaerobically in the presence of chelating agents. These results are accommodated by a scheme which incorporates autogenous repression by the apoSODs and competition between Fe(II) and Mn(II) for the metal-binding sites of the apoSODs. It is further supposed that oxygenation and intracellular O2- production favor MnSOD production because O2- oxidizes Mn(II) to Mn(III), which competes favorably with Fe(II) for the apoSODs.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Superoxide dismutases: ancient enzymes and new insights

Superoxide dismutases (SODs) catalyze the de toxification of superoxide. SODs therefore acquired great importance as O(2) became prevalent following the evolution of oxygenic photosynthesis. Thus the three forms of SOD provide intriguing insights into the evolution of the organisms and organelles that carry them today. Although ancient organisms employed Fe-dependent SODs, oxidation of the environment made Fe less bio-available, and more dangerous. Indeed, modern lineages make greater use of homologous Mn-dependent SODs. Our studies on the Fe-substituted MnSOD of Escherichia coli, as well as redox tuning in the FeSOD of E. coli shed light on how evolution accommodated differences between Fe and Mn that would affect SOD performance, in SOD proteins whose activity is specific to one or other metal ion.     

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.     

In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fe2-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Superoxide dismutase activity in Pseudomonas putida affects utilization of sugars and growth on root surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

Differences between the manganese- and the iron-containing superoxide dismutases of Escherichia coli detected through sedimentation equilibrium, hydrodynamic, and spectroscopic studies

The genome of Escherichia coli codes for two superoxide dismutases that may contain either iron (FeSOD) or manganese (MnSOD) at the active site. The crystal structures of MnSODs from two bacterial sources (but not E. coli) have been completed, and structural comparisons with the crystal structure of the FeSOD from either E. coli or Pseudomonas ovalis have been made. Despite the low degree (less than 50%) of sequence homology between the E. coli enzymes, the two proteins are suggested to be structurally homologous. Nonetheless, these enzymes exhibit absolute metal cofactor specificity in conferring enzymatic activity to the inactive apoenzyme. This observation is surprising considering the identity of the active site ligands and the similarities in their geometry and surrounding environment. Using analytical ultracentrifugation, we have determined that the solution properties of these two proteins are different. Thus dialysis of FeSOD but not of MnSOD against phosphate buffer in the presence or absence of EDTA caused dissociation of the homodimer. This dissociation appeared to be related to the loss of iron from native FeSOD. Thus, apoFeSOD but not apoMnSOD existed predominantly as a monomer at protein concentrations below 150 micrograms/mL. ApoMnSOD showed no evidence for dissociation under these conditions. Fluorescence data suggest that the tryptophan environments for the two enzymes are also different. The results of these physical measurements lead us to propose that subtle differences, perhaps at the subunit contact faces, exist in the structures of these crystallographically similar proteins.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from Mn-substituted FeSOD and its very high E(m).

Fe and Mn are both entrained to the same chemical reaction in apparently superimposable superoxide dismutase (SOD) proteins. However, neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active. We have proposed that the two SOD proteins must apply very different redox tuning to their respective metal ions and that tuning appropriate for one metal ion results in a reduction potential (E(m)) for the other metal ion that is either too low (Fe) or too high (Mn) [Vance and Miller (1998) J. Am. Chem. Soc. 120, 461-467]. We have demonstrated that this is true for Fe-substituted MnSOD from Escherichia coli and that this metal ion-protein combination retains the ability to reduce but not oxidize superoxide. We now demonstrate that the corollary is also true: Mn-substituted FeSOD [Mn(Fe)SOD] has a very high E(m). Specifically, we have measured the E(m) of E. coli MnSOD to be 290 mV vs NHE. We have generated Mn(Fe)SOD and find that Mn is bound in an environment similar to that of the native (Mn)SOD protein. However, the E(m) is greater than 960 mV vs NHE and much higher than MnSOD's E(m) of 290 mV. We propose that the different tuning stems from different hydrogen bonding between the proteins and a molecule of solvent that is coordinated to the metal ion in both cases. Because a proton is taken up by SOD upon reduction, the protein can exert very strong control over the E(m), by modulating the degree to which coordinated solvent is protonated, in both oxidation states. Thus, coordinated solvent molecules may have widespread significance as "adapters" by which proteins can control the reactivity of bound metal ions.     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location

Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity. It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms. In this work, we have targeted Escherichia coli FeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD. Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress. Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress. This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells.     

The biogenesis and physiological function of chloroplast superoxide dismutases

Iron-superoxide dismutase (FeSOD) and copper/zinc-superoxide dismutase (Cu/ZnSOD) are evolutionarily conserved proteins in higher plant chloroplasts. These enzymes are responsible for the efficient removal of the superoxide formed during photosynthetic electron transport and function in reactive oxygen species metabolism. The availability of copper is a major determinant of Cu/ZnSOD and FeSOD expression. Analysis of the phenotypes of plants that over-express superoxide dismutases in chloroplasts has given support for the proposed roles of these enzymes in reactive oxygen species scavenging. However, over-production of chloroplast superoxide dismutase gives only limited protection to environmental stress and does not result in greatly improved whole plant performance. Surprisingly, plant lines that lack the most abundant Cu/ZnSOD or FeSOD activities perform as well as the wild-type under most conditions tested, indicating that these superoxide dismutases are not limiting to photoprotection or the prevention of oxidative damage. In contrast, a strong defect in chloroplast gene expression and development was seen in plants that lack the two minor FeSOD isoforms, which are expressed predominantly in seedlings and that associate closely with the chloroplast genome. These findings implicate reactive oxygen species metabolism in signaling and emphasize the critical role of sub-cellular superoxide dismutase location. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat.

The manganese-containing isozyme of superoxide dismutase (MnSOD) is synthesized by Escherichia coli only during aerobiosis, in accordance with the fact that superoxide can be formed only in aerobic environments. In contrast, E. coli continues to synthesize the iron-containing isozyme (FeSOD) even in the absence of oxygen. A strain devoid of FeSOD exhibited no deficits during either anaerobic or continuously aerobic growth, but its growth lagged for 2 h during the transition from anaerobiosis to aerobiosis. Complementation of this defect with heterologous SODs established that anaerobic SOD synthesis per se is necessary to permit a smooth transition to aerobiosis. The growth deficit was eliminated by supplementation of the medium with branched-chain amino acids, indicating that the growth interruption was due to the established sensitivity of dihydroxyacid dehydratase to endogenous superoxide. Components of the anaerobic respiratory chain rapidly generated superoxide when exposed to oxygen in vitro, suggesting that this transition may be a period of acute oxidative stress. These results show that facultative bacteria must preemptively synthesize SOD during anaerobiosis in preparation for reaeration. The data suggest that evolution has chosen FeSOD for this function because of the relative availability of iron, in comparison to manganese, during anaerobiosis.     

Evidence that chemical modification of a positively charged residue at position 189 causes the loss of catalytic activity of iron-containing and manganese-containing superoxide dismutases

The Escherichia coli, Bacillus stearothermophilus, and human manganese-containing superoxide dismutases (MnSODs) and the E. coli iron-containing superoxide dismutase (FeSOD) are extensively inactivated by treatment with phenylglyoxal, an arginine-specific reagent. Arg-189, the only conserved arginine in the primary sequences of these four enzymes, is also conserved in the three additional FeSODs and five of the six additional MnSODs sequenced to date. The only exception is Saccharomyces cerevisiae MnSOD, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the other SODs gives very little inactivation. However, treatment with low levels of 2,4,6-trinitrobenzenesulfonate (TNBS) or acetic anhydride, two lysine-selective reagents that cause a maximum of 60-80% inactivation of the other four SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately five lysines per subunit, whereas six to seven acetyl groups per subunit are incorporated on complete inactivation with [14C]-acetic anhydride. It appears that the positive charge contributed by residue 189, lysine in yeast MnSOD and arginine in all other SODs, is critical for the catalytic function of MnSODs and FeSODs.     

Superoxide Dismutase Activity in Pseudomonas putida Affects Utilization of Sugars and Growth on Root Surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

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.     

Circadian Protection against Reactive Oxygen Species Involves Changes in Daily Levels of the Manganese- and Iron-Containing Superoxide Dismutase Isoforms in Lingulodinium polyedrum

A circadian rhythm in the total activity of superoxide dismutase (SOD; EC 1.15.1.1) from the unicellular alga Lingulodinium polyedrum is shown to be attributable to the mitochondrial MnSOD and chloroplastic FeSOD isoforms. Activity gels and labelling with polyclonal antibodies against pure CuZnSOD, MnSOD and FeSOD revealed a distinct circadian pattern in the abundance of the latter two isoforms, with peak values in early photophase 5 times greater than at the dark phase. However, no such changes were detected for the CuZnSOD isoform, which remained at constant levels over the 24-h light/dark cycle. These SOD isoforms might provide protection against damage from photochemically generated oxygen radicals, thus preventing subcellular oxidative stress.     

Circadian Protection against Reactive Oxygen Species Involves Changes in Daily Levels of the Manganese- and Iron-Containing Superoxide Dismutase Isoforms in Lingulodinium polyedrum

A circadian rhythm in the total activity of superoxide dismutase (SOD; EC 1.15.1.1) from the unicellular alga Lingulodinium polyedrum is shown to be attributable to the mitochondrial MnSOD and chloroplastic FeSOD isoforms. Activity gels and labelling with polyclonal antibodies against pure CuZnSOD, MnSOD and FeSOD revealed a distinct circadian pattern in the abundance of the latter two isoforms, with peak values in early photophase 5 times greater than at the dark phase. However, no such changes were detected for the CuZnSOD isoform, which remained at constant levels over the 24-h light/dark cycle. These SOD isoforms might provide protection against damage from photochemically generated oxygen radicals, thus preventing subcellular oxidative stress.