Manganese-containing Superoxide Dismutases Research Articles

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53.

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

Peroxomanganese complexes as an aid to understanding redox-active manganese enzymes

Over the past 7years, there have been a significant number of studies describing the structural and electronic properties, as well as the chemical reactivity, of synthetic peroxomanganese adducts. Many redox-active manganese enzymes, including manganese-containing superoxide dismutases, extradiol catechol dioxygenases, and ribonucleotide reductases, are proposed to feature peroxomanganese intermediates in their catalytic cycles. The recent efforts to model these intermediates using synthetic complexes have thus provided a strong complement to mechanistic studies of the enzymes. This review provides both a summary and a perspective of work in this area, with an emphasis on the relationship between geometric and electronic structure and chemical reactivity for η(2)-peroxomanganese(III) and η(1)-alkylperoxomanganese(III) adducts.

Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18

Many of the species of Lactobacillus can be considered to be “probiotics” with a variety of benefits, including imparting antioxidative effects to the host. Lactobacillus species evolve different mechanisms to defend themselves against oxygen toxicity, such as superoxide dismutases (SODs), hydroperoxidases and high intracellular levels of metal ions. The SODs provide a cellular defense mechanism against oxidative stress by scavenging O 2 − . Most Lactobacillus species appear to lack a sod gene. To date, only two species of Lactobacillus (Lactobacillus casei and L. sakei) may have sod genes, as evidenced by sequence analysis of the genome, but no experimental verification, including cloning and heterologous expression of the Lactobacillus sod gene, has been reported. It is therefore unknown whether these sod genes can express SOD or are functional. We have PCR-amplified the gene from L. casei Lc18 that encodes the SOD using primers designed according to the genome of L. casei ATCC 334 and ligated it into the vector pET-28a(+) for heterologous expression in Escherichia coli BL21(DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The superoxide radical scavenging activity of the recombinant strain was found to be increased relative to that of the control strain. The SOD was also purified by nickel ion affinity chromatography and found to consist of a single band, as determined by SDS-PAGE analysis, with an activity of 39.97 U/mg. N-terminal amino acid sequence analysis indicated that it may be a manganese-containing SOD. This is the first report of a sod gene from Lactobacillus spp. being expressed in other prokaryotes.

The pH-Dependent Changes of the Enzymic Activity and Spectroscopic Properties of Iron-Substituted Manganese Superoxide Dismutase

Manganese-containing superoxide dismutases (Mn-SODs) and iron-containing superoxide dismutases (Fe-SODS) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine-xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese-containing SOD (Mn-SOD) using Fe-substituted Mn-SOD prepared from apo-Mn-SOD from Serratia marcescens. The Fe3+ content of the Fe-substituted enzyme was 1.71±0.14 mol/mol dimer and the specific activity was 34.8±4.8 units · mg protein-1· mol Fe3+−1· mol subunit−1. Fe-substituted Mn-SOD was found to react with the superoxide anion at pH 8.1 with a second-order rate constant of 6×106M−1 s−1, which is approximately 1% of that of native Mn-SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5×107M−1 s−1) that of native Mn-SOD at pH 6.0 with a pK of 7.0. The visible absorption spectrum and EPR spectrum of Fe-substituted Mn-SOD also showed pH-dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe-substituted Mn-SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd =0.1 mM at pH 6.2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH-dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe-SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe-substituted Mn-SOD and Fe-SOD may cause the different pH dependencies of these changes in each SOD.

Effects of Substrate Analogues and pH on Manganese Superoxide Dismutases

The effect of the substrate analogues azide and fluoride on the manganese(II) zero-field interactions of different manganese-containing superoxide dismutases (SOD) was measured using high-field electron paramagnetic resonance spectroscopy. Two cambialistic types, proteins that are active with manganese or iron, were studied along with two that were only active with iron and another that was only active with manganese. It was found that azide was able to coordinate directly to the pentacoordinated Mn(II) site of only the MnSOD from Escherichia coli and the cambialistic SOD from Rhodobacter capsulatus. The formation of a hexacoordinate azide-bound center was characterized by a large reduction in the Mn(II) zero-field interaction. In contrast, all five SODs were affected by fluoride, but no evidence for hexacoordinate Mn(II) formation was detected. For both azide and fluoride, the extent of binding was no more than 50%, implying either that a second binding site was present or that binding was self-limiting. Only the Mn(II) zero-field interactions of the two SODs that had little or no activity with manganese were found to be significantly affected by pH, the manganese-substituted iron superoxide dismutase from E. coli and the Gly155Thr mutant of the cambialistic SOD from Porphyromonas gingivalis. A model for anion binding and the observed pK involving tyrosine-34 is presented.

Mitochondrial Manganese-Superoxide Dismutase Expression in Ovarian Cancer: ROLE IN CELL PROLIFERATION AND RESPONSE TO OXIDATIVE STRESS

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.

Manganese superoxide dismutase in pathogenic fungi: An issue with pathophysiological and phylogenetic involvements

Manganese-containing superoxide dismutases (MnSODs) are ubiquitous metalloenzymes involved in cell defence against endogenous and exogenous reactive oxygen species. In fungi, using this essential enzyme for phylogenetic analysis of Pneumocystis and Ganoderma genera, and of species selected among Ascomycota, Basidiomycota and Zygomycota, provided interesting results in taxonomy and evolution. The role of mitochondrial and cytosolic MnSODs was explored in some pathogenic Basidiomycota yeasts ( Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. gattii, Malassezia sympodialis), Ascomycota filamentous fungi ( Aspergillus fumigatus), and Ascomycota yeasts ( Candida albicans). MnSOD-based phylogenetic and pathogenic data are confronted in order to evaluate the roles of fungal MnSODs in pathophysiological mechanisms.

A biologically effective fullerene (C 60) derivative with superoxide dismutase mimetic properties

Superoxide, a potentially toxic by-product of cellular metabolism, may contribute to tissue injury in many types of human disease. Here we show that a tris-malonic acid derivative of the fullerene C60 molecule (C3) is capable of removing the biologically important superoxide radical with a rate constant (k(C3)) of 2 x 10(6) mol(-1) s(-1), approximately 100-fold slower than the superoxide dismutases (SOD), a family of enzymes responsible for endogenous dismutation of superoxide. This rate constant is within the range of values reported for several manganese-containing SOD mimetic compounds. The reaction between C3 and superoxide was not via stoichiometric "scavenging," as expected, but through catalytic dismutation of superoxide, indicated by lack of structural modifications to C3, regeneration of oxygen, production of hydrogen peroxide, and absence of EPR-active (paramagnetic) products, all consistent with a catalytic mechanism. A model is proposed in which electron-deficient regions on the C60 sphere work in concert with malonyl groups attached to C3 to electrostatically guide and stabilize superoxide, promoting dismutation. We also found that C3 treatment of Sod2(-/-) mice, which lack expression of mitochondrial manganese superoxide dismutase (MnSOD), increased their life span by 300%. These data, coupled with evidence that C3 localizes to mitochondria, suggest that C3 functionally replaces MnSOD, acting as a biologically effective SOD mimetic.

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.

Protective roles of mitochondrial manganese-containing superoxide dismutase against various stresses in Candida albicans.

Candida albicans contains copper- and zinc-containing superoxide dismutase but also two manganese-containing superoxide dismutases (MnSODs), one in the cytosol and the other in the mitochondria. Among these, the SOD2 gene encoding mitochondrial MnSOD was disrupted and overexpressed to investigate its roles in C. albicans. The null mutant lacking mitochondrial MnSOD was more sensitive than wild-type cells to various stresses, such as redox-cycling agents, heating, ethanol, high concentration of sodium or potassium and 99.9% O2. Interestingly, the sod2/sod2 mutant was rather more resistant to lithium and diamide than the wild-type, whereas overexpression of SOD2 increased susceptibility of C. albicans to these compounds. The inverse effect of mitochondrial MnSOD on lithium toxicity was relieved when the sod2/sod2 and SOD2-overexpressing cells were grown on the synthetic dextrose medium containing sulphur compounds such as methionine, cysteine, glutathione or sulphite, indicating that mitochondrial MnSOD may affect lithium toxicity through sulphur metabolism. Moreover, disruption or overexpression of SOD2 increased or decreased glutathione reductase activity and cyanide-resistant respiration by alternative oxidase, respectively. Taken together, these findings suggest that mitochondrial MnSOD is important for stress responses, lithium toxicity and cyanide-resistant respiration of C. albicans.

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26 173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.

Differential expression of superoxide dismutases containing Ni and Fe/Zn in Streptomyces coelicolor.

Streptomyces coelicolor contains two distinct superoxide dismutase (SOD) activities detected on native PAGE. The level of each changed differently depending on growth media and scarcely responded to paraquat, a superoxide-generating agent. The total SOD activity doubled in late exponential phase compared with that in mid-exponential phase and less than double upon treatment with plumbagin, another superoxide-generating agent. The two SODs from S. coelicolor ATCC 10147 (Müller) strain were purified to near homogeneity. SOD1, a tetramer of 13.4-kDa subunits, was found to be a novel type of SOD containing 0.74 mol nickel/mol subunit as determined by atomic absorption spectroscopy. SOD2, a tetramer of 22.2-kDa subunits, was found to contain 0.36 mol iron and 0.26 mol zinc/mol subunit. The N-terminal amino acid sequences of both SODs were determined. SOD2 is similar to manganese-containing superoxide dismutases (MnSODs) and iron-containing superoxide dismutases (FeSODs) from other organisms, whereas SOD1 is less similar to known SODs but still contains a few conserved amino acids. The effects of metals and chelating agents on the expression of these two SODs were examined. The presence of nickel at micromolar concentrations in growth media induced the expression of SOD1 (nickel-containing superoxide dismutase; NiSOD), whereas the expression of SOD2 (iron/zinc-containing superoxide dismutase; FeZnSOD) was repressed. The changes in SOD activities were positively correlated with the amount of each enzyme as determined by immunoblotting, suggesting that metals do not modulate the activity per se but the amount of each protein.

Peroxisomes as a source of superoxide and hydrogen peroxide in stressed plants.

Superoxide dismutases (SODs) and superoxide radicals in peroxisomes Peroxisomes are subcellular respiratory organelles that contain catalase and H202-producing flavin oxidases as basic enzymatic constituents [1,2]. In recent years, it has become increasingly clear that peroxisomes carry out essential functions in almost all eukaryotic cells [3-51, and an important characteristic of peroxisomes is that they have an essentially oxidative type of metabolism. SODs (EC 1.15.1.1) are a family of metalloenzymes that catalyse the disproportionation of superoxide (O;.) radicals, and they play an important role in protecting cells against the toxic effects of superoxide radicals produced in different cellular loci [6,7]. The presence of SODs in peroxisomes was first demonstrated in plant tissues [8-lo]. These metalloenzymes were found in peroxisomes from pea leaves [8,lO], watermelon cotyledons [9], carnation petals [ 113 , cucumber, cotton and sunflower cotyledons [ 121 and castor bean endosperm [13]. Very recently, the occurrence of SOD in peroxisomes from watermelon cotyledons has been confirmed by immunocytochemical methods (L. M. Sandalio, E. L6pez-Huertas and L. A. del Rio, unpublished work), and SOD has also been found in peroxisomes in human and animal cells. Human fibroblasts and hepatoma cells have been found to contain Cu-Zn-SOD in peroxisomes [ 14,151, and the occurrence of Cu-Zn-SOD has also been demonstrated in rat liver peroxisomes [16,17]. The type of SOD present in peroxisomes varies depending on the tissues origin. In peroxisomes from watermelon cotyledons, two SOD isoenzymes were detected: a Cu-Zn-SOD in the organelle matrix and a manganese-containing SOD bound to the external side of the peroxisomal membrane [18]. In peroxisomes from carnation flowers a Mn-SOD and an Fe-SOD have been found [l l] . Peroxisomes from pea leaves contain only a Mn-SOD, which is located in the peroxisomal matrix [8,10,18]. Recently, two peroxisomal SODs have been purified and charac-

The pH-dependent changes of the enzymic activity and spectroscopic properties of iron-substituted manganese superoxide dismutase. A study on the metal-specific activity of Mn-containing superoxide dismutase.

Manganese-containing superoxide dismutases (Mn-SODs) and iron-containing superoxide dismutases (Fe-SODs) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine-xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese-containing SOD (Mn-SOD) using Fe-substituted Mn-SOD prepared from apo-Mn-SOD from Serratia marcescens. The Fe3+ content of the Fe-substituted enzyme was 1.71 +/- 0.14 mol/mol dimer and the specific activity was 34.8 +/- 4.8 units.mg protein-1.mol Fe3+(-1).mol subunit-1. Fe-substituted Mn-SOD was found to react with the superoxide anion at pH 8.1 with a second-order rate constant of 6 x 10(6) M-1 s-1, which is approximately 1% of that of native Mn-SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5 x 10(7) M-1 s-1) that of native Mn-SOD at pH 6.0 with a pK of 7.0. The visible absorption spectrum and EPR spectrum of Fe-substituted Mn-SOD also showed pH-dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe-substituted Mn-SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd = 0.1 mM at pH 6.2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH-dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe-SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe-substituted Mn-SOD and Fe-SOD may cause the different pH dependencies of these changes in each SOD.

Superoxide dismutase ofCryptococcus neoformans: purification and characterization

We have purified to homogeneity a putative superoxide dismutase of 19.5 kDa from the pathogenic yeast Cryptococcus neoformans by homogenization, isoelectric focusing and gel filtration. The N-terminal amino acid sequence of this protein indicates a significant sequence homology with known manganese-containing superoxide dismutases (Mn-SODs) from various organisms. In addition, the presence of superoxide dismutase activity was confirmed using specific substrate gels which detect this enzyme when nitro-tetrazolium blue reduction is prevented by the photochemical source of superoxide, in the presence of riboflavin when exposed to light. Superoxide dismutase activity was also assayed using cytochrome c. The molecular weight of the native enzyme (on non-denaturing gels) is 80 kDa. The optimum pH for the enzyme is 7.5 and its pi = 6.6. The enzyme was inhibited by sodium dodecyl sulphate, sodium azide, o-phenanthroline, and EDTA, in descending order.

Maize mitochondrial manganese superoxide dismutases are encoded by a differentially expressed multigene family.

We have isolated maize cDNAs encoding three manganese-containing superoxide dismutases (MnSODs) distinct from the one previously reported. Molecular analyses indicate that multiple MnSOD transcripts are encoded by different, though similar, genes in the maize genome. A single MnSOD gene has been reported in all other organisms examined to date. The deduced amino acid sequences show that these maize MnSOD proteins have a mitochondrial transit peptide and that the first 9 amino acids (matrix-targeting sequence) in the transit peptide are conserved. This suggests that all the maize MnSOD proteins are mitochondria-associated isozymes. RNA blot analysis demonstrated that each member of the maize MnSOD multigene family is both spatially and developmentally regulated. One gene, Sod3.3, was predominantly expressed in the embryo late in embryogenesis. Patterns of increased Mn-SOD transcript accumulation are shown to be associated with increased mitochondrial activity during plant growth and development. The influence of mitochondrial metabolism on the expression of the nuclear MnSOD genes is discussed.

Myocardial Antioxidant Defenses During Cardiopulmonary Bypass

In 31 male patients undergoing coronary bypass surgery who underwent different periods of cardioplegic hypothermic arrest, the activities of glutathione peroxidase, glutathione reductase, glutathione transferase, copper/zinc-containing and manganese-containing superoxide dismutases, and catalase were studied in the right atrial myocardium, before and 5 minutes after aortic cross-clamping. The levels of thiobarbituric acid reactive substances (TBARS) and nonproteic thiol compounds (NP-SH) were also assessed. Prolonged ischemia followed by reperfusion induced activation of the major myocardial antioxidant enzymes with marked NP-SH depression and TBARS increase, despite cold crystalloid cardioplegic protection. These changes were significantly related to the duration of the ischemic arrest, suggesting: (1) that reperfusion free radical generation is dependent on the severity of the previous ischemic period; and (2) the occurrence of myocardial oxidative stress during cardiopulmonary bypass.

Iron- and manganese-containing superoxide dismutases from Methylomonas J: identity of the protein moiety and amino acid sequence

Mn-superoxide dismutase (SOD) and Fe-SOD were isolated from Methylomonas J, an aerobic methylotrophic bacterium, grown in methylamine media containing either manganese (Mn-rich medium) or iron (Fe-rich medium), respectively. The specific activity of the Mn-SOD was 2250 units mg-1 (mol of Mn)-1 (mol of dimer)-1, and the metal content of the enzyme was 0.98 mol of Mn and 0.12 mol of Fe per mole of dimer, while those of Fe-SOD were 88.5 units mg-1 (mol of Fe)-1 (mol of dimer)-1 and 1.04 mol of Fe and 0.02 mol of Mn. The electrophoretic mobilities in the presence of sodium dodecyl sulfate, with or without urea, and the chromatographic behavior on an HPLC column using an octadodecyl silicated column and a gel permeation column were identical. Amino acid compositions were practically indistinguishable in both SODs. The enzyme activity was restored by dialysis of an apoprotein obtained from the Mn-enzyme with either manganese sulfate or ferrous ammonium sulfate up to an activity level similar to that for the native Mn-SOD and the native Fe-SOD, respectively. The same result has been reported with the reconstitution using an apoprotein obtained from the Fe-enzyme [Yamakura, F., Matsumoto, T., & Terauchi, K. (1990) Free Radical Res. Commun. (in press)]. These results suggest the possibility that both types of SODs are composed of a single apoprotein synthesized in cells grown in either the Fe-rich medium or the Mn-rich medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The Positive Charge at Position 189 IS Essential for the Catalytic Activity of Iron-and Manganese-Containing Superoxide Dismutases

We have previously shown (C.L. Borders, Jr. et al., (1989) Archives of Biochemistry and Biophysics, 268, 74-80) that the iron-containing (FeSOD) and manganese-containing (MnSOD) superoxide dismutases from Escherichia coli are extensively (greater than 98%) inactivated by treatment with phenylglyoxal, an arginine-specific reagent. Examination of the published primary sequences of these two enzymes shows that Arg-189 is the only conserved arginine. This arginine is also conserved in the three additional FeSODs and seven of the eight additional MnSODs sequenced to date, with the only exception being the MnSOD from Saccharomyces cerevisiae, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the E. coli enzymes gives very little inactivation. However, treatment with low levels of 2,4,6-trinitrobenzenesulfonate (TNBS) and acetic anhydride, two lysine-selective reagents that cause a maximum of 65-80% inactivation of the E. coli SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately 5 lysines per subunit, whereas 6-7 lysines per subunit are acylated with acetic anhydride on complete inactivation. It appears that the positive charge contributed by residue 189, lysine in yeast MnSOD and arginine in all other SODs, may be critical for the catalytic activity of MnSODs and FeSODs.

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis.

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.