Structural and spectroscopic comparison of manganese-containing superoxide dismutases

Predicted secondary structures and optical properties of four manganese-containing superoxide dismutases isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, Escherichia coli and human liver are compared. The structural predictions are further compared with the known crystal structure of the manganese-containing superoxide dismutase from Thermus thermophilus HB8. The secondary structures of the four dismutases are predicted by the methods of Chou and Fasman (Adv. Enzymol. 47 (1978) 45-148), Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) and Lim (J. Mol. Biol. 88 (1974) 873-894). The three models show satisfactory agreement and predict that the enzymes have a mixed alpha-helix and beta-sheet structure, and that they have homologous structures. The former conclusion is also reached from an analysis of the hydrophobic character of the amino-acid sequences of the four proteins according to Kyte and Doolittle (J. Mol. Biol. 157 (1982) 105-132). The calculation of the secondary structure based on the 185-260 nm circular dichroism spectrum of manganese-containing superoxide dismutase from S. cerevisiae reveals that the enzyme consists of 61% alpha-helix, 13% beta-sheet, 11% turn and 8% random coil conformations, which is in good accordance with the prediction based on the amino-acid sequences. Comparison of the 400-700 nm circular dichroism spectra of manganese-containing superoxide dismutase from S. cerevisiae, E. coli and T. thermophilus demonstrates that manganese atoms have homologous coordination in the three enzymes. This investigation based on primary structures and spectral properties indicates that the four dismutases have the same overall structure. Since the structural predictions are in good agreement with the structure found for the manganese-containing superoxide dismutase from T. thermophilus HB8, it can be concluded that this structure is representative for the four enzymes and probably for manganese-containing superoxide dismutases in general.     

Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequence of an iron-containing superoxide dismutase. The iron enzyme is thought to be closely homologous to the manganese-containing superoxide dismutases. The availability of complete amino acid sequence information for four manganese superoxide dismutases and the crystal structures for two iron and two manganese superoxide dismutases prompted us to investigate the degree of homology between the two proteins at various levels. We report that it is not possible to clearly distinguish the two proteins on the basis of their secondary or tertiary structures. It would appear that a small number of single site substitutions are responsible for conferring distinguishing properties between the two proteins. Substitution of glycine 77 and glutamine 154 by a glutamine and an alanine respectively in Photobacterium leiognathi iron superoxide dismutase may distinguish the kinetic and other particular properties of this protein from the manganese protein (and other iron superoxide dismutases). Furthermore the primary structure of both the iron and manganese proteins does not appear to have any homology with any other known amino acid sequence.     

Potentiometric titrations and oxidation-reduction potentials of manganese and copper-zinc superoxide dismutases.

Bovine erythrocyte superoxide dismutase and two manganese-containing superoxide dismutases have been reduced by the indirect coulometric titration method with methylviologen as the mediator-titrant. On the basis of the titration data the manganese-containing superoxide dismutases contain 1 g-atom of metal per mol of enzyme (dimer). E0' = +0.31 V for the enzyme from Escherichia coli which exhibits a complicated pH dependence above neutral pH. The Bacillus stearothermophilus manganese-containing enzyme has an E0' = +0.26 V and delta Em/pH is 50 mV. Bovine erythrocyte superoxide dismutase exhibits anomalous behavior in the coulometric titration curves, which is indicative of two nonequivalent copper centers in the enzyme. Addition K3Fe(CN)6 or K2IrCl6 to the enzyme solution, prior to coulometric titration, indicates that these anions bind preferentially to one of the copper centers.     

A new iron-containing superoxide dismutase from Escherichia coli.

Escherichia coli B, grown under aerobic conditions, contains at least three distinct superoxide dismutases, which can be visualized on polyacrylamide gel electropherograms of crude soluble extracts of the sonically disrupted cells. Of these, the slowest migrating and the fastest migrating, respectively, have previously been isolated and characterized as manganese-containing and iron-containing enzymes. The enzyme form with medium electrophoretic mobility has now been purified to homogeneity. Its molecular weight is approximately 37,000 and it contains 0.8 atoms of iron/molecule and only negligible amounts of manganese. Like other iron-containing superoxide dismutases and unlike the corresponding manganienzymes, it is inactivated by EDTA plus H2O2. Its specific activity is comparable to that of the other superoxide dismutases of E. coli. Two types of subunits could be distinguished upon electrophoresis in the presence of sodium dodecyl sulfate. One of these migrated identically with the subunit obtained from the manganisuperoxide dismutase, while the other similarly appeared identical with the subunit from the ferrisuperoxide dismutase. This newly isolated enzyme thus appears to be a hybrid of the other two forms. In support of this conclusion, we observed that ultrafiltration or storage of the new superoxide dismutase gave rise to the mangani- and ferrienzymes on disc gel electrophoresis or isoelectric focussing.     

Superoxide Dismutase and Oxygen Metabolism in Streptococcus faecalis and Comparisons with Other Organisms

Streptococcus faecalis contains a single superoxide dismutase that has been purified to homogeneity with a 55% yield. This enzyme has a molecular weight of 45,000 and is composed of two subunits of equal size. It contains 1.3 atoms of manganese per molecule. Its amino acid composition was determined and is compared with that for the superoxide dismutases from Escherichia coli, Streptococcus mutans, and Mycobacterium lepraemurium. When used as an antigen in rabbits, the S. faecalis enzyme elicited the formation of a precipitating and inhibiting antibody. This antibody cross-reacted with the superoxide dismutase present in another strain of S. faecalis, but neither inhibited nor precipitated the superoxide dismutases in a wide range of other bacteria, including several other streptococci, such as S. pyogenes, S. pneumoniae, and S. lactis. The inhibiting antibody was used to suppress the superoxide dismutase activity present in cell extracts of S. faecalis and thus allow the demonstration that 17% of the total oxygen consumption by such extracts, in the presence of reduced nicotinamide adenine dinucleotide, was associated with the production of O(2) (-). A variety of bacterial species were surveyed for their content of superoxide dismutases. The iron-containing enzyme was distinguished from the manganese-containing enzyme through the use of H(2)O(2), which inactivates the former more readily than the latter. Some of the bacteria appeared to contain only the iron enzyme, others only the manganese enzyme, and still others both. Indeed, some had multiple, electrophoretically distinct superoxide dismutases in both categories. There was no discernible absolute relationship between the types of superoxide dismutases in a particular organism and their Gram-stain reaction.     

Purification and properties of a manganese-containing superoxide dismutase from Acholeplasma laidlawii

From the prokaryotic microorganism Acholeplasma laidlawii the major manganese-containing superoxide dismutase has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular mass of the enzyme was found to be 41 500 Da. It consists of two subunits of identical size and has an isoelectric point of 6.4. The enzyme contains 0.51 +/- 0.05 atoms of manganese per subunit. Its amino-acid composition and light absorption spectra are presented and compared with Mn- and Fe- containing superoxide dismutases from other prokaryotic organisms.     

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 26173 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.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

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.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus. A kinetic model for the enzyme action.

The enzymic reaction mechanism of a manganese-containing superoxide dismutase from Bacillus stearothermophilus was studied by using pulse radiolysis. During catalysis (pH 8.9; 25 degrees C), changes occurring in the kinetics of substrate disappearance and in the visible absorption of the enzyme at 480 nm established that the simple two-step mechanism found for copper- and iron-containing superoxide dismutases is not involved. At a low ratio (less than 15) of substrate concentration to enzyme concentration the decay of O2--is close to exponetial, whereas at much higher ratios (greater than 100) the observed decay is predominantly zero-order. The simplest interpretation of the results invokes a rapid one-electron oxidation-reduction cycle ('the fast cycle') and, concurrently, a slower reaction giving a form of the enzyme that is essentially unreactive towards O2-- but which undergoes a first-order decay to yield fully active native enzyme ('the slow cycle'). The fast cycle involves the native enzyme EA and a form of the enzyme EB which can be obtained also by treating the form EA with H2O2. Computer calculations made with such a simple model predict behaviour in excellent agreement with the observed results.     

Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures,evolution, and expression

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date. SOD1, or CuZn-SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces. SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. What role(s) these SODs play in both normal and disease states is only slowly beginning to be understood. A molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles. For example, a variety of single amino acid mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis. Knocking out the SOD2 gene in mice results in a lethal cardiomyopathy. A single amino acid mutation in human SOD3 is associated with 10 to 30-fold increases in serum SOD3 levels. As more information is obtained, further insights will be gained.     

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.     

从超氧化物歧化酶的分布和结构看其分子进化

超氧化物歧化酶(SOD)是一种催化超氧化物阴离子自由基发生歧化反应, 生成氧和过氧化氢的金属酶. 按其结合的金属离子, 区分为Fe-SOD, Mn-SOD和CuZn-SOD三种. Fe-SOD主要存在于原核细胞中;Mn-SOD在原核和真核细胞中都存在;CuZn-SOD主要存在于真核细胞中. Fe, Mn-SOD的一级结构, 空间结构及其性质很相似, 来自一个共同的祖先; CuZn-SOD的结构与前两者相差较大, 是在以后的发展中单独进化的.     

Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons

The iron- and manganese-containing superoxide dismutases (Fe/Mn-SOD) share the same chemical function and spatial structure but can be distinguished according to their modes of oligomerization and their metal ion specificity. They appear as homodimers or homotetramers and usually require a specific metal for activity. On the basis of 261 aligned SOD sequences and 12 superimposed x-ray structures, two phenetic trees were constructed, one sequence-based and the other structure-based. Their comparison reveals the imperfect correlation of sequence and structural changes; hyperthermophilicity requires the largest sequence alterations, whereas dimer/tetramer and manganese/iron specificities are induced by the most sizable structural differences within the monomers. A systematic investigation of sequence and structure characteristics conserved in all aligned SOD sequences or in subsets sharing common oligomeric and/or metal specificities was performed. Several residues were identified as guaranteeing the common function and dimeric conformation, others as determining the tetramer formation, and yet others as potentially responsible for metal specificity. Some form cation-pi interactions between an aromatic ring and a fully or partially positively charged group, suggesting that these interactions play a significant role in the structure and function of SOD enzymes. Dimer/tetramer- and iron/manganese-specific fingerprints were derived from the set of conserved residues; they can be used to propose selected residue substitutions in view of the experimental validation of our in silico derived hypotheses.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

Immunocytochemical evidence for a peroxisomal localization of manganese superoxide dismutase in leaf protoplasts from a higher plant

The controversial question of the intracellular location of manganese-containing superoxide dismutase in higher plants was examined under a new experimental approach by applying the more rigorous and specific methods of immunocytochemistry to protoplasts isolated fromPisum sativum L. leaves. Manganese superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from 15 kg of leaves ofPisum sativum L. Rabbits were immunized with the mangano enzyme and the antibody specific for pea manganese superoxide dismutase was purified and found not to contain antigenic sites in common with (i) human manganese superoxide dismutase, (ii) iron superoxide dismutase from eitherEscherichia coli or higher plants, or (iii) plant or animal cuprozinc-superoxide dismutase.Pisum sativum L. manganese superoxide dismutase only appears to have antigenic determinants similar to other manganese superoxide dismutases from higher land plants. The antibody to pea Mn-superoxide dismutase was used to locate the enzyme in protoplasts isolated from young pea leaves by indirect immunofluorescence, and by electron microscopy using the unlabelled antibody peroxidase-antiperoxidase method. Results from immunofluorescence showed that chloroplasts were devoid of specific fluorescence which appeared scattered over the cytosolic spaces among chloroplasts, and demonstrate the absence of manganese superoxide dismutase inside chloroplasts. The metalloenzyme was found to be localized only in peroxisomes, whereas mitochondria, the traditionally accepted site for this enzyme in many eukaryotic organisms, did not show any specific staining. The possible subcellular roles of manganese superoxide dismutase inPisum sativum L. leaves are discussed in the light of its peroxisomal location.     

Chemical modification of iron- and manganese-containing superoxide dismutases from Escherichia coli

The manganese-containing (MnSOD) and iron-containing (FeSOD) superoxide dismutases from Escherichia coli are extensively (greater than 95%) inactivated by treatment with phenylglyoxal. The relatively high concentrations of phenylglyoxal and high pH required for optimal inactivation suggest that inactivation may be due to modification of an arginine with a "normal" elevated pKa, i.e., one not in an active site cavity where the pKa is likely to be lowered because of lower solvent accessibility and decreased polarity of the local environment. Treatment of either enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-hydroxy-5-nitrobenzyl bromide, m-chloroperoxybenzoate, or tetranitromethane causes no inactivation, while 2,4,6-trinitrobenzenesulfonate, N-acetylimidazole, or diethyl pyrocarbonate cause 55-75% inactivation of each enzyme. Failure of hydroxylamine to reverse inactivation by the latter two suggests that in each instance loss of activity is due to lysine modification. The previously reported inactivation of FeSOD by H2O2 was further investigated, and no evidence was found for an affinity mechanism, i.e., a reversible binding of peroxide that precedes inactivation.     

Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases

Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.