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.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Interaction of superoxide dismutase with phospholipid liposomes. An uptake, spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu2+, Zn2+ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7 degrees C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.     

Interaction of superoxide dismutase with phospholipid liposomes an uptake,spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu²⁺, Zn²⁺ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7°C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.     

Primary structure of human copper-zinc superoxide dismutase. Cysteine - and tryptophan - containing peptides

Analysis of soluble peptides derived from tryptic and chymotryptic digestions of carboxymethylated human superoxide dismutase gives primary structural information for approximately 67% of the protein. Regions so far elucidated appear to be highly homologous to the corresponding bovine enzyme; in particular Cys 6 and the two cysteine residues 55 and 144, which form the intrasubunit disulfide bond of the bovine enzyme, are conserved. A cluster of three substitutions including the fourth cysteine residue unique to the human enzyme has been found in positions 107–109 and may be related to the presence of persulfide groups in the human enzyme. The single tryptophan residue of the human protein is not homologous to the single tyrosine residue of the bovine protein.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

Porcine superoxide dismutase

Summary A cuprozinc superoxide dismutase has been isolated from pig liver. The enzyme is similar to previously described cuprozinc superoxide dismutases in that it is a dimer of about 32 000 molecular weight consisting of approximately two equally sized subunits, and 2 atoms of copper and two atoms of zinc per molecule. It differs, however, from previously described cuprozinc superoxide dismutases because of its higher isoelectric point; pI 6.8 vs 4.9 for bovine enzyme. The diffusion coefficient for the porcine enzyme was determined to be 7.53×10⁻⁷ cm²s⁻¹, while the equivalent spherical hydrodynamic radius was computed as 28.5 ?. The enzyme was observed to undergo self-association with time. Sulfhydryl interaction is postulated to be involved.     

Cu,Zn superoxide dismutase from chicken erythrocytes.

1. Copper, zinc superoxide dismutase (Cu,Zn SOD) has been purified to homogeneity from chicken erythrocytes by anion-exchange, immobilized metal affinity and size exclusion chromatography. 2. Molecular properties (amino acid composition, molecular mass, subunit composition and spec. act.) of the chicken enzyme are similar to those of a bovine erythrocyte Cu,Zn SOD. 3. The chicken and bovine enzymes are immunologically similar since antisera raised against each enzyme are cross-reactive.     

Modelling the three-dimensional structure and electrostatic potential field of the two Cu,Zn superoxide dismutase variants from Xenopus laevis

The crystallographic structure of bovine superoxide dismutase has been used as a template for the graphic reconstruction of the three-dimensional structures of the two Xenopus laevis variants (Schininà, M.E. et al. Arch. Biochem. Biophys. 272:507-515, 1989). In these models the structure-essential residues maintain their position and their structural role, and the interactions between the subunits and the close packing within the beta-barrel are maintained with conservative substitutions and even increased with "aromatic pairs." Because of the same topological motif and surface location of charges, arising from the model building of the two variants with respect to the bovine enzyme, we have calculated the electrostatic potential fields around the models of the two Xenopus laevis variants by numerically solving the Poisson-Boltzmann equation. We show that conservation of a specific space-relationship of charges maintains the potential field pattern already observed in the bovine enzyme, where a negative potential field surrounds the protein surface and specific positive regions wrap up the copper center active site. This electrostatic potential field distribution supports the idea that electrostatic interactions control, like in the bovine enzyme, the mechanism of enzyme-substrate recognition in the Xenopus laevis Cu,Zn superoxide dismutases, suggesting that coordinated mutation of charged residues has occurred in the evolution of this enzyme.     

Zeta-crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts

Exposure to 2,4,6-trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS-PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to zeta-crystallin. Superoxide anions were formed during reduction of TNT by zeta-crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

Some sulfhydryl properties and primary structure of human erythrocyte superoxide dismutase

Human Cu-Zn superoxide dismutase prepared by different methods shows varying properties relevant to its sulfhydryl chemistry. A cysteine residue not found in the analogous bovine enzyme appears to be responsible for its unusual lability. Alkylation of this cysteine results in a marked increase in stability, and this form of the protein may be readily crystallized. The primary structure of the 153 amino acid residues found in the human protein has been determined, and 82% of the residues are identical with those of the bovine enzyme. A significant variation is seen in the portion of those proteins comprising residues 17-36, with eleven changes being noted.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Investigation of zinc-deprived bovine superoxide dismutase

Zinc-deprived bovine superoxide dismutase and its adducts with azide and thiocyanate ions have been investigated through water ¹H NMR relaxation measurements. The affinity constants of the anions for the modified protein have been determined and compared with those for the native enzyme. The results suggest that a histidine different from the bridging one is displaced upon anion coordination.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Stabilization of L-ascorbic acid by superoxide dismutase and catalase

The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

Room temperature electron spin resonance of superoxide dismutase-loaded liposomes and erythrocytes. A direct approach to the interaction of O2- with cells

Human erythrocytes were enriched with bovine superoxide dismutase by fusion with liposomes containing the entrapped enzyme. Liquid solution ESR of intact cells at room temperature was used to measure directly the increase in the superoxide dismutase content. From the spectral characteristics (g-value and hyperfine splitting tensor), the structural integrity of the Cu site of the enzyme was found to be unaffected by the liposome preparation procedure or the incubation with cells. Changes in the ESR signal size were used to test directly the interaction of superoxide with the enzyme entrapped in liposomes or delivered to erythrocytes. It was found that the liposome-entrapped enzyme does not react with externally generated O2-, but once delivered to red blood cells this reaction can take place. This is the first demonstration of O2- -scavenging activity by superoxide dismutase delivered into an intact cell structure and is therefore to be considered as strong evidence for activity of this enzyme under in vivo conditions.     

High density lipoprotein exchange reactions

The Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein. The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively. According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested. The catalytic constants were found to be higher at pH = 7.6 than at pH 10.0 for all the three enzymes. This relative increase was much more pronounced in the case of the porcine enzyme. The KM value at pH = 10.0 was also significantly higher for the porcine enzyme. Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.