1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.     

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.     

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.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.     

X-ray absorption investigation of a unique protein domain able to bind both copper(I) and copper(II) at adjacent sites of the N-terminus of Haemophilus ducreyi Cu,Zn superoxide dismutase

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Isolation and characterization of Cu,Zn superoxide dismutase of the shark Prionace glauca

A Cu,Zn superoxide dismutase was purified for the first time from an elasmobranch species (Prionace glauca) and showed the following differences with respect to other animal superoxide dismutases. The enzyme displays a low isoelectric point. The enzyme activity is unusually independent of ionic strength. The isolated enzyme has 30% of its copper in the reduced state.     

Superoxide dismutase, a study of the electronic properties of the copper and zinc by X-ray absorption spectroscopy

The x-ray absorption for copper and zinc in oxidized and reduced superoxide dismutase, as well as in various model compounds, was studied. Upon reduction of the protein, the added electron affects the copper site almost exclusively, while the zinc remains virtually unchanged. Reduction decreases the charge on the copper atom [toward Cu(I)] and changes the configuration of the copper site so that it becomes less symmetric. An analysis of the copper absorption observed with the oxidized enzyme and a comparison with that for Cu(II)(imid)4 suggests that the copper is not simply ligated to four imidazoles. The addition of H2O2 to superoxide dismutase reduces the copper to Cu(I), while oxygen addition to the peroxide-reduced protein restores the copper to Cu(II).     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase.

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.     

Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase

The reaction of the copper of (Cu,Zn)-superoxide dismutase with diethyldithiocarbamate was studied at pH = 7.4 and the results obtained led to a reaction scheme basically different from the conclusion of a previous study (Misra, H. P. (1979) J. Biol. Chem. 254, 11623-11628). The analysis of optical and ESR spectra at 9 and 35 GHz, at different ligand/protein ratios and reaction times, showed that a ternary diethyldithiocarbamate. Cu(II).protein complex never formed in spectroscopically detectable amounts. The system is described in any condition as the mixture, in variable proportions, of only two components, that is the diethyldithiocarbamate-free (Cu(II) chelate and the copper-depleted protein. The formation of a catalytically active copper-diethyldithiocarbamate intermediate with distinct optical and ESR spectra was also ruled out by kinetic studies, which demonstrated that enzyme inactivation strictly parallels the binding of diethyldithiocarbamate as monitored by optical absorption and ESR. Separation of the copper complex from the protein was obtained for the first time, and the procedure was suitable for rapid preparation of reconstitutable copper-free superoxide dismutase.     

Cyanide binding to Cu, Zn superoxide dismutase. An NMR study of the Cu(II), Co(II) derivative

The Cu,Co superoxide dismutase derivative, in which the native Zn(II) was replaced by Co(II), was investigated by 1H NMR spectroscopy at pH 7.0 in the presence of CN- and N-3. Addition of either anion produced large but remarkably different variations in the position of the histidine proton signals bound to the metal cluster. The resonances of the histidines bound to the copper broadened at low CN- concentrations (6 X10(-5)-16.5 X 10(-3) M KCN, in the presence of 1.5 mM protein) and narrowed again, with changed chemical shifts at [KCN] greater than 10(-2) M. At 7 degrees C two resonances split into two pairs of lines as a function of [CN-]. The temperature dependence of these resonances, in the presence of nonsaturating [CN-], suggests a slow exchange between two forms of the protein-bound copper in the presence of the anion. The apparent activation parameters associated with the interconversion of the two species indicate a local conformational change in the presence of CN-. No evidence of temperature dependence was seen in the spectrum in the presence of N-3, which, on the other hand, was fully removed from the copper by addition of CN-. No evidence was obtained for removal by CN- of a histidine bound to the copper as previously reported for low affinity anions at pH 5.5 (Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monanni, R., and Scozzafava, A. (1985) J. Am. Chem. Soc. 107, 4391-4396). These results indicate that CN- has a unique pattern of binding to the enzyme copper. Since catalytic and structural data indicate that CN- is the only appropriate substrate analogue for the Cu,Zn superoxide dismutase, data from anions with much less affinity may lead to misleading conclusions on the mechanism of anion and substrate binding to the enzyme.     

Toward the engineering of a super efficient enzyme

The catalytic activity of a mutant of Photobacterium leiognathi Cu, Zn superoxide dismutase in which the Glu59 residue, conserved in most bacterial variants of the enzyme, has been replaced by glutamine was investigated by pulse radiolysis. At neutral pH the enzyme was found to have a kcat/KM of 1.0 +/- 0.1 x 10(10) M-1s-1 the highest value ever found for any superoxide dismutase. Brownian dynamics simulation suggests that such a high value is due to an enhanced substrate attraction by the modified electric field distribution. The mutant is also characterized by an active-site widely accessible for the solvent, since iodide is able to interact with the copper atom with an affinity constant twice as high as that found in the native enzyme. The large solvent accessible surface of the copper site together with a favorable distribution of the protein-generated electric field gives rise to the most efficient enzyme ever found with activity close to the diffusion limit.     

113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.     

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.     

Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat

Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.     

Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear Overhauser enhancement of the isotropically shifted 1H-NMR lines of the Cu,Co derivative

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu,Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X-ray crystallography.