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.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.     

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.     

113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase

Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M. (1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase. The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR. Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer. This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate. The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102. The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As) complex. Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate. The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts. Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis. The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.     

Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor

The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR(440-525)] with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

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.     

Structural properties of the zinc site in Cu,Zn-superoxide dismutase; Perturbed angular correlation of gamma ray spectroscopy on the Cu, 111Cd-superoxide dismutase derivative

The Zn(II) site of the dimeric Cu(II),Zn(II)-superoxide dismutase from Saccharomyces cerevisiae has been examined by means of perturbed angular correlation of gamma rays (PAC) on the Cu(II),Cd(II)- and Cu(I),Cd(II)-superoxide dismutase. The PAC spectrum for the Cu(II),Cd(II) enzyme reveals two different, pH independent, coordination geometries for the Cd site. Removal of Cu(II) does not affect the PAC spectrum, which suggests that Cu(II) and Cd(II) do not share a common histidine side chain as ligand. The results are consistent with either an equilibrium between two coordination geometries for Cd(II) in each subunit or a difference in the structure of the Cd(II) site in the two subunits. In contrast, in the reduced enzyme only one structure is present, identical for the two subunits.     

A ferrocyanide charge-transfer complex of bovine superoxide dismutase. Relevance of the zinc imidazolate bond to the redox properties of the enzyme.

Ferrocyanide does not reduce the bovine superoxide dismutase copper at pH 3.0 as it does at higher pH (1,2) but binds at the copper site giving a pink-violet charge-transfer complex. Similar reactions occur between ferrocyanide and Cu(II) bovine carbonic anhydrase or Cu(II) diethylenetriamine near neutral pH. The non-reducibility of superoxide dismutase Cu(II) at low pH suggests that its redox potential depends on the conformation of the site and on the presence of the zinc-imidazolate bond.     

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.     

Oxidation of histidine residues in copper-zinc superoxide dismutase by bicarbonate-stimulated peroxidase and thiol oxidase activities: pulse EPR and NMR studies

In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. 1D NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and Cu(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H(2)O(2)) or cysteine in the absence of bicarbonate anion (HCO(3)(-)), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(II) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO(3)(-) protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 ? away from the Cu(II) site, were completely abolished during both HCO(3)(-)-stimulated peroxidase activity and thiol oxidase activity in the presence of HCO(3)(-) . Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO(3)(-) revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO(3)(-), these modifications were absent. HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities.     

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.     

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identifiedin the liver of rats made copper-deficient by dietary restriction. In homogenates ofsuch livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoreticprofile with respect to the native enzyme. When rat liver extracts were treated withexogenous copper an electrophoretic pattern resembling the native one was observed.Enzyme purified by chromatography on DE-52 resin shows two major components, onecorresponding to genuine, native enzyme and another one, eluting at higher ionicstrength. The latter protein (Fraction II) consists of several isoforms which showthe same characteristics of the native superoxide dismutase as far as immunoreactivityand molecular weight are concerned, but with decreased contents of copper and zinc. Itscatalytic constant, referring to copper content, was 15 times lower than that obtainedfor the native enzyme. Moreover, the catalytic power of purified Fraction II was notregained upon incubation with copper. The occurrence of a superoxide dismutase voidof metals confirms the hypothesis that this protein plays a dual physiological role:in metal metabolism and in superoxide anion dismutation.     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

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.     

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.     

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.     

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.