Inhibition of erythrocyte superoxide dismutase by diethyldithiocarbamate also results in oxyhemoglobin-catalyzed glutathione depletion and methemoglobin production

N,N-Diethyldithiocarbamate (DDC), a copper-chelating agent, not only inhibits superoxide dismutase activity in the red cell, but also depletes glutathione and promotes the production of methemoglobin, sulfhemoglobin, and small amounts of lipid peroxidation products. DDC reacts with oxyhemoglobin to yield disulfiram, hydrogen peroxide, and methemoglobin. Disulfiram and hydrogen peroxide both convert GSH to GSSG, while DDC reduces methemoglobin to oxyhemoglobin. Although disulfiram also reacts with the hemoglobin sulfhydryl groups, this reaction does not play a role in the conversion of GSH to GSSG. Other hemoglobin derivatives, ferrous, and ferric ions do not catalyze the oxidation of GSH by DDC. These results support the conclusion that DDC reacts with the super-oxo-ferriheme complex of oxyhemoglobin to generate hydrogen peroxide and disulfiram and that the cyclic conversion of oxyhemoglobin to methemoglobin and DDC and disulfiram results in the net oxidation of GSH. Thus, damage to DDC-treated erythrocytes exposed to a putative superoxide-generating toxin, such as 1,4-naphthoquinone-2-sulfonate, may actually be due to diminished GSH concentration and hemoglobin oxidation rather than to superoxide radicals. Glucose added to the incubation medium of DDC-treated erythrocytes fully prevented glutathione depletion but not the oxidation of oxyhemoglobin to methemoglobin. Several other copper-chelating agents either failed to inhibit the activity of purified superoxide dismutase or when incubated with erythrocytes produced more extensive GSH depletion and hemoglobin oxidation than DDC. It is concluded that the interpretation of results with erythrocytes exposed to copper-chelating agents must consider their effects on GSH and hemoglobin as well as on superoxide dismutase inhibition. Moreover, one must be mindful of the interference by DDC in the analysis of GSH with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of sufficient quantities of metaphosphoric acid to destroy DDC and that contamination of DDC with trace quantities of disulfiram may be a significant problem.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

Dual effects of copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((.)OH). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (.)OH formation. This dCuZnSOD-dependent (.)OH generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-independent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (.)OH generation, implying that Cu may be responsible for dCuZnSOD-induced (.)OH formation. Catalase eliminated ()OH generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (.)OH production. Furthermore, nitric oxide ((.)NO) completely inhibited dCuZnSOD-induced (.)OH radical generation, indicating that (.)NO is an important (.)OH radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (.)OH formation in biological systems.     

Metal sites of copper-zinc superoxide dismutase.

Silver-copper and silver-cobalt proteins have been prepared in which Ag+ resides in the native copper site of superoxide dismutase and either Cu2+ of Co2+ reside in the zinc site. The electron paramagnetic resonance (EPR) spectrum of the copper and the visible absorption spectrum of the cobalt greatly resemble those of either Cu4 of Cu2,Cu2,Co2 proteins, respectively, in which the copper of the native copper sites has been reduced. It was found that, unlike cyanide, azide anion would not perturb the EPR spectrum of Ag2,Cu2 protein. Since azide produces the same perturbation upon the EPR spectrum of native and Cu2 proteins, it must bind to the copper and not the zinc of superoxide dismutase. A model of the metal sites of the enzyme has been fitted to a 3-A electron-density map using an interactive molecular graphics display. The model shows that histidine-61, which appears to bind both copper and zinc, does not lie in the plane of the copper and its three other histidine ligands, but occupies a position intermediate between planar and axial. This feature probably accounts for the rhombicity of the EPR spectrum and the activity of the enzyme.     

Purification and characterization of iron superoxide dismutase and copper-zinc superoxide dismutase from Acanthamoeba castellanii

Two superoxide dismutases (SOD I and SOD II) were purified from Acanthamoeba castellanii and characterized for several biochemical properties. Analysis of the primary structure and inhibition studies revealed that SOD I is iron SOD (Fe-SOD), with a molecular mass of 50 kDa, and SOD II is copper-zinc SOD (Cu,Zn-SOD), with a molecular mass of 38 kDa. Both enzymes have a homodimeric structure consisting of 2 identical subunits, each with a molecular mass of 26 and 19 kDa for SOD I and SOD II, respectively. The isoelectric points of SOD I and SOD II were 6.4 and 3.5, respectively, and there were no isoenzyme forms detected. Both enzymes show a broad optimal pH of 7.0-11.0. Because no differences were observed in the apparent molecular weight of SOD I after addition of the reducing agent 2-mercaptoethanol, the subunits do not appear to be linked covalently by disulfide bonds. However, the subunits of SOD II were covalently linked by intra- and interdisulfide bonds. Western blot analyses showed that the 2 enzymes have different antigenicity. Both enzymes occur as cytoplasmic and detergent-extractable fractions. These enzymes may be potential virulence factors of A. castellanii by acting both as antioxidants and antiinflammatory agents. These enzymes may be attractive targets for chemotherapy and immunodiagnosis of acanthamoebiasis.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Heterodimer formation between superoxide dismutase and its copper chaperone

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers. We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1). According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were detected. Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS. The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.     

Reaction of copper-zinc superoxide dismutase with diethyldithiocarbamate.

Diethyldithiocarbamate reacted with superoxide dismutase from bovine erythrocytes. Changes in both optical and esr spectra, which accompanied this reaction, indicated involvement of the Cu(II). The reaction was accelerated by raising the concentrations of the reactants, elevating the temperature, and lowering the pH, in the range 10.2 to 5.5, and it was independent of the presence of oxygen. During the first phase of this reaction the Cu(II).diethyldithiocarbamate complex remained bound to the enzyme and the catalytic activity did not diminish. There followed a second and slower process which was accompanied by the appearance of colloidal Cu(II).chelate complex and by a loss of activity which could be restored by the addition of CuSO4. All of the observations are accomodated by a model in which 1 diethyldithiocarbamate molecule reacts/copper center, with retention of activity, in Phase I, while a second diethyldithiocarbamate displaces the copper, with a loss of activity, in Phase II.     

Phosphate is an inhibitor of copper-zinc superoxide dismutase

The superoxide dismutase (SOD) activity of bovine copper-zinc superoxide dismutase (Cu,Zn-SOD) in 50 mM Hepes [4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid], pH 7.4, was decreased by approximately 50% when the solution was made 10 mM in phosphate, in spite of the fact that the ionic strength of both solutions was adjusted to be equal. A similar experiment was carried out with bovine Cu,Zn-SOD chemically modified at Arg-141 with phenylglyoxal, which consequently had approximately 20% of the activity of the unmodified protein. (This activity was shown not to be due to residual unmodified protein.) Addition of 10 mM phosphate to solutions of the modified protein caused only a small decrease (less than 5%) in the SOD activity. The presence of phosphate also caused the affinity of Cu,Zn-SOD for binding azide or cyanide anions to be reduced; this effect of phosphate was also much less for the arginine-modified protein. We conclude that the inhibitory effect of phosphate on bovine Cu,Zn-SOD is due primarily to the neutralization of the positive charge on the side chain of Arg-141. The effect of increasing ionic strength on the activities of the native and arginine-modified proteins was also investigated. We found that at high concentrations of phosphate (greater than or equal to 10 mM), the SOD activities of native and arginine-modified Cu,Zn-SOD were inhibited comparably when the ionic strength was increased. This effect is presumably due to the lysine residues near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD)

Haddad, Imad Y., Bedford Nieves-Cruz, and Sadis Matalon.Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD). J. Appl.Physiol. 83(5): 1545-1550, 1997.The efficacy ofantioxidant enzymes to limit oxidant lung injury by instillation withsurfactant mixtures in preterm infants with hyaline membrane disease isunder investigation. However, there is concern that instillation ofproteins in the alveolar space may inactivate pulmonary surfactant. Westudied the effects of bovine copper-zinc superoxide dismutase(CuZn-SOD) on the biophysical properties of two distinct surfactantpreparations. Incubation of calf lung surfactant extract (CLSE, 1 mgphospholipid/ml) and Exosurf (0.1 mg phospholipid/ml) with CuZn-SOD(1-10 mg/ml) prevented the fall of surface tension at minimalbubble radius (T_min) to lowvalues with dynamic compression in a pulsating bubble surfactometer. CuZn-SOD also enhanced the sensitivity to inactivation by albumin, normal human serum, and after treatment with peroxynitrite. The inhibitory effects of CuZn-SOD on CLSE, but not Exosurf, were abolishedat high lipid concentrations (3 mg/ml) and after the addition of humansurfactant protein A (by weight). We conclude that CuZn-SOD mayinterfere with the surface activity of surfactant mixtures, leading todecreased effectiveness of surfactant replacement therapy.

     

A time-resolved fluorescence study of human copper-zinc superoxide dismutase

The intrinsic fluorescence decay of human Cu,Zn superoxide dismutase was measured by frequency-domain techniques. The protein consists of two subunits, each containing one tryptophan and no tyrosine residues. Using a synchrotron radiation source, which allows facile selection of the excitation wavelength, the dependence of the emission decay upon excitation was studied. No significant excitation wavelength effects were found. The two tryptophans contained in the dimer, although fully equivalent and exposed to solvent, showed a fluorescence decay that cannot be described by a single lifetime. Either two lifetimes, or one Lorentzian-shaped continuous distribution of lifetimes, are needed to obtain a good fit. Under identical experimental conditions, control experiments showed that N-acetyltryptophanamide, an analogue of tryptophanyl residues in proteins, decays with a single lifetime. The heterogeneous decay of tryptophan fluorescence in superoxide dismutase is interpreted as due to the presence of static and/or dynamic conformers in the protein that decay with different lifetimes. The two models of discrete lifetimes and continuous distribution of lifetimes are discussed with reference to measurements on holo- and apo-human superoxide dismutase.     

Primary structure of copper-zinc superoxide dismutase from Neurospora crassa

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.     

Immunolocalization of copper-zinc superoxide dismutase. II. Rat

Copper-zinc superoxide dismutase (CuZn SOD) has been localized in formalin-fixed rat tissues. Staining with a modified immunoenzyme bridge technique using the avidin-biotin-peroxidase complex revealed abundant endogenous CuZn SOD in cells that function in transporting ions, either cellularly, as in the case of tracheal, bronchiolar, and colonic epithelial cells, gastric oxyntic cells, and cells lining the salivary ducts and proximal convoluted tubules in the nephron, or intracellularly, as exemplified by skeletal muscle and neurons. Additionally, the enzyme was consistently demonstrable in hepatocytes, endocrine cells of the islets of Langerhans, and the highly membranous oligodendrocytes in the central nervous system. Cellular processes that maintain high ionic gradients appear especially vulnerable to the superoxide anion, thus necessitating the presence of CuZn SOD to scavenge toxic free radicals of oxygen. Comparison of these observations with other immunocytochemical reports indicates that the cellular distribution of CuZn SOD varies between different species.     

Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and formation of histidinyl radical

Human recombinant copper-zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC(50)) was approximately 100 microM at 5 microM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 +/- 1.0) x 10(3) M(-1) s(-1) per monomer at pH 7.5 and 37 degrees C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical.     

Alteration of endogenous glutathione peroxidase, manganese superoxide dismutase, and glutathione transferase activity in cells transfected with a copper-zinc superoxide dismutase expression vector. Explanation for variations in paraquat resistance

Transfection of a human pSV2 (copper-zinc) superoxide dismutase expression vector into murine fibroblasts resulted in stable clones producing increased amounts of copper-zinc superoxide dismutase. A marked increase in endogenous glutathione peroxidase activity (up to 285%) and a smaller increase in glutathione transferase activity (up to 16%) also occurred. Manganese superoxide dismutase activity was decreased in all clones, whereas catalase and NADPH reductase activities were not affected. Alterations in glutathione peroxidase and manganese superoxide dismutase activities correlated with increases in copper-zinc superoxide dismutase activity. Whereas all clones were resistant to paraquat, a direct correlation between copper-zinc superoxide dismutase activity and resistance to paraquat did not exist. In agreement with previous reports clones expressing the highest copper-zinc superoxide dismutase activity did not display the highest resistance to paraquat. However, there was a direct correlation between the increase in glutathione peroxidase activity and paraquat resistance (p less than 0.002).     

Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.     

Function of periplasmic copper-zinc superoxide dismutase in Caulobacter crescentus.

Caulobacter crescentus is one of a small number of bacterial species that contain a periplasmic copper-zinc superoxide dismutase (CuZnSOD). A C. crescentus mutant, with the CuZnSOD gene interrupted by a promoterless cat gene, was constructed and characterized to analyze CuZnSOD function. Periplasmic SOD does not protect against oxyradical damage in the cytosol or play a major role in maintaining the integrity of the cell envelope. Studies of the effect of sodium citrate on plating efficiency suggest that CuZnSOD protects a periplasmic or membrane function(s) requiring magnesium or calcium.