Differential binding of anions to the active site of Cu,Zn superoxide dismutase. A study of the Co,Zn enzyme derivative

The reaction of N3- with Co,Zn superoxide dismutase, a good analogue of the native Cu,Zn enzyme, was studied in the presence and absence of phosphate, which is known to perturb the spectroscopic properties of the cobalt chromophore in the Co,Zn enzyme. EPR, NMR, and optical titrations demonstrated the formation of different adducts for N3- depending on the presence of phosphate, at variance with results previously obtained with CN- [3]. This evidence indicates that the mechanism of anion binding to Cu,Zn superoxide dismutase cannot be described on the basis of data obtained with a single type of anions.     

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.     

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.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.     

Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.     

A secreted protein from the human hookworm necator americanus binds selectively to NK cells and induces IFN-gamma production

Parasitic helminths induce chronic infections in their hosts although, with most human helminthiases, protective immunity gradually develops with age or exposure of the host. One exception is infection with the human hookworm, Necator americanus, where virtually no protection ensues over time. Such observations suggest these parasites have developed unique mechanisms to evade host immunity, leading us to investigate the role of the excretory/secretory (ES) products of adult N. americanus in manipulating host immune responses. Specifically, we found that a protein(s) from ES products of adult N. americanus bound selectively to mouse and human NK cells. Moreover, incubation of purified NK cells with N. americanus ES products stimulated the production of augmented (4- to 30-fold) levels of IFN-gamma. This augmentation was dependent on the presence of both IL-2 and IL-12 and was endotoxin-independent. This is the first report of a pathogen protein that binds exclusively to NK cells and the first report of a nematode-derived product that induces abundant levels of cytokines from NK cells. Such an interaction could provide a means of cross-regulating deleterious Th2 immune responses in the host, thereby contributing to the long-term survival of N. americanus.     

Antigenicity of filarial superoxide dismutase in human bancroftian filariasis

Superoxide dismutase activity was measured in different stages of growth of filarial parasites (human and cattle). The activity was almost undetected or very low in microfilarial stage but in adult worms, the enzyme activity was high. The enzyme was characterized to be a Cu/Zn superoxide dismutase. Most of the enzyme activity was associated with a detergent extractable fraction of adult (Setaria) parasite. The enzyme was also detected in thein vitro released products of adult worms. The superoxide dismutase activity was completely inhibited with IgG antibody from chronic filarial patients in contrast to IgG from normal people. Filarial patients particularly have high IgG and IgM antibody levels to purified enzyme. However, individuals from non-filarial regions of Orissa are sero-negative for superoxide dismutase antibodies. Antibody response to superoxide dismutase could thus be used for filarial diagnosis.     

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.     

Identification of superoxide dismutase in rat liver peroxisomes.

In this paper we have investigated whether or not superoxide dismutase is localized in peroxisomes from rat liver. Using an improved method to prepare peroxisomes from clofibrate induced rat livers, we identified superoxide dismutase activity in peroxisomes. This activity was found to be predominantly of the copper-zinc type. The finding of superoxide dismutase activity in peroxisomes makes sense since peroxisomes also contain superoxide generating enzyme activities such as xanthine oxidase.     

Evaluation of a new copper(II)-curcumin complex as superoxide dismutase mimic and its free radical reactions

A mononuclear (1:1) copper complex of curcumin, a phytochemical from turmeric, was synthesized and examined for its superoxide dismutase (SOD) activity. The complex was characterized by elemental analysis, IR, NMR, UV-VIS, EPR, mass spectroscopic methods and TG-DTA, from which it was found that a copper atom is coordinated through the keto-enol group of curcumin along with one acetate group and one water molecule. Cyclic voltammetric studies of the complex showed a reversible Cu(2+)/Cu(+) couple with a potential of 0.402 V vs NHE. The Cu(II)-curcumin complex is soluble in lipids and DMSO, and insoluble in water. It scavenges superoxide radicals with a rate constant of 1.97 x 10(5) M(-1) s(-1) in DMSO determined by stopped-flow spectrometer. Subsequent to the reaction with superoxide radicals, the complex was found to be regenerated completely, indicating catalytic activity in neutralizing superoxide radicals. Complete regeneration of the complex was observed, even when the stoichiometry of superoxide radicals was 10 times more than that of the complex. This was further confirmed by EPR monitoring of superoxide radicals. The SOD mimicking activity of the complex was determined by xanthine/xanthine oxidase assay, from which it has been found that 5 microg of the complex is equivalent to 1 unit of SOD. The complex inhibits radiation-induced lipid peroxidation and shows radical-scavenging ability. It reacts with DPPH radicals with rate constant 10 times less than that of curcumin. Pulse radiolysis-induced one-electron oxidation of the complex by azide radicals in TX-100 micellar solutions produced strongly absorbing ( approximately 500 nm) phenoxyl radicals, indicating that the phenolic moiety of curcumin remained intact on complexation with copper. The results confirm that the new Cu(II)-curcumin complex possesses SOD activity, free radical neutralizing ability, and antioxidant potential. Quantum chemical calculations with density functional theory have been performed to support the experimental observations.     

Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia

The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

Zinc deficiency enhances ozone toxicity in bush beans (Phaseolus vulgaris L. cv. Saxa)

In zinc-deficient bush beans (Phaseolus vulgaris L. cv. Saxa)ozone sensitivity was enhanced compared to plants sufficientlysupplied with this nutrient. This was correlated with reducedlevels of Cu/ZnSOD activity, but was unrelated to effects ofzinc deficiency on transpiration, rates of ethylene formation,ascorbic acid content or levels of MnSOD and ascorbatedependentperoxidase activities. Thus, these results show that detoxificationof superoxide anions by Cu/ZnSOD is important in plant resistanceto ozone. Additionally, this also indicates the in vivo formationof superoxide anions when plants are exposed to ozone. Key words: Ethylene, ozone, ascorbate peroxidase, superoxide dismutase, zinc deficiency     

Involvement of the Copper in the Inhibition of Cu,Zn Superoxide Dismutase Activity at high pH

The alkaline spectroscopic transition of the copper at the active site of Cu, Zn superoxide dismutase has been reexamined by room temperature EPR. in order to correlate it with the inhibition of the enzyme activity at high pH. The EPR transition is governed by a single prototropic equilibrium, with pK values of 11.3 and 11.1 for ox and shark superoxide dismutase. respectively. This result suggests possible contributions of changes of the copper environment to the higher pK of the activity/pH curve.

When Arg141 was cheniically modified by phenylglyoxal treatment of the ox protein. a lower pK value (10.8) was obtained, indicating that Arg141 is involved in the observed modifications of the EPR spectra.     

Effects of the generation of superoxide anion on permeability of liposomes.

Multilamellar liposomes released previously sequestered chromate ions when these artificial membrane structures were suspended in reaction mixtures containing hypoxanthine and xanthine oxidase. Release of chromate was reduced significantly if active superoxide dismutase and/or catalase were added to these reaction mixtures. These findings suggest that superoxide anion and/or related reactive molecules are capable of perturbing lipid bilayers sufficiently to cause leakage of relatively impermeant anions.     

Electrochemical Sensors for Direct Reagentless Measurement of Superoxide Production by Human Neutrophils

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.     

Studies on indoleamine 2,3-dioxygenase. I. Superoxide anion as substrate.

Indoleamine 2,3-dioxygenase purified to apparent homogeneity from rabbit intestine was inhibited by scavengers for superoxide anion such as superoxide dismutase and 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron). On the other hand, beta-carotene and 1,4-diazobicyclo-(2,2,2)-octane, scavengers for singlet oxygen, did not affect the enzyme activity significantly. The degree of inhibition of the dioxygenase by superoxide dismutase preparations from bovine erythrocytes, green peas, spinach leaves, and Escherichia coli paralleled that observed with these dismutase preparations on the aerobic reduction of cytochrome c by xanthine oxidase and its substrate. The pH profiles of the inhibition by dismutase of the dioxygenase and cytochrome c reduction were also similar and the maximal inhibition was observed around pH 10 in both cases. The degree of inhibition was not affected by the concentration of substrate but was a function of the concentration of dismutase. It was inversely related to the concentrations of the dioxygenase and its cofactors, ascorbic acid and methylene blue, both of which were required for maximum activity. Ascorbic acid could be replaced either by xanthine oxidase and its substrate, or by tetrabutylammonium superoxide prepared by electrolytic reduction of molecular oxygen, or by potassium superoxide. When limited amounts of superoxide anion were added to the reaction mixture containing a substrate amount of the dioxygenase, the ratio of the amount of superoxide anion added to that of the product formed was approximately unity both under aerobic and anaerobic conditions. Taken together, these findings indicate that superoxide anion, rather than molecular oxygen, is utilized as substrate by indoleamine 2,3-dioxygenase.