Superoxide dismutases in photosynthetic organisms: absence of the cuprozinc enzyme in eukaryotic algae.

Superoxide dismutases in photosynthetic organisms at different evolutionary levels were characterized using the criterion that the Cu,Zn-enzyme is sensitive to cyanide while the Mn- and Fe-enzymes are insensitive. The effect of the antibody against spinach Cu,Zn-superoxide dismutase was also tested as a means of distinguishing the several forms of the enzyme. Superoxide dismutase activity in extracts from photosynthetic bacteria, prokaryotic algae (blue-green algae), and eukaryotic algae (red, green, and brown algae, diatoms, Euglena, and Charophyta) were insensitive to cyanide and to the antibody, suggesting the presence of the Fe- and/or Mn-enzymes and the absence of the Cu,Zn-enzyme. In contrast, ferns, mosses, and seed plants including gymnosperms and angiosperms contained the Cu,Zn-superoxide dismutase in addition to the cyanidein-sensitive enzyme in soluble or bound form. Although an aerial green alga lacks the Cu,Zn-superoxide dismutase, aquatic angiosperms and ferns, like other land plants, contain this form of superoxide dismutase. Thus the distribution of the Cu,Zn-superoxide dismutase does not reflect the habitat but, rather, the phylogeny of the organism. The relation between the oxygen concentration in the atmosphere and the appearance of various forms of superoxide dismutase during the evolution of photosynthetic organisms is discussed.     

Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice

Mitochondrial respiratory chain dysfunction, impaired intracellular Ca2+ homeostasis and activation of the mitochondrial apoptotic pathway are pathological hallmarks in animal and cellular models of familial amyotrophic lateral sclerosis associated with Cu/Zn-superoxide dismutase mutations. Although intracellular Ca2+ homeostasis is thought to be intimately associated with mitochondrial functions, the temporal and causal correlation between mitochondrial Ca2+ uptake dysfunction and motor neuron death in familial amyotrophic lateral sclerosis remains to be established. We investigated mitochondrial Ca2+ handling in isolated brain, spinal cord and liver of mutant Cu/Zn-superoxide dismutase transgenic mice at different disease stages. In G93A mutant transgenic mice, we found a significant decrease in mitochondrial Ca2+ loading capacity in brain and spinal cord, as compared with age-matched controls, very early on in the course of the disease, long before the onset of motor weakness and massive neuronal death. Ca2+ loading capacity was not significantly changed in liver G93A mitochondria. We also confirmed Ca2+ capacity impairment in spinal cord mitochondria from a different line of mice expressing G85R mutant Cu/Zn-superoxide dismutase. In excitable cells, such as motor neurons, mitochondria play an important role in handling rapid cytosolic Ca2+ transients. Thus, mitochondrial dysfunction and Ca2+-mediated excitotoxicity are likely to be interconnected mechanisms that contribute to neuronal degeneration in familial amyotrophic lateral sclerosis.     

Increase of Cu,Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein.

Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.     

Developmental expression of Cu,Zn superoxide dismutase in Xenopus. Constant level of the enzyme in oogenesis and embryogenesis

cDNA clones for Xenopus laevis Cu,Zn-superoxide dismutase were isolated, sequenced and used as probes to study the expression of the corresponding gene during oogenesis and embryogenesis; Cu,Zn-superoxide dismutase activity was also monitored throughout development. It has been observed that its mRNA is actively synthesized during early oogenesis, reaching a maximum level at stage II, and is utilized through oogenesis. This results in an accumulation of enzyme activity during oocyte growth, paralleling the accumulation of the several other cellular components which are stored in the oocyte to be utilized later on by the developing embryo. In fact, Cu,Zn-superoxide dismutase activity is present at an approximately constant level until late embryonic development, while its mRNA disappears soon after fertilization to be accumulated again only during the last part of embryogenesis. This developmental expression behaviour can be viewed as typical of an housekeeping function and suggests that Cu,Zn-superoxide dismutase activity is a constant need of the cell rather than being subject to regulation by oxygen metabolism.     

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.     

Studies of the Cellular Distribution of Superoxide Disrnutases in Adult and Fetal Rat Tissues

Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cyto-plasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.     

A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.     

Effect of the H, K-ATPase inhibitor, esomeprazole magnesium, on gut total antioxidant capacity in mice

Antioxidant depletion is believed to be a mechanism involved in the pathophysiology of several upper gastrointestinal disorders, and H, K-ATPase inhibitors can alter free radical production by neutrophils. We hypothesized that the H, K-ATPase inhibitor esomeprazole magnesium would decrease gut free radical production with a concomitant increase in gut total antioxidant capacity. A/J mice (n = 10/group) received either vehicle (control) or one of three concentrations of esomeprazole magnesium in vehicle by once-daily gavage for 10 days. Using tissue extracts from stomach and colon, total antioxidant capacity, lipid peroxide levels, and constitutive Cu/Zn-superoxide dismutase were measured using validated assays. There was a dose-related increase in total antioxidant capacity (analysis of variance, P < 0.001) in stomach, but there was no change in the colon. In the assessment of free radical production, there was a trend toward decreased lipid peroxide levels in stomach from mice receiving esomeprazole. In stomach, Cu/Zn-superoxide dismutase activity was increased (ANOVA: p=.03) in mice receiving esomeprazole. In conclusion, gastric total antioxidant capacity and Cu/Zn-superoxide dismutase activity are increased by esomeprazole, and these changes may result in part from decreased free radical production. The present results support the notion that the pharmacological effects of this agent on upper intestinal tissue are more complex than previously thought, and appear to involve both enzymatic and nonenzymatic tissue antioxidants.     

Determination of Manganese Superoxide Dismutase Activity By Direct Spectrophotometry

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O₂^- in a spectrophotometer, is described. Decay of O₂^- generated by KO₂ at pH 9.5, was monitored as the fall in absorbance (A_250nm-A_360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O₂^- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu, Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu, Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.     

Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

Genetic mapping of tomato cDNA clones encoding the chloroplastic and the cytosolic isozymes of superoxide dismutase

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.     

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.     

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H₂O₂ and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H₂O₂-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol. Ekaterina Krumova and Alexander Dolashki equally contributed to this work.     

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.     

Human <Emphasis Type="Italic">CCS</Emphasis> gene: genomic organization and exclusion as a candidate for amyotrophic lateral sclerosis (ALS)

Background  

Amyotrophic lateral sclerosis (ALS) is a progressive lethal disorder of large motor neurons of the spinal cord and brain. In approximately 20% of the familial and 2% of sporadic cases the disease is due to a defect in the gene encoding the cytosolic antioxidant enzyme Cu, Zn-superoxide dismutase (SOD1). The underlying molecular defect is known only in a very small portion of the remaining cases and therefore involvement of other genes is likely. As SOD1 receives copper, essential for its normal function, by the copper chaperone, CCS (Copper Chaperone for SOD), we considered CCS as a potential candidate gene for ALS.     

Nitrated and oxidized products of a single tryptophan residue in human Cu,Zn-superoxide dismutase treated with either peroxynitrite-carbon dioxide or myeloperoxidase-hydrogen peroxide-nitrite

We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.     

Indices of iron and copper status during experimentally induced,marginal zinc deficiency in humans

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.