Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

Copper,zinc superoxide dismutase and H2O2. Effects of bicarbonate on inactivation and oxidations of NADPH and urate,and on consumption of H2O2

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO(3)(-)-dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H(2)O(2), at the active site copper. This bound oxidant then oxidizes HCO(3)(-) to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H(2)O(2). It is now demonstrated that the rates of HCO(3)(-)-dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by approximately 100-fold. Cu,Zn-SOD is also seen to catalyze a HCO(3)(-)-dependent consumption of the H(2)O(2) and that HCO(3)(-) does not protect Cu,Zn-SOD against inactivation by H(2)O(2). A scheme of reactions is offered in explanation of these observations.     

Mechanism of hydrogen peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations

We have reinvestigated the biochemistry of H2O2-induced Cu,Zn-superoxide dismutase (SOD1)-centered radicals, detecting them by immuno-spin trapping. These radicals are involved in H2O2-induced structural and functional damage to SOD1, and their mechanism of generation depends on copper and/or (bi)carbonate (i.e., CO2, CO3(-2), or HCO3-). First, in the absence of DTPA and (bi)carbonate, Cu(II) was partially released and rebound at His, Cys, and Tyr residues in SOD1 with the generation of protein-copper-bound oxidants outside the SOD1 active site by reaction with excess H2O2. These species produced immuno-spin trapping-detectable SOD1-centered radicals associated with H2O2-induced active site ( approximately 5 and approximately 10 kDa fragments) and non-active site (smearing between 3 and 16 kDa) copper-dependent backbone oxidations and subsequent fragmentation of SOD1. Second, in the presence of DTPA, which inhibits H2O2-induced SOD1 non-active site fragmentation, (bi)carbonate scavenged the enzyme-bound oxidant at the SOD1 active site to produce the carbonate radical anion, CO3*-, thus protecting against active site SOD1 fragmentation. CO3*- diffuses and produces side chain oxidations forming DMPO-trappable radical sites outside the enzyme active site. Both mechanisms for generating immuno-spin trapping-detectable SOD1-centered radicals were susceptible to inhibition by cyanide and enhanced at high pH values. In addition, (bi)carbonate enhanced H2O2-induced SOD1 turnover as demonstrated by an enhancement in oxygen evolution and SOD1 inactivation. These results help clarify the free radical chemistry involved in the functional and structural oxidative damage to SOD1 by H2O2 with the intermediacy of copper- and CO3*--mediated oxidations.     

1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.     

Kinetics of the oxidation of reduced Cu,Zn-superoxide dismutase by peroxymonocarbonate

Kinetic evidence is reported for the role of the peroxymonocarbonate, HOOCO(2)(-), as an oxidant for reduced Cu,Zn-superoxide dismutase-Cu(I) (SOD1) during the peroxidase activity of the enzyme. The formation of this reactive oxygen species results from the equilibrium between hydrogen peroxide and bicarbonate. Recently, peroxymonocarbonate has been proposed to be a key substrate for reduced SOD1 and has been shown to oxidize SOD1-Cu(I) to SOD1-Cu(II) much faster than H(2)O(2). We have reinvestigated the kinetics of the reaction between SOD1-Cu(I) and HOOCO(2)(-) by using conventional stopped-flow spectrophotometry and obtained a second-order rate constant of k=1600±100M(-1)s(-1) for SOD1-Cu(I) oxidation by HOOCO(2)(-). Our results demonstrate that peroxymonocarbonate oxidizes SOD1-Cu(I) to SOD1-Cu(II) and is in turn reduced to the carbonate anion radical. It is proposed that the dissociation of His61 from the active site Cu(I) in SOD-Cu(I) contributes to this chemistry by facilitating the binding of larger anions, such as peroxymonocarbonate.     

Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374) and functions as a proton pump. It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria. In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine. Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability. A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted. The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes. Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme. These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations. Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely. Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme. His-333, -334, and -419 residues are attributed to the ligands of CuB. We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model.     

Bicarbonate-enhanced peroxidase activity of Cu,Zn SOD: is the distal oxidant bound or diffusible?

The Cu,Zn SOD catalyzes the bicarbonate-dependent oxidation of a wide range of substrates by H2O2. A mechanism in accord with this activity has been described. It involves the generation of a strong oxidant (Cu(I)O, Cu(II)OH, or Cu(III)) by reaction of the active site Cu with H2O2, followed by oxidation of bicarbonate to CO3-* that in turn diffuses from the active site to oxidize the various substrates in free solution. Recently, an alternative mechanism, entailing firmly bound HCO3- and CO3-*, has been proposed [J. Biol. Chem. 278 (2003) 21032-21039]. We present data supporting the diffusible CO3-* and discuss the properties of this system that can be accommodated in this way and that preclude bound intermediates.     

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).     

Electron transfer from cytochrome b5 to iron and copper complexes

The rates of electron transfer from the tryptic fragment of bovine liver cytochrome b5 to FeIIINTA, FeIIIATP, CuIINTA, CuIIATP, and CuIIHis have been measured by anaerobic stopped-flow techniques. The rates of reduction of the Fe(III) complexes are independent of ionic strength, enhanced at low pH, and slightly inhibited by ZnIINTA. Saturation kinetics are observed with CuIINTA (kappa et = 0.05 s-1, K = 8.6 M-1), CuIIHis (kappa et = 0.2 s-1, K = 2.6 X 10(3) M-1), and CuIIATP (kappa et = 0.6 s-1, K = 4.5 X 10(3) M-1), thereby indicating that binding of Cu(II) to the protein occurs prior to electron transfer. 1H NMR resonances of the three surface histidines and some neighboring residues have been assigned by two-dimensional NMR techniques. NMR titration experiments show unequivocally that CuIINTA binds preferentially at a site near His-26 and Tyr-27.     

An ENDOR study of human and bovine erythrocyte superoxide dismutase: 1H and 14N interactions

Hyperfine interactions (1H and 14N) with the paramagnetic Cu(II)-site obtained from frozen solutions of human and bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) as well as from their derivatives produced by anion binding (N3-, CN-) and by depletion of the Zn(II) site were studied using electron nuclear double resonance (ENDOR) spectroscopy at about 15 K. Both interactions were found to be identical in human and bovine erythrocyte superoxide dismutase. In all compounds, an anisotropic, exchangeable 1H interaction with a nearly constant coupling value (approximately 3 MHz along g perpendicular ) was observed which is due to either histidine NH- or water protons. Other proton interactions were tentatively assigned to H beta 1 of His-44, H delta 2 of His-46 and to H beta 2 of His-44. Depletion of the Zn(II) site did not alter appreciably the pattern of the proton interactions. The 14N couplings of the native specimen indicated equivalent coordination, whereas Zn(II) depletion and CN- addition were found to produce either some or drastic inequivalences, respectively. For N3- addition to either the native or the Zn(II)-depleted sample only minor effects on the respective 14N coupling pattern were observed.     

The mechanism of azide activation of polyphenol oxidase II from tobacco

So far, azide has been consistently reported to act as an inhibitor of metal enzymes, especially copper proteins. The present work shows that azide can also act as an activator of polyphenol oxidase II (PPO II) from tobacco leaves. From the square-wave voltammetry of native PPO II, peroxide-PPO II complex and azide-PPO II complex, the reduction of nitro blue tetrazolium by the enzymes and activation of PPO II by peroxide it follows that the binding of azide to PPO II induces the formation of CuO(2)(2-)Cu in the active site of PPO II from CuO(2)(-)Cu in native PPO II. The reason for azide acting as an activator can be attributed to azide complexing with PPO II, thus inducing the formation of CuO(2)(2-)Cu, which is the active site of the peroxide-PPO II complex in which peroxide plays the role of activator.     

Investigations of the alkaline and acid transitions of umecyanin, a stellacyanin from horseradish roots

The effect of pH on Cu(I) and Cu(II) umecyanin (UCu), a phytocyanin obtained from horseradish roots, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pK(a) value of approximately 9.5-9.8 is observed for the alkaline transition in UCu(II), and this leads to a slightly altered active site structure, as indicated by the changes in the paramagnetic 1H NMR spectrum. Electrochemical studies show that the pK(a) value for this transition in UCu(I) is 9.9. The alkaline transition is caused by the deprotonation of a surface lysine residue, with Lys96 being the most likely candidate. The isotropically shifted resonances in the (1)H NMR spectrum of UCu(II) also shift upon lowering the pH (pK(a) 5.8), and this can be assigned to the protonation of the surface (noncoordinating) His65 residue. This histidine titrates in UCu(I) with a pK(a) of 6.3. The reduction potential of the protein in this range is also dependent on pH, and pK(a) values matching those from NMR, for the two oxidation states of the protein, are obtained. There is no evidence for either of the active site histidines (His44 and His90) titrating in UCu(I) in the pH range studied (down to pH 3.7). Also highlighted in these studies are the remarkable active site similarities between umecyanin and the other phytocyanins which possess an axial Gln ligand.     

1H NMR study on the interaction of cupric ion with ribonuclease A.

The reassignment of the 1H NMR C-2 histidine signals of the bovine pancreatic ribonuclease A has required a revision of the 1H NMR data on the role of the different histidines in their interaction with the Cu2+. The results of our measurements carried out at p2H 5.5 and 7.0 reduce the importance of His-12 as main site of interaction. At p2H 5.5 a very strong binding site involves His-119, while a weaker one contains certainly His-105. On the contrary, at p2H 7.0 the histidines 105 and 119 seem to possess binding constants of the same order of magnitude and in addition they provide stronger ligands for the Cu2+ than His-12. The comparison with X-ray data in the crystal shows numerous analogies. Finally, preliminary results on the competitive inhibition effect between the Cu2+ and 2',3'-cytidine monophosphoric acid are discussed.     

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.     

Effects of aortic coarctation on aortic antioxidant enzymes and NADPH oxidase protein expression

Abdominal aortic coarctation above the renal arteries leads to severe hypertension above the stenotic site and provides a model for simultaneous testing of the effects of increased and decreased pressure and consequently shear stress in the same animal. The effects of increased pressure, per se, on oxidative stress and antioxidant enzyme expression is unknown. We studied the protein expressions of antioxidant enzymes and NADPH oxidase (gp91phox subunit) in the aortic segments above and below the stenosis site in sham-operated control and aortic-banded rats at four weeks postoperatively. Compared with the control group, the banded group showed significant up-regulation of NADPH oxidase, catalase (CAT), Cu/Zn superoxide dismutase (SOD) and Mn SOD protein content in the thoracic aorta. In contrast, Mn SOD, Cu/Zn SOD and NADPH oxidase protein abundance were unchanged in the abdominal aortic segment below the stricture where blood pressure is not elevated, whereas CAT protein abundance was also elevated in the abdominal aorta. No changes were noted for glutathione peroxidase (GPX) protein content either in the thoracic or abdominal aortic segments. Coarctation-induced hypertension is associated with increased aortic CAT, Cu/Zn SOD, Mn SOD and NADPH oxidase protein expression. The up-regulation of NADPH oxidase increases reactive oxygen species (ROS) generation noted in the present study and contributes to inactivation of nitric oxide (NO) as shown previously in this model. Upregulation of antioxidant enzymes may be a compensatory response in the face of elevated pressure and oxidative stress. The normality of protein abundance in the abdominal aorta wherein blood pressure is not elevated points to the role of baromechanical factors, as opposed to circulating humoral factors that were similar in both segments, as a mechanism responsible for increased antioxidant enzyme expression.     

Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.