Multiple replacements of glutamine 143 in human manganese superoxide dismutase: effects on structure, stability, and catalysis

Glutamine 143 in human manganese superoxide dismutase (MnSOD) forms a hydrogen bond with the manganese-bound solvent molecule and is investigated by replacement using site-specific mutagenesis. Crystal structures showed that the replacement of Gln 143 with Ala made no significant change in the overall structure of the mutant enzyme. Two new water molecules in Q143A MnSOD were situated in positions nearly identical with the Oepsilon1 and Nepsilon2 of the replaced Gln 143 side chain and maintained a hydrogen-bonded network connecting the manganese-bound solvent molecule to other residues in the active site. However, their presence could not sustain the stability and activity of the enzyme; the main unfolding transition of Q143A was decreased 16 degrees C and its catalysis decreased 250-fold to k(cat)/K(m) = 3 x 10(6) M(-)(1) s(-)(1), as determined by stopped-flow spectrophotometry and pulse radiolysis. The mutant Q143A MnSOD and other mutants at position 143 showed very low levels of product inhibition and favored Mn(II)SOD in the resting state, whereas the wild type showed strong product inhibition and favored Mn(III)SOD. However, these differences did not affect the rate constant for dissociation of the product-inhibited complex in Q143A MnSOD which was determined from a characteristic absorbance at 420 nm and was comparable in magnitude ( approximately 100 s(-)(1)) to that of the wild-type enzyme. Hence, Gln 143, which is necessary for maximal activity in superoxide dismutation, appears to have no role in stabilization and dissociation of the product-inhibited complex.     

Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.     

Kinetic analysis of product inhibition in human manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) states during the catalyzed disproportionation of O(2)(*-), a catalysis that is limited at micromolar levels of superoxide by a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibition of the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showed significant conformational changes on adjacent residues near the active site, particularly Gln143 and Tyr34 which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate constants for the catalytic dismutation of superoxide. In addition, the rates of formation and dissociation of the product-inhibited complex of these mutants were determined by direct observation of the characteristic visible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway leading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of the inhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of this complex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing proton transfer; however, the dissociation of the complex requires proton transfer to generate HO(2)(-) or H2O2.     

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.     

Manganese superoxide dismutase, MnSOD and its mimics

Increased understanding of the role of mitochondria under physiological and pathological conditions parallels increased exploration of synthetic and natural compounds able to mimic MnSOD - endogenous mitochondrial antioxidant defense essential for the existence of virtually all aerobic organisms from bacteria to humans. This review describes most successful mitochondrially-targeted redox-active compounds, Mn porphyrins and MitoQ(10) in detail, and briefly addresses several other compounds that are either catalysts of O(2)(-) dismutation, or its non-catalytic scavengers, and that reportedly attenuate mitochondrial dysfunction. While not a true catalyst (SOD mimic) of O(2)(-) dismutation, MitoQ(10) oxidizes O(2)(-) to O(2) with a high rate constant. In vivo it is readily reduced to quinol, MitoQH(2), which in turn reduces ONOO(-) to NO(2), producing semiquinone radical that subsequently dismutes to MitoQ(10) and MitoQH(2), completing the "catalytic" cycle. In MitoQ(10), the redox-active unit was coupled via 10-carbon atom alkyl chain to monocationic triphenylphosphonium ion in order to reach the mitochondria. Mn porphyrin-based SOD mimics, however, were designed so that their multiple cationic charge and alkyl chains determine both their remarkable SOD potency and carry them into the mitochondria. Several animal efficacy studies such as skin carcinogenesis and UVB-mediated mtDNA damage, and subcellular distribution studies of Saccharomyces cerevisiae and mouse heart provided unambiguous evidence that Mn porphyrins mimic the site and action of MnSOD, which in turn contributes to their efficacy in numerous in vitro and in vivo models of oxidative stress. Within a class of Mn porphyrins, lipophilic analogs are particularly effective for treating central nervous system injuries where mitochondria play key role. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Manganese superoxide dismutase V16A single-nucleotide polymorphism in the mitochondrial targeting sequence is associated with reduced enzymatic activity in cryopreserved human hepatocytes

Mitochondrial manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, represents a major cellular defense against environmental carcinogens that cause oxidative stress. Two single-nucleotide polymorphisms -9 T>C (V16A in the MnSOD mitochondrial targeting sequence) and -102 C>T (in the SOD2 promoter sequence) modify risk toward various types of malignancies and overall survival. Since little is known about the effects of these polymorphisms on overall enzyme function in normal human tissue, the goal of this study was to evaluate their functional effects in cryopreserved human hepatocytes. Cryopreserved human hepatocytes were genotyped for the MnSOD -9 T>C and -102 C>T polymorphisms by TaqMan allelic discrimination assays. MnSOD catalytic activities were determined in vitro in lysates derived from the hepatocytes. In random samplings of cryopreserved hepatocytes, 16% possessed the -9 T>C and 6% possessed polymorphism on at least one of the two alleles. -9 T>C (V16A) significantly (p < 0.02) reduced MnSOD catalytic activity whereas -102 C>T did not (p > 0.05). The -9 T>C (V16A) polymorphism in the MnSOD mitochondrial targeting sequence significantly reduced MnSOD catalytic activity in cryopreserved hepatocytes, consistent with its reported associations with cancer risk and treatment.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Dismutation of superoxide radicals by ceruloplasmin--details of the mechanism

Like superoxide dismutase (SOD), human ceruloplasmin (Cp) scavenges superoxide anion radicals injected into the solution with the aid a high-voltage generator, hydrogen peroxide being the product of reaction. The O2-/H2O2 ratio is close to 2:1. The dismutase activity of Cp is about 1500 times lower than that of Cu, Zn-SOD isolated from human erythrocytes. The dismutation of O2- accomplished by SOD, "free" copper ions, native Cp or partly copper-depleted Cp, is inhibited with equal efficiency by cyanide. All the copper ions of the multicopper catalytic center of Cp are not essentially required for the dismutation of O2-, since the enzyme depleted of all type 2 Cu2+ and partly of type 1 Cu2+ lost none of its dismutase activity. Type 1 copper ions of Cp seem to play the leading role in the one-electron transfer occurring upon dismutation of O2-.     

The interface behavior and biocatalytic activity of superoxide dismutase at carbon nanotube

The interface behavior and biocatalytic activity of superoxide dismutase (SOD) were studied at carbon nanotube (CNT) surface with cyclic voltammetry (CV). The results show that SOD participates in a rapid exchange with CNT and the process is a single-electron-single-proton process. The electron-transfer coefficient was calculated to be 0.52, the electron-transfer rate constant was 1.4s(-1) and the average surface coverage was measured to be 6.93 x 10(-11)+/-4.2 x 10(-12) mol cm(-2). The bioactive measurements show that SOD keeps its bioactivity at the CNT surface. SOD's remarkable ability to catalyze the dismutation of the superoxide anion (O (2)(-)) has been demonstrated.     

Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase

The geometric and electronic structures of the six-coordinate azide adduct of oxidized manganese superoxide dismutase (Mn3+ SOD) that is formed at low temperatures, LT N3-Mn3+ SOD, has been examined in detail through a combined spectroscopic/computational approach. Electronic absorption, circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD spectroscopies were used to determine electronic transition energies and to obtain an estimate of zero-field splitting parameters for LT N3-Mn3+ SOD. These experimental data were utilized in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization-configuration interaction (INDO/S-CI) and time-dependent density functional theory (TD-DFT) computations to evaluate hypothetical active-site models of LT N3-Mn3+ SOD generated by constrained DFT geometry optimizations. Collectively, our spectroscopic/computational results indicate that N3- binding to Mn3+ SOD at low temperatures promotes neither protonation of the axial solvent ligand nor reorientation of the redox-active molecular orbital, both of which had been previously suggested. Using the same experimentally validated computational approach, models of the product-inhibited form of MnSOD were also developed and evaluated by their relative energies and TD-DFT-computed absorption spectra. On the basis of our computational results as well as previously published kinetic data, we propose that the product-inhibited form of MnSOD is best described as a side-on peroxo-Mn3+ adduct possessing an axial H2O ligand. Notably, attempts to generate a stable hydroperoxo-Mn3+ SOD species by protonation of the proximal O atom of the hydroperoxo ligand resulted in dissociation of HOO- and eventual H+ transfer from Tyr34 to HOO-, generating deprotonated Tyr34 and H2O2. The implications of these results with respect to the mechanism of O2*- dismutation by MnSOD are discussed.     

A facilitated electron transfer of copper--zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper--zinc superoxide dismutase (Cu(2)Zn(2)SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65 +/- 3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k(s) is 1.2 +/- 0.2 s(-1) and the anodic (alpha(a)) and cathodic (alpha(c)) transfer coefficients are 0.39 +/- 0.02 and 0.61 +/- 0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu(2)Zn(2)SOD), its Cu- or Zn-free derivatives (E(2)Zn(2)SOD and Cu(2)E(2)SOD, E designates an empty site) and the SOD reconstituted from E(2)Zn(2)SOD and Cu(2+). The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

Nitroxyl (NO-): a substrate for superoxide dismutase

The interactions of Cu, Zn superoxide dismutase (SOD) with nitroxyl (NO-) and nitric oxide (NO), both of which are thought to be biologically significant, have been studied but remain undefined. Having previously noted that NO- can reduce Cu (II), Zn SOD aerobically, we now report that it also can do so anaerobically and that Cu, Zn SOD can catalyze the elimination of NO(-) in the absence of O2.NO- acts as a reductant of ferricytochrome c anaerobically, but in the presence of O2 causes the oxidation of ferrocytochrome c and NADPH. Equivalent fluxes of NO-, and NO + O2- were able to comparably oxidize NADPH, but the oxidation by NO + O2- was more than fivefold more sensitive to inhibition by Cu, Zn SOD than was the oxidation by NO-. Thus Cu, Zn SOD inhibited NADPH oxidation by NO- by a route independent of catalyzing the dismutation of O2. Plausible mechanisms for those observations are offered and rate constants are estimated.     

Vasomotor responses in MnSOD-deficient mice

MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD(-/-) mice die soon after birth, and MnSOD(+/-) mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD(+/-) mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2alpha (PGF2alpha), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD(+/-) mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2alpha and impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD(+/-) mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD(+/-) mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD(+/-) mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2alpha similarly in aortas of WT and MnSOD(+/-) mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD(+/-) mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.     

Reduction of antigen-induced respiratory abnormalities and airway inflammation in sensitized guinea pigs by a superoxide dismutase mimetic

Reactive oxygen species have been implicated in the pathogenesis of asthma and, in atopic asthmatics, endogenous superoxide dismutase (SOD) enzyme levels are known to decrease. This suggests that replacing a failed endogenous SOD enzyme system with a mimetic of the endogenous enzyme would be beneficial and protective. In this study we demonstrate that removal of superoxide by the SOD mimetic (SODm) M40403 reduces the respiratory and histopathological lung abnormalities due to ovalbumin (OA) aerosol in a model of allergic asthma-like reaction in sensitized guinea pigs. Both respiratory abnormalities and bronchoconstriction in response to OA challenge are nearly absent in na?ve animals, while they sharply became severe in sensitized animals. In addition, OA aerosol induced a reduction of MnSOD activity which was paralleled with bronchiolar lumen reduction, pulmonary air space hyperinflation, mast cell degranulation, eosinophil infiltration, bronchial epithelial cell apoptosis, increase in myeloperoxidase activity, malonyldialdehyde production and 8-hydroxy-2'-deoxyguanosine formation in the lung tissue, as well as elevation of PGD2 in the bronchoalveolar lavage fluid. Treatment with M40403 restored the levels of MnSOD activity and significantly reduced all the above parameters. In summary, our findings support the potential therapeutic use of SOD mimetics in asthma and anaphylactic reactions and account for a critical role for superoxide in acute allergic asthma-like reaction in actively sensitized guinea pig.     

The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations

Deinococcus radiodurans (Drad), a bacterium with an extraordinary capacity to tolerate high levels of ionizing radiation, produces only a manganese-containing superoxide dismutase (MnSOD). As MnSOD has been shown to remove superoxide radical with varying efficiency depending upon its cellular origin, a comparison of the Drad MnSOD efficiency with that of both human and Escherichia coli MnSODs was undertaken. Pulse radiolysis studies demonstrate that, under identical ratios of enzyme to superoxide radical, the dismutation efficiencies scaled as Drad MnSOD > E. coli MnSOD > human MnSOD. Further, Drad MnSOD is most effective at high superoxide fluxes found under conditions of high radioactivity. A mechanism is postulated to account for the differences in the activities of the MnSODs that considers the release of peroxide as not always an optimal process.     

Managing odds in stem cells: insights into the role of mitochondrial antioxidant enzyme MnSOD

Reactive oxygen species (ROS) have been poised at a straddled state of being beneficiary as well detrimental depending on its threshold levels. Maintaining the homeostasis of ROS is imperative for normal cellular physiology, wherein physiological concentrations of ROS are involved in cell signaling and elevated ROS contribute to the development of various diseases. Superoxide dismutases (SODs), enzymes involved in dismutation of superoxide anion to hydrogen peroxide, arrive as a first line of defense when there is perturbation in the homeostasis of ROS. As mitochondria are the main site of superoxide production, among SODs, mitochondrial manganese SOD (MnSOD) is the primary antioxidant enzyme that protects cells from ROS. Most importantly, knockout of MnSOD leads to postnatal lethality and tissue-specific conditional knockout in brain resulted in death of mice, conclusively portraying the essential role of MnSOD in development. Although MnSOD has been extensively discussed with the purview of tumor biology and aging, understanding the crucial role of MnSOD in stem cell physiology is still at its infant stage. Ever increasing progress in stem cell research has recently unveiled the essential role of MnSOD in self-renewal and differentiation of stem cells. In this review, we will conglomerate the current aspects by which MnSOD can contribute to embryonic stem cells’ and adult stem cells’ functions and interpret the necessity of understanding MnSOD for further stem cell mediated applications.     

Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine C gamma into the substrate access channel

Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.     

Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12.

Superoxide dismutases are enzymes that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous superoxide dismutases, one containing manganese (MnSOD) and the other iron (FeSOD). Although E. coli Mn and FeSOD catalyze the dismutation of superoxide with comparable rate constants, it is not known if they are physiologically equivalent in their protection of cellular targets from oxyradical damage. To address this issue, isogenic strains of E. coli containing either Mn or FeSOD encoded on a plasmid and under the control of tac promoter were constructed. SOD specific activity in the Mn and FeSOD strains could be controlled by the concentration of isopropyl beta-thiogalactoside in the medium. The tolerance of these strains to oxidative stress was compared at equal Mn and FeSOD specific activities. Our results indicate that E. coli Mn and FeSOD are not functionally equivalent. The MnSOD is more effective than FeSOD in preventing damage to DNA, while the FeSOD appears to be more effective in protecting a cytoplasmic superoxide-sensitive enzyme. These data are the first demonstration that Mn and FeSOD are adapted to different antioxidant roles in E. coli.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.