Battles with iron: manganese in oxidative stress protection

The redox-active metal manganese plays a key role in cellular adaptation to oxidative stress. As a cofactor for manganese superoxide dismutase or through formation of non-proteinaceous manganese antioxidants, this metal can combat oxidative damage without deleterious side effects of Fenton chemistry. In either case, the antioxidant properties of manganese are vulnerable to iron. Cellular pools of iron can outcompete manganese for binding to manganese superoxide dismutase, and through Fenton chemistry, iron may counteract the benefits of non-proteinaceous manganese antioxidants. In this minireview, we highlight ways in which cells maximize the efficacy of manganese as an antioxidant in the midst of pro-oxidant iron.     

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

The many highways for intracellular trafficking of metals

Metal ions such as copper and manganese represent a unique problem to living cells in that these ions are not only essential co-factors for metalloproteins, but are also potentially toxic. To aid in the homeostatic balance of essential but toxic metals, cells have evolved with a complex network of metal trafficking pathways. The object of such pathways is two-fold: to prevent accumulation of the metal in the freely reactive form (metal detoxification pathways) and to ensure proper delivery of the ion to target metalloproteins (metal utilization pathways). Much of what we currently know regarding these complex pathways of metal trafficking has emerged from molecular genetic studies in baker's yeast, Saccharomyces cerevisiae. In this review, we shall briefly highlight the current understanding of factors that function in the trafficking and handling of copper, including copper detoxification factors, copper transporters and copper chaperones. In addition, very recent findings on the players involved in manganese trafficking will be presented. The goal is to provide a paradigm for the intracellular handling of metals that may be applied in a more general sense to metals that serve essential functions in biology.Electronic Supplementary Material Supplementary material is available in the online version of this article Abbreviations CTR cell surface transporter - GSH glutathione - MCF mitochondrial carrier family - mito mitochondria - MT metallothionein - SOD superoxide dismutase     

Macrocyclic copper(II) complexes: superoxide scavenging activity, structural studies and cytotoxicity evaluation

Synthetic superoxide dismutase mimetics have emerged as a potential novel class of drugs for the treatment of oxidative stress related diseases. Among these agents, metal complexes with macrocyclic ligands constitute an important group. In this work we synthesized five macrocyclic copper(II) complexes and evaluated their ability to scavenge the superoxide anions generated by the xanthine-xanthine oxidase system. Two different endpoints were used, the nitro blue tetrazolium (NBT) reduction assay (colorimetric method) and the dihydroethidium (DHE) oxidation assay (fluorimetric method). IC(50) values in the low micromolar range were found in four out of five macrocyclic complexes studied, demonstrating their effective ability to scavenge the superoxide anion. The IC(50) values obtained with the NBT assay for the macrocyclic copper(II) complexes, were consistently higher, approximately threefold, than those obtained with the DHE assay. Spectroscopic and electrochemical studies were performed in order to correlate the structural features of the complexes with their superoxide scavenger activity. Cytotoxicity assays were also performed using the MTT method in V79 mammalian cells and we found that the complexes, in the range of concentrations tested in the superoxide scavenging assays were not considerably toxic. In summary, some of the presented macrocyclic copper(II) complexes, specially those with a high stability constant and low IC(50), appear to be promising superoxide scavenger agents, and should be considered for further biological assays.     

Borrelia burgdorferi, a pathogen that lacks iron, encodes manganese-dependent superoxide dismutase essential for resistance to streptonigrin

Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H(2)O(2) and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.     

Antioxidant Systems in Tumour Cells: The Levels of Antioxidant Enzymes, Ferritin, and Total Iron in a Human Hepatoma Cell Line

The human hepatoma cell line Hep 3B, which has the hepatitis B virus genome, shows over 80% decrease of copper/zinc superoxide dismutase activity, over 90% decrease of manganese superoxide dismutase activity, over 70% decrease of catalase activity, absence of glutathione peroxidase and glutathione S-transferase activities, over 270-fold increase of ferritin content and 25-fold increase of total iron compared to normal autopsy liver. These conditions of low antioxidant enzyme activities and iron overload are those which support the accumulation of oxygen free-radicals and DNA damage commonly considered to be carcinogenic mechanisms.     

Antioxidant Systems in Tumour Cells: The Levels of Antioxidant Enzymes,Ferritin, and Total Iron in a Human Hepatoma Cell Line

The human hepatoma cell line Hep 3B, which has the hepatitis B virus genome, shows over 80% decrease of copper/zinc superoxide dismutase activity, over 90% decrease of manganese superoxide dismutase activity, over 70% decrease of catalase activity, absence of glutathione peroxidase and glutathione S-transferase activities, over 270-fold increase of ferritin content and 25-fold increase of total iron compared to normal autopsy liver. These conditions of low antioxidant enzyme activities and iron overload are those which support the accumulation of oxygen free-radicals and DNA damage commonly considered to be carcinogenic mechanisms.     

Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity

Dyshomeostasis of extracellular zinc and copper has been implicated in β-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the β-amyloid precursor protein to release β-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-month-old presenilin 1 heterozygous knock-out (PS1(+/-)) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1(+/-) brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact β-amyloid aggregation through metal ion clearance.     

Mice Transgenic for Copper/Zinc Superoxide Dismutase Exhibit Increased Markers of Biogenic Amine Function

Abstract: Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two- to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over-expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.     

The overexpression of major antioxidant enzymes does not extend the lifespan of mice

We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.     

Activation of superoxide dismutases: putting the metal to the pedal

Superoxide dismutases (SOD) are important anti-oxidant enzymes that guard against superoxide toxicity. Various SOD enzymes have been characterized that employ either a copper, manganese, iron or nickel co-factor to carry out the disproportionation of superoxide. This review focuses on the copper and manganese forms, with particular emphasis on how the metal is inserted in vivo into the active site of SOD. Copper and manganese SODs diverge greatly in sequence and also in the metal insertion process. The intracellular copper SODs of eukaryotes (SOD1) can obtain copper post-translationally, by way of interactions with the CCS copper chaperone. CCS also oxidizes an intrasubunit disulfide in SOD1. Adventitious oxidation of the disulfide can lead to gross misfolding of immature forms of SOD1, particularly with SOD1 mutants linked to amyotrophic lateral sclerosis. In the case of mitochondrial MnSOD of eukaryotes (SOD2), metal insertion cannot occur post-translationally, but requires new synthesis and mitochondrial import of the SOD2 polypeptide. SOD2 can also bind iron in vivo, but is inactive with iron. Such metal ion mis-incorporation with SOD2 can become prevalent upon disruption of mitochondrial metal homeostasis. Accurate and regulated metallation of copper and manganese SOD molecules is vital to cell survival in an oxygenated environment.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Purification and characterization of Cu-Zn superoxide dismutases from mungbean (Vigna radiata) seedlings

Mungbean contains three isoenzymes of superoxide dismutase designated isoenzyme I, II and III. The two cytosolic superoxide dismutases (I and II) were purified to homogeneity by ammonium sulphate fractionation, ion exchange chromatography on diethylaminoethyl cellulose, gel filtration and preparative polyacrylamide.gel electrophoresis. The molecular weights of isoenzyme I and isoenzyme II were determined to be 33,000 and 31,600 respectively. The subunit molecular weight was approximately 16,000 indicating that the isoenzymes contained two identical subunits. The ultra-violet absorption spectra revealed a maximum at 258–264 nm for the two isoenzymes. Superoxide dismutase I and II were inhibited to different extents by metal chelators. Isoenzyme I was more sensitive to inhibition by cyanide and azide, while isoenzyme II was more susceptible to inhibition by diethyldithiocarbamate ando-phenanthroline. Both the isoenzymes exhibited similar denaturation profiles with heat, guanidinium chloride and urea. The denaturation with urea and guanidinium chloride was reversible. The two copper-zinc enzymes were more stable towards thermal inactivation compared to manganese and iron superoxide dismutases from other sources. The results indicate that the two isoenzymes differ from each other only with respect to charge and sensitivity towards metal chelators.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

过氧化氢对铜锌超氧化物歧化酶活性及某些理化性质的影响

用H_2O_2作用于牦牛红细胞铜锌超氧化物歧化酶。观察到酶活性随H_2O_2浓度升高及作用时间增加而下降;酶分子连接的铜和锌有所丢失;PAGE图谱中三条酶活性带成为四条酶活性带;等电点下降;680nm处表征二价铜光学性质的可见光吸收减弱;紫外吸收增加,表现为增色效应;内源性荧光减弱;在含有3.0mol/LKCl的PH3.8—5.4琥珀酸缓冲液中溶解度下降;酶对胰蛋白酶水解的敏感性增加。     

Determination of superoxide dismutase-like activity in some divalent metal saccharinates

The superoxide-dismutase-like activity of a series of divalent metal saccharinates of general stoichiometry [M^II(Sac)₂(H₂O)₄]·2H₂O (with M^II=Mn,Fe,Co,Ni,Cu,Zn) has been investigated using the nitroblue tetrazolium O ₂ ⁻ reduction assay. The results show that all these complexes possess the capability to dismutate the superoxide anion generated in the xanthine/xanthine oxidase system. Interestingly, the greatest activity is shown by the corresponding copper complex. The results are discussed and compared with those obtained for native superoxide dismutase, which was tested under the same experimental conditions. Dedicated to Prof. Pedro J. Aymonino on the occasion of his 65^th birthday.     

A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.     

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.