Superoxide Dismutase and Catalase*

Superoxide Dismutase and Catalase*

4.12 Superoxide Dismutase and Catalase J F Turrens, University of South Alabama, Mobile, AL, USA ª 2010 Elsevier Ltd. All rights reserved. This articl...

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4.12 Superoxide Dismutase and Catalase J F Turrens, University of South Alabama, Mobile, AL, USA ª 2010 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by J M McCord, J C Marecki, J A Handler and R G Thurman, Volume 3, pp 199–228, ª 1997, Elsevier Ltd.

4.12.1 4.12.2 4.12.2.1 4.12.2.2 4.12.3 4.12.3.1 4.12.4 4.12.5 References

Introduction Superoxide Dismutases (SOD, EC 1.15.1.1) Structure Regulation of Gene Expression Catalases (EC 1.11.1.6) Structures Clinical Scenarios Related to Changes in SOD and/or Catalase Activity Future Directions

220 222 222 223 223 224 224 226 226

Glossary antioxidant enzymes A group of enzymes that evolved to eliminate directly or indirectly potentially damaging oxidants generated in various metabolic reactions. Examples of antioxidant enzymes that directly eliminate ROS include superoxide dismutases, catalases, and various peroxidases. Other enzymes usually included in this group are accessory enzymes such as glutathione reductase, glucose-6-phosphate dehydrogenase, thioredoxins reductase, and other enzymes involved in the production of cosubstrates needed in these reactions. antioxidants Also known as small molecular weight antioxidants, they are a group of compounds that react directly with free radicals preventing the propagation of chain reactions causing oxidation of biomolecules. Examples include vitamin E, vitamin C, reduced glutathione, coenzyme Q, etc. dismutation A chemical reaction in which one species acts both as an oxidant and as a reductant. lipid peroxidation Chain reaction started by free radicals and propagated by oxygen, which leads to oxidation of unsaturated fatty acids. The process starts with a radical (e.g., hydroxyl radical) abstracting a H atom from a lipid, to form a lipid radical. This secondary radical binds molecular oxygen to make a peroxyl radical, which in turn abstracts a H atom from another lipid, to start the cycle again.

oxidative stress A phrase used to group a variety of oxidative reactions, usually started by free radicals and propagated by molecular oxygen, which results in the oxidation of lipids, proteins, and nucleic acids. oxygen free radicals See reactive oxygen species. peroxidases A group of enzymes required for the elimination of hydrogen peroxide and/or other hydroperoxides. peroxides A group of molecules in which two oxygen atoms are bound together through a single bond. The simplest peroxide is hydrogen peroxide (H–O–O–H). radical A molecular species containing an unpaired electron. Many of these species are highly reactive, and stabilize the orbital containing a free electron by either losing the electron or incorporating a second electron from another molecule. In general, bigger molecules are able to stabilize a radical through resonance while smaller radicals tend to be more reactive. The molecule of oxygen in its ground state (triplet) has two unpaired electrons and therefore is usually referred to as a ‘bi-radical.’ reactive nitrogen species (RNS) A group of reactive molecules derived from nitric oxide (?NO). Some of these are strong nitrosylating agents of proteins and lipids. One of the best studied products in these reactions is the formation of nitrotyrosine derivatives in proteins.

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220 Superoxide Dismutase and Catalase

reactive oxygen species (ROS) Expression used to define a variety of oxidants derived from molecular oxygen. Many of these species result from the partial reduction of oxygen including superoxide anion, hydrogen peroxide, hydroxyl radical, alcoxyl radical, and peroxyl radical. Other

nonreduced ROS include singlet oxygen and ozone. Originally, the phrase ‘free radicals’ was used to describe all these species, but since not all of them are ‘radicals’ the term ROS has become more popular and is obviously more appropriate.

Abbreviations

FALS H2O2 RNS ROS SOD

?NO ?O2 ?OH ALS

nitric oxide superoxide anion hydroxyl radical amyotrophic lateral sclerosis

4.12.1 Introduction Approximately 2.2 or 2.3 billion years ago, a biochemical event dramatically modified the atmospheric conditions of planet Earth: cyanobacteria (blue-green algae) developed the ability to oxidize water introducing oxygen (Kasting and Siefert 2002; Raymond and Segre 2006). This change had an enormous impact. First, as a result of this change, those organisms were able to colonize almost all environments on the planet since water was already abundant. Second, this new metabolic pathway resulted in a massive production of oxygen, which initially accumulated as a waste product into the environment. This new oxidizing atmosphere provided primitive organisms with new ways to produce energy (Raymond and Segre 2006), but the increase in oxygen concentration exposed those primitive organisms to a new family of toxic by-products, commonly known as reactive oxygen species (ROS). ROS are an unavoidable consequence of the electronic configuration of molecular oxygen in its ground state, which has two unpaired electrons in its outer layer (Pauling 1949). As a result of this ‘bi-radical’ electronic configuration, the reduction of oxygen to water occurs through four consecutive single-electron steps, producing a variety of partially reduced intermediates of different reactivity (Figure 1). When molecular oxygen accepts the first electron, it is converted into superoxide anion (?O2). The second electron produces hydrogen peroxide (H2O2). Addition of a third electron breaks the

familiar amyotrophic lateral sclerosis hydrogen peroxide reactive nitrogen species reactive oxygen species superoxide dismutase

bond between both oxygen atoms, generating a molecule of water and a very powerful oxidant called hydroxyl radical (?OH). The fourth electron reduces ?OH producing OH, which is then protonated to produce a second molecule of water. In addition, one of the two unpaired electrons in the outer shell may also be excited to a new spin level, resulting in the formation of singlet oxygen (Figure 1), a very reactive form of oxygen capable of reacting with most biomolecules, particularly with double bonds producing dioxetanes (cyclic peroxides) (Foote and Clennan 1995). Some of the reactions between singlet oxygen and molecules such as proteins, lipids, and DNA may affect gene expression, triggering or blocking various intracellular responses (Klotz et al. 2003). The reactivity (and therefore stability) of partially reduced ROS varies substantially, with ?OH being the strongest oxidant while ?O2 and H2O2 are far more stable. In fact, ?O2 can spontaneously dismute into H2O2 with a rate constant around 105 mol l1 s1 (Liochev and Fridovich 2007; McCord and Fridovich 1969). However, in the presence of transition metals (such as iron) these two species become the main physiological source of ?OH through the ‘metal-catalyzed Haber–Weiss reaction’ (reactions [1] and [2]): ?O2 – þ Fe3þ ! O2 þ Fe2þ

ð1Þ

H2 O2 þ Fe2þ ! OH – þ ?OH þ Fe3þ

ð2Þ

Superoxide Dismutase and Catalase

σ∗

O2 +1e– –

O2

+1e–

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H2O2 +1e–

π∗



σ∗



π∗

1ΔgO 2

π∗

σ∗ π∗

σ∗

1Σg+O

2

H2O + .OH

σ∗

σ∗

π∗

π∗

+1e– 2H2O

σ∗ π∗

Figure 1 Electronic configuration of the outer orbitals in an oxygen molecule. The asterisk by the vacant orbitals indicates that those are ‘anti-ligand’ orbitals. Once all those orbitals are filled up, the atoms in the original molecule break apart. In the ground state for molecular oxygen (triplet) the two electrons occupy different orbitals. Excitation of one of these electrons causes it to change spins, producing ‘singlet’ oxygen. Of the two forms of singlet oxygen, only 1g has a half-life long enough to reach other biomolecules. The two unpaired electrons in the outer layer of triplet oxygen cause its complete reduction to water to occur in single-electron steps. The figure shows the electronic distribution of individual intermediates formed during the reduction of oxygen to water (superoxide, hydrogen peroxide, and hydroxyl radical).

Since ?OH may oxidize any biomolecule it comes in contact with, there are no enzymes capable of eliminating this species. Instead, ?OH is scavenged through its reaction with small molecular weight antioxidants such as glutathione, vitamin E, and uric acid. In vivo, ?O2 can also react with nitric oxide (?NO) in radical–radical annihilation reaction to produce peroxynitrite, a powerful oxidant with a reactivity similar to ?OH (Beckman et al. 1994). Thus, enzymes that eliminate ?O2 will prevent both ?OH and peroxynitrite formation in vivo, while the elimination of H2O2 will further contribute to preventing ?OH production.

In summary, aerobic organisms adapted to this ‘oxygen paradox’ by developing specific antioxidant enzymes to eliminate ?O2 and H2O2, thus preventing the formation of secondary oxidants. This chapter describes the enzymes that eliminate ?O2 (superoxide dismutases) and a group of peroxidases known as catalases involved in the elimination of H2O2 (Table 1). Although their substrates are totally different, catalases and superoxide dismutases work in tandem to prevent oxidative stress and also share a couple of unique biochemical features. First, both enzymes catalyze ‘dismutations,’ a type of redox reaction in which two molecules of the same

Table 1 Different types of catalases and superoxide dismutases Enzyme

Active site

Species

Structure

Catalases Heme catalase Catalase–peroxidase Mn catalase

Heme-Fe Heme-Fe 2 Mn

Eukaryotes Prokaryotes Prokaryotes (thermophilic bacteria)

Homotetramer Homodimer Homohexamer

Cu

Eukaryotes and some prokaryotes Eukaryotes (extracellular spaces) Eukaryotes (mitochondria) and prokaryotes Plant chloroplasts, trypanosomes, prokaryotes Streptomyces

Dimer Tetramer Homodimer or homotetramer Homodimer or homotetramer Homohexamer

SODs CuZnSOD EC-CuZnSOD MnSOD FeSOD NiSOD

Mn Fe Ni

222 Superoxide Dismutase and Catalase

substrate exchange electrons, thus becoming both an oxidant and a reductant. Second, these enzymes cannot be saturated under physiological conditions. This allows them to respond to increases in the production of their substrates by increasing their activity, thus protecting the cell over a wide range of concentrations.

4.12.2 Superoxide Dismutases (SOD, EC 1.15.1.1) In 1969 McCord and Fridovich discovered that a protein thought to be involved in copper metabolism was in fact a superoxide dismutase (McCord and Fridovich 1969). The enzyme speeds the rate of ?O2 dismutation by around five orders of magnitude, close to a diffusion-controlled process. The rate constant for the spontaneous process of superoxide anion is approximately 104 mol l1 s1 and in the presence of SOD the rate is close to 109 mol l1 s1 (Liochev and Fridovich 2007; McCord and Fridovich 1969). There are several isoforms of SOD found across the biosphere which have evolved from different genes. The mammalian forms of these enzymes include a copper–zinc form (CuZnSOD, found in the cytoplasm of the cell and in the mitochondrial intermembrane space), a manganese isozyme (MnSOD, of bacterial origin, located in the mitochondrial matrix), and an extracellular form of SOD (EC-SOD), a glycoprotein which also contains Cu and Zn. These three enzymes are coded by three different nuclear genes known as SOD1, SOD2, and SOD3 (Zelko et al. 2002). For many years it was thought that bacterial isozymes included only a MnSOD and an iron form (FeSOD). In fact, when a CuZnSOD was first found in bacteria, it was proposed that it represented the first example of gene transfer from eukaryotes to prokaryotes. This hypothesis was later reevaluated and now we know that CuZnSODs independently evolved in bacteria (Leunissen and de Jong 1986; Zelko et al. 2002). Since then, though, the gene for CuZnSOD was also identified in many prokaryotes. Recently, two new SOD isozymes (a Ni-containing SOD as well as a NiFe containing SOD) have been isolated from Streptomyces (Kim et al. 1996, 1998; Youn et al. 1996) and from a few other prokaryotes (Barondeau et al. 2004). The genes coding for these bacterial isoforms are known as sodA (MnSOD), sodB (FeSOD), sodC (CuZnSOD), and sodN

(NiSOD) (Kim et al. 1998; Lynch and Karumitsu 2000). Furthermore, although FeSOD was originally thought to be only an enzyme found in bacteria, it has since been detected in plant chloroplasts, in the cytoplasm of some plants (Mun˜oz et al. 2005), and in mitochondria and glycosomes of tryapanosomatids (Dufernez et al. 2006; Turrens and McCord 2006; Wilkinson et al. 2006). Superoxide dismutases evolved from different ancestor genes. There is a substantial degree of homology among all eukaryotic CuZnSODs including EC-SOD, although the latter is a much longer polypeptide and is a glycoprotein (Zelko et al. 2002). On the other hand, MnSOD and FeSOD are coded by two different genes which evolved from a single progenitor gene and therefore have high degree of homology (Smith and Doolittle 1992). In some cases the same apoenzyme incorporates either Fe or Mn, although in most cases this is not the case (Wintjens et al. 2008). The Ni-containing forms of SOD do not have any homology with either Cu/Zn or Mn/Fe forms of SOD (Kim et al. 1998). The mechanism of action is the same for all SODs: a metal ion located in the active site is responsible for catalyzing the electron transfer between two molecules of ?O2 (reactions [3] and [4]). In CuZnSODs, the catalytic metal is Cu, and it changes from cupric (Cu2þ) to cuprous (Cuþ) during the reaction. Ironcontaining SODs contain ferric ions (Fe3þ) which are reduced to ferrous (Fe2þ) in the first half of the reaction. In MnSODs the metal ion redox cycles between þ4 and þ3 while in NiSODs nickel redox cycles between þ2 and þ1. ?O2 – þ Meþn ! O2 þ Meþn – 1

ð3Þ

2Hþ þ ?O2 – þ Meþn – 1 ! H2 O2 þ Meþn

ð4Þ

4.12.2.1

Structure

The final structure of oligomeric proteins is the result of four levels of organization: (1) amino acid sequence (primary structure), (2) regular arrangements of the polypeptide chain in either -helices or -pleated sheets resulting from interactions between groups in peptide bonds (secondary structure), (3) folding of the polypeptide chain as a result of interactions among amino acid side chains (tertiary structure), and (4) association of more than one polypeptide chain (quaternary structure). The amazing catalytic activity of CuZnSOD is the result of optimizing all levels of protein organization.

Superoxide Dismutase and Catalase

Mammalian CuZnSOD is a dimer and each of its subunits contains one Cu and one Zn ion. The copper ion (positively charged) is located at the end of a Greek key -barrel. This rigid configuration is responsible for the high thermostability of CuZnSODs. The interior surface of the barrel has a critical arginine (Arg 141 in bovine and Arg 143 in humans) that guides each superoxide toward the copper ion. Acidic amino acids on the outer surface and positive amino acids near the entrance to the active site (Glu 131 and Lys 134 in bovine SOD) create an electric field which repels superoxide toward the positive entry to the active site (Getzoff et al. 1983, 1992). Finally, the dimeric quaternary structure places the two active sites at both ends of the molecule. As a result, any ?O2 that approaches CuZnSOD is electrostatically guided into the active site while both products in the reaction (H2O2 and oxygen), by not being charged, leave the active site without interference (Getzoff et al. 1983; Tainer et al. 1983). Bacterial CuZnSODs are also homodimers which show a Greek key -barrel comprised of eight antiparallel strands even though they are not related to the mammalian isoforms (Desideri and Falconi 2003). MnSODs and FeSODs have an entirely different structure, although they also contain a critical arginine near the active site, needed to guide ?O2 to the active site (Arg 180 in Thermus thermophilus) (Borders et al. 1994). The tertiary structure of these SODs involves primarily -helices and also -pleated sheets and the quaternary structure is either a homodimer (bacteria) or a homotetramer (eukaryotes) (McCord and Marecki 1997; Mun˜oz et al. 2005). The gene sequence for NiSOD from several prokaryotes has been identified and the crystallography data (1.3 A˚) shows that the active site has nickel bound to two cysteines, which is somehow surprising given the fact that in other proteins these amino acids are targets for ROS. The enzyme is a homohexamer which only contains -helices, and again a positive amino acid is located near the active site for guidance although in this case the amino acid is lysine instead of arginine (Barondeau et al. 2004). 4.12.2.2

Regulation of Gene Expression

Mammalian CuZnSODs appear to be constitutive enzymes, although some investigators have reported scenarios associated with increased transcription. Since it is a constitutive enzyme and the gene is located in the 21st chromosome, tissues from Down

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syndrome patients (trisomy 21) contain 50% more CuZnSOD than that from normal individuals. MnSOD both in bacteria and mammalian cells is highly inducible. In mammalian cells increased activity of MnSOD has been reported under a variety of scenarios associated with oxidative stress (hyperoxia, exposure to radiation, increased interleukins, etc.) in response to the activation of the nuclear factor NFB (Mattson et al. 1997, 2000; McCord and Marecki 1997; Wong et al. 1989). In bacteria FeSOD appears to be a constitutive enzyme while MnSOD is also induced during oxidative stress. In prokaryotes an iron–sulfur clustercontaining protein present in very small concentrations (SoxR) is reduced by ?O2, which in turn induces the expression of SoxS. SoxS activates the expression of several enzymes including MnSOD (Fridovich 1997; McCord and Marecki 1997; Pomposiello 2001).

4.12.3 Catalases (EC 1.11.1.6) Catalases were among the first enzymes to be characterized in biochemistry. In the year 1900, Loew named this ubiquitous enzyme catalase because of ‘‘its catalytic activity on hydrogen peroxide’’ (Loew 1900). After more than 100 years, the volume of new information concerning these enzymes does not show signs of slowing down (Chelikani et al. 2004; Kirkman and Gaetani 2006). Catalases may be classified into three distinct groups. Heme-catalases (also known as typical catalases) are the best studied enzymes and include homotetramers from various species of prokaryotes and eukaryotes containing either small (60 Kd) or large (>75 Kd) subunits. The second group includes catalase-peroxidases (KatG), a group of catalases found in bacteria and in fungi which share high homology with plant peroxidases but at the same time catalyze the dismutation of H2O2. The third group includes some species of bacteria that have a homohexameric catalase in which manganese dimers replace the heme group found in other catalases (Chelikani et al. 2004; Switala and Loewen 2002; Za´moky´ and Koller 1999). Free heme as well as many hemoproteins (myoglobin, cytochrome oxidase, etc.) depict low catalase-like activity in that they may catalyze H2O2 dismutation but they are not considered catalases. In all heme-containing catalases, the heme group is responsible for the enzyme’s catalytic activity

224 Superoxide Dismutase and Catalase

through an unusual chemistry in which the iron is oxidized to an oxyferryl state also known as Compound I (reaction [5]). Catalase-mediated decomposition of H2O2 may follow two different mechanisms. The dismutation reaction in which both the oxidant and the reductant are H2O2 is known as the ‘catalatic’ reaction. In this case, the oxyferryl heme is then reduced back to the ferric form by the second H2O2 (reaction [6]). Alternatively, mammalian catalases may utilize other electron donors (e.g., ethanol) oxidizing it in a two-electron reaction to reduce Compound I (reaction [7]). This is known as the ‘peroxidatic’ mechanism of catalase, because it resembles all other peroxidases and provides a route for the elimination of ethanol (Chance et al. 1979). H2 O2 þ catalase-hemeðFe3þ Þ ! H2 O þ catalase-hemeþ ðFe4þ ¼ OÞ

ð5Þ

H2 O2 þ catalase-hemeþ ðFe4þ ¼ OÞ ! O2 þ H2 O þ catalase-hemeðFe3þ Þ

ð6Þ

CH3 CH2 OH þ catalase-hemeþ ðFe4þ ¼ OÞ ! CH3 CHO þ catalase-hemeðFe3þ Þ þ H2 O

ð7Þ

The formation of Compound I is accompanied by a decrease in light absorption at 405 nm (Soret band). In 1947, Chance used this spectral change to study the formation of Compound I, discovering one of the first enzymatic intermediates (Bonnichsen et al. 1947; Chance 1947, 1949). Compound I may also be partially reduced by one electron, becoming Compound II, an inactive form of catalase (reaction [8]). e – þ catalase-hemeþ ðFe4þ ¼ OÞ ! catalase-hemeFe4þ OH

ð8Þ

Many catalases, including all mammalian forms, include a molecule of NADPH as part of the complex and its role appears to be the prevention of Compound I to Compound II conversion (Chelikani et al. 2004; Kirkman and Gaetani 2006). Yet, the stoichiometry of NADPH oxidation is not clear, and given the very positive reduction potential of Compound I, part of the NADPH may be reacting directly with it rather than just preventing Compound II formation (Kirkman and Gaetani 2006). In mammalian tissues, the intracellular localization of catalase varies substantially from organ to organ. In some organs, including liver and kidney,

the enzyme is primarily localized in specific organelles known as peroxisomes while in erythrocytes, catalase in localized in the cytoplasm (Chance et al. 1979). Catalase is not usually found in mitochondria with the exception of heart tissue where it is also present in the matrix (Radi et al. 1991). Interestingly, mitochondria from skeletal muscle tissues do not contain catalase (Phung et al. 1994). The apparent KM of catalase for H2O2 in the ‘catalatic’ reaction is between 30 and 600 mM, more than a 1000-fold higher than the physiological intracellular concentration of its substrate (Switala and Loewen 2002). This makes the ‘catalatic’ reaction very slow at low H2O2 concentrations, even though the rate constant is very high (2.6  107 mol l1 s1, Chance et al. 1979). On the other hand, these unusually high apparent KM values make catalases able to increase their activity linearly as their substrate concentration increase, making it a perfect protective enzyme during oxidative stress. Moreover, because the rate of H2O2 decomposition in the absence of other hydrogen donors follows a first-order kinetics, it is important to precisely define the concentration of H2O2 when catalase activity is determined spectrophotometrically. 4.12.3.1

Structures

Typical catalases show the heme in the active site located in a -barrel surrounded by various -helical segments. There is too much detailed information concerning the crystallographic properties of catalases. For details, readers are referred to two excellent reviews (Chelikani et al. 2004; Za´moky´ and Koller 1999). The secondary structure of catalase-peroxidases (KatG) and Mn catalases involves primarily helices (Smulevich et al. 2006; Za´moky´ and Koller 1999).

4.12.4 Clinical Scenarios Related to Changes in SOD and/or Catalase Activity Oxidative stress is a very complex process that may be triggered either by increased steady state concentrations of ROS or by a decreased antioxidant enzyme activity. On one hand, ROS are produced at various rates depending on the tissue as well as the compartment in each tissue. In most tissues the mitochondrion is the primary ROS-producing organelle

Superoxide Dismutase and Catalase

(Turrens 2003). In addition, the NADPH oxidase in endothelial cell membrane and leukocytes can produce variable amounts of ROS affecting all tissues, particularly during inflammation (Babior 1999, 2000; Brown 2007). The proportion and distribution of antioxidant enzymes and other low molecular weight antioxidants also vary from tissue to tissue. This combination of possibilities has made it very difficult to unequivocally establish the role of each component in cell toxicity. Not surprisingly, the literature includes a lot of contradictory results. Over the last 40 years, since the discovery of SOD (McCord and Fridovich 1969) most studies have reinforced the idea that antioxidant enzymes are required for life in an aerobic environment. For example, bacteria and yeast lacking SOD cannot survive in an oxygen-containing environment, and even transformation of these cells with SOD genes from other species restores protection (Touati 1989). This experiment alone elegantly proves the requirement of SOD for life in an aerobic envriornment. Almost 25 years ago, additional evidence further contributed to the idea that catalase and SOD protect against oxidative stress. In this study the effect of hyperoxia on rats injected with liposomes containing either SOD or catalase, or liposomes containing both enzymes was tested. The results showed that only the animals treated with both enzymes together were more resistant to 100% oxygen (Turrens et al. 1984). More recently, transgenic flies overexpressing both SOD and catalase show an increased lifespan than the wild-type counterpart (Orr et al. 2003). Some investigators have proposed that part of the symptoms in Down syndrome patients (which include a shorter lifespan) may be related to the increased CuZnSOD activity. Although this hypothesis is highly controversial, McCord has proposed an interesting model that suggests that high levels of SOD may be deleterious because it may scavenge radicals that otherwise would be involved in the termination of chain reactions (McCord and Marecki 1997; Omar et al. 1990). A few years ago I proposed a new model to explain why an increased CuZnSOD could increase the chances of having a Down syndrome child as mothers became older (Turrens 2001). The model is based on the fact that females are born with all the eggs they will produce in their lifetime, although most of them will be eliminated via apoptosis prior to ovulation. Those eggs that kept a second copy of chromosome 21 during meiosis should already have higher SOD since CuZnSOD is a constitutive

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enzyme and, therefore, be more resistant to apoptosis, resulting in a relative increase in their proportion with the age of the mother, increasing the mother’s chances of having a Down syndrome child. There are several other diseases that appear to be related to SOD mutations. One of these diseases is amyotrophic lateral sclerosis (ALS). In 90% of the patients, the disease appears spontaneously. For the remaining 10% of patients, the disease is hereditary and is known as familiar amyotrophic lateral sclerosis (FALS). About 20% of the patients diagnosed with FALS have mutations in the gene for CuZnSOD (Deng et al. 1993; Rosen et al. 1993), although the types of mutations vary (Jacobsson et al. 2001; Suzuki et al. 2008). MnSOD deficiencies have also been associated with a variety of diseases (Macmillan-Crow and Cruthirds 2001). The location of MnSOD in the mitochondrial matrix is critical for the scavenging of radicals generated by the respiratory chain (Turrens 2003). Knockout mice for this enzyme do not survive. In cell lines isolated from patients suffering from progeria, Rosenblum and collaborators found a mutation in the signal polypeptide responsible for the transport of MnSOD into the mitochondrial matrix. The mutation leads to a decreased activity of MnSOD in the mitochondrial matrix, which is the compartment where the enzyme should reside (Rosenblum et al. 1996). This observation also supports the idea that aging may be, at least in part, a result of a continuous production of ROS by mitochondria. The expression of CuZnSOD in bacteria has been associated with pathogenicity, probably by providing protection against ?O2 produced by macrophages and polymorphonuclear cells during phagocytosis (Lynch and Karumitsu 2000). This phenomenon has been observed in Nocardia asteroides (Beaman et al. 1985) and in Mycobacterium tuberculosis (Piddington et al. 2001). In humans, changes in EC-SOD activity have been implicated in the pathogenesis of diabetes. This protein is present in different extracellular fluids, particularly in the lumen of arteries and capillaries. It has high affinity for heparin and is rapidly released from the vessels’ surface upon injection of relatively small doses of heparin. In humans, an injection of only 50 IU kg1 body weight led to a 2.5-fold increase in plasma EC-SOD with a half-life of about 90 min (Qin et al. 2008). In diabetic patients, glucosedependent glycosylation of EC-SOD results in lower binding of EC-SOD to the endothelial surface, which may be a contributing factor to the vascular

226 Superoxide Dismutase and Catalase

complications observed in these patients (Fattman et al. 2003). On the other hand, catalases are not essential for life under normoxic conditions. In fact, acatalasemic individuals live normal lives with no change in their lifespan. Yet, transgenic mice overexpressing catalase targeted to the mitochondrion appear to have an increased lifespan (Schriner et al. 2005), again supporting a connection between mitochondrial ROS formation and lifespan.

4.12.5 Future Directions The formation of ROS may impact cell homeostasis at many levels, which may or may not involve gene expression. On one hand, minor fluctuations in the intracellular steady state concentration of ROS may modify certain molecules involved in the intracellular signaling and gene expression. In addition, their indiscriminate reactivity may either protect the cell or exacerbate cell damage depending on whether they annihilate a harmful oxidant or form stronger oxidant species such as peroxynitrite. From a pharmacological standpoint, learning more about the role of ROS in cell signaling will open new fields and targets toward the development of new chemotherapies. Cell permeable ROS scavengers or enzyme mimetics could be useful in modulating the intracellular steady state concentration of ROS. Quite a few of them have been used in experimental settings but their application in clinical scenarios is still in the early stages (Sampayo et al. 2003).

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