Proceedings of Bioc 462a Honors

Abstract

The superoxide dismutase (SOD) family of enzymes is vital to maintaining the antioxidant-oxidant balance in many organisms. These enzymes play an important role in inflammation and neurodegenerative diseases, among others. This report reviews the normal and pathologic structure and function of the human Cu,Zn-SOD (SOD1). It is hoped that new treatment pathways will become apparent through the clarification of the mechanism of pathogenesis. SOD1 is immunoglobulin-like, and oxidizes superoxide radicals (O₂^-) into hydrogen peroxide through the reduction of copper. It is composed primarily of beta-sheets in a beta-sandwich formation, with two subunits, each with 8 anti-parallel beta-sheets and a short, five-residue alpha-helix (132-137). Each subunit has 153 residues, two copper ions and one zinc ion. The beta-sheets are linked by seven loops to form two Greek key structures. The most notable SOD1 conformational shift occurs in the active site between the oxidized form to the reduced form of the enzyme. As His61 is protonated, the Copper(II) ion is oxidized to Copper(I). This breaks the coordination bond between the unprotonated His61 residue and the copper ion and uncharged O₂ is released. The SOD1 dimer tends to coordinate to other dimers through Asn86 hydrogen bonding Lys128 and Gly130, forming extended structures. This sort of aggregation is the focus of many researchers at the present time, as an explanation of the acquired toxicity of SOD1 mutants in familial amyotrophic lateral sclerosis (FALS). Mutant SOD1 also exhibits a decline in antioxidant activity, however, this does not explain FALS pathogenesis as evidenced by the survival of genetically engineered mice completely lacking SOD1 (Lindberg et al., 2002).

Introduction

As the AIDS epidemic rages on, scientists continue to search for answers to this most plaguing problem. Over the years, critical steps have been made on the journey toward finding a cure, and progress is still being made: research is probing into the physical structure of the HIV virus, the mechanisms of infection are under constant scrutiny, and the body’s response to the foreign invader is a subject that still has much to be discovered. The ultimate goal in AIDS research would be to find a cure and rid the world of this horrible disease. While ongoing research is definitely making progress on that journey, scientists may be close to achieving an important intermediate – prevention.
The HIV virus attacks the human immune system. There are two components involved in joining of host and viral cells – glycoprotein 120 and CD4 receptors. CD4 receptors are glycoproteins expressed on the surface of a variety of human immune cells, such as lymphocytes (most notably T lymphocytes, or T cells), monocytes, and macrophages (Bour et al, 1995). The other component of binding is gp120, part of a glycoprotein spike that project from the surface of the virus. Most of HIV’s surface proteins are constantly evolving, which is part of the reason it remains elusive to the human immune system. Gp120 differs because a part of it must always remain the same in order to “fit” with non-changing human immune cells. Since this site is the only unchanging, and therefore recognizable, unit on the surface of the virus, it is the location of greatest vulnerability.
Until recently, many thought that this site of attachment was hidden deep within the protein until just before binding with human immune cells. The hidden binding site created an enormous challenge for antibody mediation; namely that an antibody couldn’t bind to a site it couldn’t access, thus rendering it nearly impossible to use an antibody to block HIV infection. Researchers at the US National Institute of Allergy and Infectious Diseases, however, have now shown that the gp120 molecule doesn’t change its shape until after it has bound to human immune cells (Kwong et.al., 2007). If no shape change occurs to make the binding site available, it must be accessible all along.
Additionally, the HIV virus has been shown to bind to a human antibody called b12. The discovery of an antibody that can neutralize the HIV virus coupled with the knowledge that the antibody is able to access its site of action in the body leads to the exciting possibility of an HIV/AIDS vaccine. The structure and function of the HIV/b12 complex, both by itself and compared to the complex of HIV and human immune cells, is critical to understanding the components of infection and prevention and possibly developing a vaccine. Although more work is necessary before a vaccine becomes a reality, a more complete knowledge of the virus and its interactions with human cells is an important step toward achieving that goal.

Cellular processes and environmental conditions give rise to radical species that initiate damaging and uncontrollable reactions in cells, leading to DNA and other cell component damage. This oxidative damage and the oxidative stress that it produces have been implicated in many diseases, from cancer to diabetes. Cells mediate this damage through repair mechanisms but also by prevention via antioxidants, including antioxidant enzymes.
This paper concerns the cytosolic human copper-zinc superoxide dismutase (SOD1) enzyme. Mann and Keilin purified this metalloenzyme, which they called hemocuprein, in 1938. After the discovery of copper in plants and non-human animals, researchers were trying to discover where in erythrocytes this element was sequestered. This first discovery utilized bovine erythrocytes and researchers have since found SOD in many different species. The analogous human form of SOD was named erythrocuprein, and it wasn’t until 30 years later that an enzymatic function was discovered.
In 1969 McCord and Fridovich published a paper on the superoxide dismutase function of SOD. These two researchers found that the superoxide anion was inactivated in the presence of the erythrocuprein enzyme, and found that the reaction proceeded according to the following:

2O₂^- + 2H⁺ --> O₂ + H₂O₂

At the same time, McCord and Fridovich found that the copper ionis essential to the function of the enzyme. One of the superoxide anion oxygen atoms binds to the copper ion, while the other hydrogen-binds to Arg141 (Tainer et al., 1983). The copper ion is reduced, then oxidized according to the following reactions (Banci et al., 2002):

Cu²⁺+ O₂^- --> Cu⁺ + O₂

2H⁺ + Cu⁺ + O₂^---> Cu²⁺(O₂^2-) --> Cu²⁺ + H₂O₂

There have been various efforts at using SOD1 as a drug, in transplant patients to inhibit rejection and to break down scar tissue. However, being a protein, SOD1 makes a poor drug in terms of clearance time and delivery to tissues. Additionally, it has recently been realized that the antioxidant-oxidant balance is important to health and disease, and that more SOD1 is not always better (McCord & Edeas, 2005).
SOD1 is a stable, homodimeric metalloenzyme of about 32 kDa and 153 residues per subunit. It has a Greek-key beta-barrel conformation. There is an intrasubunit disulfide bond between Cys57 and Cys146 that adds to stability of the subunits. The overall enzyme is shown in Figure 1. Each subunit binds two copper ions and one zinc ion so that there are two identical active sites.

Mutations in SOD1 have been implicated in familial amyotrophic lateral sclerosis (FALS, Lou Gehrig’s disease), which accounts for about 10 percent of all ALS. This autosomal dominant disease manifests as its carriers age, through progressive loss of muscle control and tone as a result of the death of spinal motor neurons. The disease is nearly always fatal, usually within five years of diagnosis (Ray et al 2005, Rosen et al., 1993). Genetic SOD1 mutations have been associated with ALS since 1993. About 20 percent of those with FALS have mutations in their SOD1 gene on chromosome 21 (Ray et al., 2004).

Figure 1. Dimer of human SOD1. Human SOD1 is a homodimeric metalloenzyme. The copper ions (orange) and zinc ions (blue) are shown as spheres. The active channel sidechains are shown in stick form in the green-colored side of the dimer. This figure was created using PyMOL, with PDB accession code 2v0a.

There are over 100 known point mutations now associated with the disease, most of which are missense. For example, one mutation causes a substitution of arginine for glycine at residue number 85 (G85R). The resulting mutant SOD1 is inactive. Overall FALS usually results in 50-65 percent reduction in SOD activity (Borchelt et al., 1994). This large activity reduction may be due to the mutations found in FALS affecting several aspects of SOD1 function, including substrate binding, copper binding, thermodynamic and/or kinetic stability, zinc binding, and intersubunit interactions (Ray et al., 2004).

However, research since 1993 has indicated that loss of SOD1 function is not responsible for FALS. Studies with genetically engineered SOD1-null mice have shown that such animals have normal motor function, while mice with both the normal SOD1 and mutant SOD1 forms have neural damage (Lindberg et al., 2002). Thus it is indicated that pathogenesis is not from a loss of SOD1 function but rather from SOD1’s acquisition of toxic function. The mechanism of pathogenesis is a subject of current research, with a focus on the aggregation of SOD1 monomers into multimers.
General structure and function
SOD1 has two subunits composed primarily of beta-sheets in a beta-sandwich formation, each with 8 anti-parallel beta-sheets and a short five-residue alpha-helix (residues 132-137). Each subunit has 153 amino acid residues, two copper ions and one zinc ion. The beta-sheets are linked by seven loops to form two Greek key structures. Arg143 and Thr137 are at the outer edge of the active site, effectively limiting the size of anions that can gain entry. Arg143 points toward the copper atom and hydrogen bonds to Lys57 and Cys146, functioning to orient the oxygen radical in the proper direction. The short alpha-helix is oriented such that the dipole of the helix stabilizes the zinc ion. Meanwhile, His61 binds to the zinc ion, further stabilizing it.
The stable wild type SOD1 homodimer forms out of the association between two monomers, with the lower limit of the association constant being K_a > 3 x 10⁶ M^-1 and the upper limit K_a < 2 x 10² M^-1(Arnesano et al., 2004). The crucial forces driving the association are hydrophobic interactions. The intrasubunit disulfide bond seems to be critical to the dimerization, despite the apparent lack of significant change to the secondary structure, according to Arnesano et al. (2004). Monomers with the disulfide bond favor the formation of dimers by at least four orders of magnitude. Zinc ion binding also favors the dimerization, as it reduces the mobility of loop IV, which is in the area of the monomer interface (Arnesano et al., 2004).
The active channel is positively charged, including the crucial residues Arg141, His120 and Ser134 (Figure 2) (Yim et al., 1993). The active site channel has residues 131, 134-139, and 141 comprising one rim, 56, 58-60, and 63 the other rim, while the metal ions and ligands make up the channel floor. Copper is liganded by His118, His44, His61, and His46. His61 forms a bridge to the zinc ion (Tainer et al., 1983). Zinc is liganded by His61, His71 and His80. The mechanism of dismutation with coordinating residues is shown in Figure 3, also from Tainer et al. (1983). His61 is protonated in the reduced form but connects the copper and zinc ions in the oxidized form (Banci et al., 2002). The protonation of His61 breaks the coordination bond with Copper(II) as that ion is oxidized to Copper(I). The oxidized oxygen radical is released in this step as uncharged O₂.

Figure 2. SOD1 active site. The human SOD1 active site channel includes four critical histidine sidechains and one arginine. The zinc (blue) and copper (orange) ions are shown as spheres. This figure was created using PyMOL, with PDB accession code 2v0a.

Figure 3. Schematic dismutation of superoxide in SOD1. Created with ChemDraw following the original figure by Tainer et al.(1983).

SOD1 requires the copper ion to function, but such ions are not found free in the cytosol. In order to function, SOD1 must acquire the copper ion from the copper chaperone for superoxide dismutase (CCS) (Wong et al., 2000; Lindberg et al., 2002). The mechanism by which SOD1 acquires the zinc ion is not yet known.
SOD1 undergoes three additional reactions besides the superoxide dismutation: anion binding, inactivation by hydrogen peroxide, and peroxidase function. The anions that SOD1 can bind include cyanide, azide, halides, phosphate, and vanadate. These ions bind to either the copper ion or Arg141 (Yim et al., 1993).
To return to the primary function of SOD1, oxygen radical dismutation, the superoxide radical (O₂^-) itself can reduce or oxidize other species. In the dismutation reaction, this function is utilized by the enzyme as half of the radical is oxidized by the other half, which is reduced. The products are O₂and H₂O₂. This reaction is second order, and proceeds spontaneously at neutral pH with a rate of 2 x 10⁷M^-1s^-1(McCord & Fridovich, 1969). More recently, Yim and colleagues (1993) found a different, faster rate of enzyme-catalyzed superoxide dismutation that approaches the limit of enzyme rate enhancement: 2 x 10⁹M^-1s^-1.
SOD1 is deactivated by hydrogen peroxide, as well as the neurotransmitters taurine and glutamate. However, as the enzyme catalyzes the formation of H₂O₂, the channel has a very low affinity for this molecule and it is released to the surroundings. Consequently, the rate of inactivation by H₂O₂is low, at 6.7M^-1s^-1 at pH 10 and 3.1 M^-1s^-1at pH 9. Because of the release of H₂O₂to the cellular environment, it appears that an increase in SOD activity is accompanied by an increase in glutathione peroxidase, which neutralizes H₂O₂into water (Yim et al., 1993).
Pathogenic structure and function
That SOD1 mutations are linked to FALS is accepted, however, the mechanism by which SOD1 mutations influence the course of neural cell death is not yet decided. Lindberg et al. (2002) list several models for the genesis of the neurodegenerating toxicity: “ (i) enzymatic side-reactions in catalytically promiscuous SOD mutants producing increased levels of oxidative compounds such as hydroxyl radicals and peroxynitrite, (ii) release of free Cu ions, (iii) aberrant binding of SOD to other proteins, (iv) binding of mutant SOD to heat shock proteins with the subsequent prevention of their antiapoptotic function, (v) altered redox regulation, and (vi) formation of toxic SOD aggregates.” Of these multiple models, the scope of this paper encompasses only the formation of cytotoxic SOD1 aggregates and altered redox regulation.
Loss of dismutation function
The reduced dismutation activity of the mutant SOD1 forms found in FALS is proposed as an FALS disease mechanism. An individual heterozygous for any of six common mutant SOD1 forms should lose about 20-50% of total SOD activity, according to in vitro experiments. However, FALS usually causes a 50-65 percent reduction of SOD activity (Borchelt et al., 1994). Specific SOD1 mutants have different degrees of activity loss. One common SOD1 mutation is G37R (Rosen et al., 1993), which is less stable than the wild type SOD1, with 40-60 percent of normal activity. The H46R mutant has the substitution in the active site, but nonetheless it has 80 percent of normal activity (Borchelt et al., 1994). In contrast, Wiedau-Pazos et al. (1996) found that mutants A4V and G93A catalyze the oxidation of a model substrate to H₂O₂ at a higher rate than the wild type. These oxidative reactions start neuropathologic changes in FALS.
To account for the inconsistencies between actual and predicted mutant SOD1 activity, other aspects of the mutant forms must be considered. Banci et al. (2002) found that the active site (residues 131-142) is less mobile in the mutant than in the dimeric wild type. This loss of flexibility, while on the nanosecond to picosecond timescale, may make it impossible for the superoxide anion to reach the active site (Banci et al., 2002). However, Borchelt et al. (1994), found that there was no evidence for increased free radical damage in FALS, as would be expected with decreased antioxidant activity. There are many other cellular enzymes that neutralize free radicals and could compensate for SOD1 activity reduction, including glutathione peroxidase, manganese-SOD, and extracellular SOD. Consensus seems to be that loss of function alone cannot account for FALS pathogenesis as related to SOD1.
Aggregation
Recently there has been much debate over the mechanism of SOD1 mutations and neuronal cell death in FALS, especially in light of the unexpectedly large reduction in SOD1 activity mentioned above. One idea is that pathogenic SOD1 toxicity arises through aggregation of SOD1 into polymers (Arnesano et al., 2004; Elam et al., 2003; Hart 2006, Ray et al., 2004; Strange et al., 2004; Strathopulos et al., 2003). There is currently no consensus on the mechanism of mutant SOD1 aggregation. Some of the ideas are that decreased stability leads to monomerization then multimerization (Arnesano et al., 2004; Strathopulos et al., 2003; Ray et al., 2004), reduction of disulfide bond in the apoprotein mutant leads to monomers then multimers (Hart 2006), and the exposure of the beta-sheet to the environment in the apoprotein mutant leads to monomerization then multimerization (Strange et al., 2007; see Figure 4).

Figure 4. The apo form of human SOD1 mutant H46R. This mutant occurs in FALS, aggregated into a pathogenic 8 subunit form. These beta-sheet associated dimers can form amyloid-like strands and nanotubes leading to neuronal cell dysfunction and death This figure was reated using PyMOL with PDB accession code 1ozt.

The A4V mutant forms aggregates similar to the amyloid pores found in Alzheimer’s disease via the formation of SOD1 monomers. The A4V mutant monomerizes at low concentrations, aggregates, is found in the most aggressive FALS, and is less stable than the wild-type (Ray et al., 2004; see Figure 5). Hart (2006) classified A4V as a wild-type-like mutant, with nearly normal metal content. Nonetheless, the A4V mutant leads to apoptosis in neural cell culture (Ghadge et al., 1997).

Figure 5. The holo form of human SOD1 mutant H46R. This mutant SOD1 is bound to zinc but not copper, and is shown with four associated subunits in a pathogenic aggregation. This figure was created using PyMOL with PDB accession code 1oez.

Arnesano et al. (2004) also found that FALS SOD1 mutants are less stable than wild type, and have cysteine residues that are normally involved in the intrasubunit disulfide bond exposed to the cytosolic environment. The disulfide bond is reduced on exposure to the cytosol, then disulfide-linked multimers can form (Arnesano et al., 2004). Mutants A4V, G93A, G93R and E100G all decrease protein stability, creating aggregates that are cytotoxic (Strathopulos et al., 2003). These aggregates form via partly folded or unfolded states of the protein. Strathopulos et al. (2003) suppose that the small aggregates are toxic by overwhelming protein chaperones and degradation pathways, leading to deregulation of many cellular functions, and ultimately to cell death.

However, other researchers have found that simple removal of the metal ions from the wild-type enzyme causes significant and possibly pathogenic changes in the SOD1 conformation (Hart 2007; Strange et al., 2007; see Figure 4). Hart divides FALS mutants into two groups: wild-type like and metal-binding region (MBR). The mutations involved in the MBR are H46R, D125H, and S143N (Hart 2007). These mutations lead to apo enzyme forms (with no bound metals), which then undergo disulfide reduction followed by aggregation (Hart 2007). The beta-sheet is more exposed to the surrounding environment when metals are lost, leading to monomerization, then aggregation. The aggregation is edge-to-edge, stabilized by hydrogen bonds, specifically between Asn86, Lys128 and Gly130 (Strange et al., 2007). Rodriguez et al. (2005) however find that this explanation of the destabilization of apoproteins is not enough to explain FALS pathogenesis, because not all ALS mutant types exist as apoproteins with reduced stability compared to wild type. Particularly D124V and H46K are not more thermally stable than the wild type apoprotein (Rodrigues et al., 2005).

Not all researchers find that mutant SOD1 aggregates form from apoproteins. Elam et al. (2003) found that mutant SOD1 dimers (S143N and H46R) aggregate into cytotoxic hollow fibrils that resemble the protein aggregations found in Alzheimer's, Parkinson’s, prion-type, and other neurodegenerative diseases. The H46R mutant forms polymers in the zinc-bound holo form. The association of dimeric mutant SOD1 enzymes into linear polymers is thought to occur via the non-native conformation of the electrostatic loop (residues 125 - 131) interacting with a deprotected cleft between beta-strand 5 and beta-strand 6 in another mutant dimer (Elam et al., 2003).

Discussion
The human antioxidant enzyme SOD1 is a homodimeric Greek-key beta-barrel metalloenzyme found in erythrocytes that requires both copper and zinc ions to function (see Figure 1). The copper ion is reduced, then oxidized in the dismutation reaction.
SOD1 is ubiquitous and useful for reducing the amount of oxidative damage in cells via the conversion of the superoxide anion to hydrogen peroxide (O₂^- to H₂O₂; see Figure 3). The existence superoxide radical was discovered by Linus Pauling in the 1930s (Biomedicine 2004) but only in the past few decades has the importance of this charged molecule to redox reactions in the cell, in signaling and in genetic damage, been realized. The antioxidant-oxidant balance in the body mediates changes between normal and pathologic forms, and has been implicated in diseases as diverse and wide-ranging as ischemia-reperfusion injury, cancer, and diabetes.
In particular, SOD1 is found in mutant forms in about 20 percent of FALS patients. Some of the more common mutations are A4V, C6F, D90A, G93A and G93C, though there are many more (Lindberg et al., 2002). Initially, it was thought that the loss of antioxidant function was responsible for FALS pathogenesis, but mouse studies have shown that the animals are able to survive without neural damage even when completely lacking SOD1 (Lindberg et al., 2002). This indicates that SOD1 is somehow gaining toxicity in the mutant form rather than causing disease by loss of function.
SOD1 mutants aggregate into multimers, possibly causing FALS pathogenesis (Arnesano et al., 2004; Elam et al., 2003; Hart 2006; Ray et al., 2004; Strange et al., 2007; Strathopulos et al., 2003). There are several proposals for the mechanism of aggregation, including (i) decreased stability leading to first monomerization then multimerization (Arnesano et al., 2004; Strathopulos et al., 2003; Ray et al., 2004), (ii) reduction of disulfide bond in the apoprotein mutant leading to monomers aggregating into multimers (Hart 2006), and (iii) the exposure of the beta-sheet to the environment in the apoprotein mutant leading to monomerization then multimerization (Strange et al., 2007).
The SOD1 multimers can form into fibrils that resemble the amyloid fibrils characteristic of Alzheimer’s disease. The manner in which these large protein aggregates cause neural cell death is, as of yet, unknown. The aggregates may physically obstruct normal cell processes, but more research is required.
Much more research remains to be done on SOD1, on the mechanism of SOD1 multimer formation, as well as on the cellular response to SOD1 mutant multimers. This future research may lead to therapies that could help to prevent the neural cell degeneration that is at the root of FALS.

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