Cu/Zn Superoxide Dismutase: A Key Player in the Antioxidant Defense System

Genevieve Houston-Ludlam
Genetics BSCI 230, Spring 2003
University of Maryland

Printed with the permission of the author

Introduction:

All living cells, from the smallest bacterium to the longest nerve cell or the biggest ovum, must carry on a variety of cellular processes to be able to stay alive. Some of these activities are proactive in nature, such as gaining nourishment, and reproducing, whereas other actions are defensive in nature. Every cell is susceptible to harm from a variety of sources, both activities within the cell, and activities outside the cell. One of the activities that happens as a normal part of living for any cell, can also be something that causes damage- generation of reactive oxygen species, or ROS. In fact, these ROS are used by many types of cells as part of their ability to defend themselves, stored in the lysosomes of cells.
This paper focuses on one particular enzyme which deals with one particular ROS and the mechanisms within the cell to protect against it. The ROS in question is superoxide, and the defense mechanism is the enzyme superoxide dismutase (SOD) followed either by the enzyme catalase or glutathione peroxidase. The overall process will be described, and then the specifics of SOD will be discussed.
The superoxide radical (O₂^-·) is formed in the mitochondria as a byproduct of electron transport. (1) They are very dangerous to the cell because they steal electrons from neighboring molecules, starting a cascade of electron stealing, the end result being damaging of the cell. The superoxide dismutase enzyme is the first step that cells use to stop this process.
SOD converts superoxide radicals to a less toxic ROS, hydrogen peroxide (H₂O₂), as follows:

	Cu²⁺ + O₂^-· → Cu⁺ + O₂
	Cu⁺ + O₂^-· + 2H⁺ → Cu²⁺ + H₂O₂

Net Reaction:	2 O₂^-· + 2H⁺ → H₂O₂ + O₂

The hydrogen peroxide molecule, which is still a danger to cells, is then further processed to nontoxic by-products. In an aqueous environment, the enzyme catalase degrades the hydrogen peroxide as follows:

2 H₂O₂ → 2H₂O + O₂

The oxygen gas created in this reaction accounts for the bubbles seen when hydrogen peroxide is poured on a skin injury. Many bacteria do not have catalase, and the cells are destroyed, which is why hydrogen peroxide is effective against bacteria.
In a lipid environment, the body uses glutathione peroxidase to degrade the H₂O₂ as follows:

H₂O₂ + 2H⁺ → 2H₂O

Copper/Zinc Superoxide Dismutase:

Superoxide dismutase is categorized as an oxidoreductase class of enzyme, and has the Enzyme Commission identifier of EC1.15.1.1. SOD is a metalloenzyme, meaning that in addition to amino acids, it contains metal ions. There are several forms of superoxide dismutase, including MnSOD, which contains a manganese ion and is located exclusively in the mitochondria, FeSOD which contains an iron ion and is generally found in some prokaryotes, and CuZnSOD, which is active in the cytoplasm of eukaryotic cells. (2) This paper will be looking specifically at CuZnSOD.
CuZnSOD occurs as a dimer of identical 16 KDa subunits. Each subunit is 151 amino acids long, and the total protein weighs 32 KDa. The motif is an eight stranded greek-key beta-barrel connected by three external loops. A Protein Explorer rendering of the protein structure from the Protein Database is shown in Figure 1 (3). The two metal ions are shown and labeled on the upper subunit.

Figure 1: Protein Structure of CuZnSOD

The active site of the enzyme is shown in Figure 2. As might be inferred from its name, CuZnSOD has both a copper ion and a zinc ion embedded in its structure. The zinc ion is bound to three histidine residues and one aspartate residue. The copper atom is bound to four histidine residues. The two metal ions are connected via a histidine bridge.

Figure 2. Active Site of SOD

Superoxide is not thought to actually bind with the active site, but is guided near the active site using electrostatic attraction. The specific form of electrostatic attraction for SOD is referred to as a cationic funnel. (4) When the first superoxide is drawn near the enzyme, then an electron jumps from the superoxide to the active site, the histidine bridge "breaks", and the copper ion moves slightly away while the rings on the copper structure rotate about 20º towards the zinc. (5) When the second superoxide is drawn near the active site, the reduced copper is re-oxidized, the original electron jumps back to the second superoxide molecule along with two protons, and one molecule of hydrogen peroxide is formed. Because the substrate never actually binds to the enzyme, and is actively drawn in to the "funnel" the catalytic reaction can occur very quickly. The Kcat for Cu/Zn SOD is approximately 1 x 10⁹/M*sec which is much faster than the random diffusion/collision model would predict. An animation of the SOD catalytic reaction, a kinetics demonstration and details about the structure of the enzyme is given at http://www.nottingham.ac.uk/biochemcourses/students/sod/ [No longer available].

SOD and Human Health:

The CuZnSOD gene, SOD1, is located on human Chromosome 21 at location 21q22.1. (6). It has been associated with two specific medical conditions: Amyotrophic Lateral Sclerosis (ALS) and Down syndrome, also known as Trisomy-21.
ALS, also known as Lou Gehrig's disease, is a progressive, fatal disease where the motor neurons progressively degenerate. The disease usually starts out with muscle weakness, followed by progressive loss of muscle control and muscle wasting. The actual cause of death is usually an infection, due to the weakened condition of the body. The association of one form of ALS with mutations in the SOD1 gene was made in 1993. (7) This form of ALS is caused by inadequate activity by the mutated SOD, leaving the motor neurons open to oxidative damage and death.
Individuals with Down syndrome suffer the exact opposite fate. Down syndrome is caused by 3 copies of chromosome 21 being present in each (or most in the case of mosaic Trisomy-21) cell. SOD1, being located on Chromosome 21, is therefore triplicated. Measurements of SOD activity in individuals with Down syndrome show that there is overexpression of this gene, with an activity level of about 150% of normal. (8) There is a corresponding upregulation of either catalase or glutathione peroxidase in some tissues, but not in neurons (9). While there is still some disagreement in the scientific community about the implication of this overexpression, many scientists believe that the overexpression of SOD can be linked directly to the premature aging, neuronal degeneration, early-onset Alzheimer's disease, and other clinical features of Down syndrome. (10) In fact, one case study identified a boy with a micro-duplication of SOD1 with many of the clinical features of Down syndrome, but without a duplication of any other part of chromosome 21. (11)
Another very interesting area of research into CuZnSOD is being done in the area of Alzheimer's disease. The brain lesions that are characteristic of Alzheimer's disease show evidence of oxidative processes as well as other damage, such as inflammation. (12) Estevez et al. (13) examined how zinc-deficient CuZnSOD not only did not function as an antioxidant, but instead behaved like a pro-oxidant compound. They observed that the loss of zinc from CuZnSOD which still had its copper was sufficient to induce apoptosis in cultured motor neurons. This finding is significant not only in the disease of ALS which has been directly tied to this enzyme, but also to Alzheimer's disease, where pathological behavior of this enzyme is suspected. (14)

Conclusion:

Copper/Zinc Superoxide Dismutase is an enzyme that appears to play a very vital role in health, both at the tissue level and at the whole-body level. Research into the functions of this enzyme is ongoing, and may provide the key to slowing or reversing the neuropathy of Alzheimer's disease.

References:

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