The role of catalase in protecting cells from oxidative damage
Department of Chemistry, Berea College, Berea, KY 40404
CHM340 Biochemistry I Research Project, Spring 2016
Professor: Dr. Matthew Saderholm
Keywords: enzyme, hydrogen peroxide (H2O2), heme group, nicotinamide adenine dinucleotide phosphate (NADPH), oxidation
Investigation: the role of catalase enzyme in resistance to oxidative damage
Results: catalase is an enzyme with an efficient mechanism of hydrogen peroxide breakdown through an interaction with an evolutionary conserved heme group
Conclusion: catalase enzyme plays an important role of protecting various types of cells from reactive oxygen species
Enzymes are incredible biological molecules which are extremely important for life. Life is made possible by an enormous number of biochemical reactions that happen continuously in living organisms. Constant turnover of biomolecules produces many reactive byproducts which have no physiological function and need to be neutralized immediately to prevent them from damaging cells and tissues. Hydrogen peroxide is one of such reactive species produced by aerobic cells in huge amounts. Many of us are familiar with hydrogen peroxide as it is very commonly used as a disinfectant to treat cuts and abrasions. The same hydrogen peroxide is produced by the cells to fight infections. Hydrogen peroxide is a very powerful disinfectant and in small concentrations it is not damaging to human cells. When present in large concentrations, however, hydrogen peroxide tends to form very reactive species which, if not neutralized, will have detrimental effects on various cellular components. The ability of these reactive hydrogen peroxide species to oxidize the active sites of protein residues is especially dangerous and jeopardize healthy functioning of cells. Catalase is an enzyme with an incredibly important function. It catalyzes the decomposition of the excess of hydrogen peroxide to water and oxygen and protects cells from oxidative damage. Catalase has one of the fastest catalytic rates currently known. It converts millions of hydrogen peroxide molecules to water and oxygen per second preventing them from causing significant damage. The ability of catalase to perform its functions is derived from its unique structure which evolved due to the necessity of aerobic organisms to get rid of hydrogen peroxide in order to preserve life. This paper will look deeper at the structure of the catalase enzyme and explain how the structure of catalase allows it to achieve an efficient reaction mechanism of hydrogen peroxide neutralization.
The structural peculiarities of catalase and their important roles.
Catalase is located in the cellular organelles involved in important biochemical reactions that can be compromised by oxidizing agents. In mammals, catalase has been found in the matrix of peroxisomes, in cytosol and in mitochondrial matrix. In mammalian catalases even if a small difference in the sequence of amino acids is present, functionally important groups are conserved (1). Hence, all mammalian catalases, including human catalase, are considered to be the same enzyme (2). One might think that an enzyme should not be very complicated to be able to catalyze a single reaction of breaking down H2O2. So, a monomer with a couple of reactive sites somewhere on the surface of the protein would be able to do the job quite well. Catalase, however, is a tetramer composed of four identical monomeric subunits each over 500 amino acids (Fig.1). The active site of the catalase enzyme, heme group (red on Fig.1), located not only far away from the surface, but moreover, is buried deep inside the monomer and surrounded primarily by hydrophobic amino acids (Fig. 3). Two monomers in catalase are connected through the N-terminal threading arm that attaches to a long wrapping loop around another subunit (3). Monomers of catalase are illustrated on top of Fig.1.
Click threading arm yellow/wrapping loop red on the left to see these parts on the 3D model
The outer surface of the protein is mostly made of hydrophilic α-helices, while the extensive hydrophobic core is generated by an eight-stranded antiparallel beta-barrel, a large closed structure of twisted beta-sheets connected through hydrogen bonds (3). In monomers and dimers, the heme group, surrounded by the beta-barrel, α-helices and loops, is exposed to the outer surface. Only when two dimers come together to make a tetramer, is when the heme group gets buried deep inside the protein structure forming a hydrophobic pocket (3).
Click hydrophobic yellow/polar grey on the left to look at the 3D model showing location of hydrophobic and hydrophilic parts
Use Slab to cut the outer part of molecule and see the hydrophobic core (yellow) inside
The catalase structure is an example of how nature took into account various factors that might be in the way for the enzyme to perform its functions. One of the most important of these factors, is the necessity to selectively bind hydrogen peroxide. The location of the heme group solved this problem. In order to approach an active heme group of catalase, a molecule would have to travel deep inside the protein through a narrow hydrophobic passageway. This position of an active site deep inside the protein is an important evolutionary adaptation since this structure allows it to selectively interact with only small molecules, such as hydrogen peroxide (1). Another interesting peculiarity of the catalase structure that came to researches as a surprising discovery is the presence of NADPH molecule impeded among α-helices in the back part of each monomer (dark green of Fig.1). Currently, evidence suggests that NADPH plays an integral role in protecting the heme group from oxidation (4).
Click show ligands on the left to look at the 3D model showing heme and NADPH ligands in catalase structure
Figure 1. Monomers, dimers and a tetramer of human catalase (3)
Click secondary structure view on the left to look at the 3D model
How is the heme group involved in H2O2 breakdown? The enzymatic mechanism of catalase.
Different types of the heme group are known (A, B, C, O). All these heme groups are slightly different in their molecular structure (5). The most abundant heme B group is evolutionary conserved and is present in a number of proteins (1). This evolutionary conservation suggests its importance and high efficiency. The oxygen transporting proteins hemoglobin and myoglobin are examples of proteins that also utilize heme group to bind molecular oxygen. Since the vital function of oxygen transport relies on the chemical reactivity of the heme group, this kind of mechanism must have proven evolutionarily efficient and reliable. A classic heme group consists of an iron atom in the center with an organic component bounded to it (Fig 2). The organic component in a heme group is called protoporphyrin which is a structure of four pyrrole rings linked by methane bridges forming a tetrepyrrole ring (6). The iron atom in a heme group forms six coordinated bonds: with four pyrole nitrogen atoms, with a new-coming hydrogen peroxide molecule and a tyrosine residue of the protein on the opposite from the hydrogen peroxide binding site.
Click conserved residues on the left to look at the 3D model of a monomer showing conservation. Dark red colored parts are the most conserved areas and blue are the least conserved.
Use Slab to cut the outer part of molecule and see the conserved part (dark red) inside
Click heme active site on the left to look at the 3D model
Figure 2. Four subunits of Helicobacter pylori catalase colored in blue, red, yellow and green. heme binding pocket of one of the subunits (blue) and molecular structure of Heme (7)
The heme group is the center where the catalysis of the H2O2 breakdown takes place. As previously mentioned, the position of the active heme group away from the enzyme surface in a deep hydrophobic construct is critical for the molecular recognition mechanism of hydrogen peroxide (Fig.3) (3). The narrow pathway leading to the active site allows only small molecules such as water and hydrogen hydroxide pass through it. Water molecules, however, are being trapped early on before they reach the active site through the so called molecular ruler mechanism (8). Histidine (His56) and asparagine (Asn129) residues form hydrogen bonds with incoming water molecules and do not allow them to pass through the channel and reach the active site (Fig. 2). The presence of hydrogen-bonded water molecules is essential, since they form bridges allowing hydrogen peroxide molecules pass through the hydrophobic channel. After these hydrogen bonded water molecules built hydrogen bonds with His and Asn, they block part of the pathway preventing other water molecules from entering the channel. Also, water molecules cannot bridge the channel with water-water hydrogen bonds since the distance to form a hydrogen bond is out of reach of their bonding capacity (3). Hydrogen peroxide molecules that are larger due to the presence of two bonded oxygens would be able to satisfy the hydrogen bonding network with the water molecules trapped in the hydrophobic channel. Since they also have a lower dipole moment than water, they will be more stable surrounded by nonpolar hydrophobic protein residues (3). Due to the electrical potential in the channel created between the conserved negatively charged aspartate and the positively charged iron, newly coming H2O2 are oriented in a certain way to the heme group that their orientation is favorable for the rapid reaction to take place (8).
Figure 3. A heme group (red) buried inside hydrophobic channel of catalase structure of Human erythrocyte catalase. Amino acids along the channel (green) make the construct hydrophobic (3)
The heme binding site is so specific that even the amino acid residues around the heme group play a role in the catalytic reaction. A proximal tyrosine (bacterial catalase Tyr 339 on Fig. 2, mammalian Tyr 358 on Fig.3) donates electrons to the central iron atom contributing to its +3 oxidation state necessary for the reaction with H2O2 (7). The histidine (His75) and asparagine (Asn148) residues contributing to the hydrogen bonding with incoming H2O2 molecules are essential in the heterolytic cleavage of peroxide bond (3) (Fig. 4).
Figure 4. The mechanism of the initial step of the hydrogen peroxide enzymatic breakdown. Formation of the main reaction intermediate - heme group with the oxidized form of central iron (Compound I) (3).
The proposed mechanism of hydroxide bond cleavage (Fig. 4) shows the formation of the reaction intermediate, compound I (Cmpd I). The overall reaction can be summarized as follows:
Catalase Fe3+ + H2O2→catalase FeO3+ (Cmpd I) + H2O (9)
As you can see from the mechanism, incoming polar H2O2 molecules form hydrogen-bond bridges with water molecules trapped in the hydrophobic channel by amino acid residues Gln168 and Asp128. This bonding places the reactive oxygen of peroxide in close proximity to the heme iron atom. As a result, iron atom is oxidized by the H2O2 oxygen and forms Cmpd I (9).
Figure 5. The mechanism of the final step of the hydrogen peroxide enzymatic breakdown. The reduction of compound I back to the heme group with Fe3+ with a free binding site (3)
Kinetics studies have shown that once catalase Cmpd I forms, it rapidly reacts with another molecule of H2O2 to generate a water molecule and O2 (7) (Fig. 5). This brings the heme group back to its resting state (3). The second step of the mechanism can be summarized by the following reaction:
Catalase FeO3+ (Cmpd I) + H2O2 → Catalase Fe3+ + O2 + H2O (9)
The mechanism above is extremely fast and efficient. This efficiency is achieved via the structural peculiarities of catalase that allow the enzyme to selectively target H2O2 molecules and rapidly initiate their binding to the active site. As shown, catalase structure is well suited to its performed function.
Another ligand? The role of NADPH
Click NADPH ligand on the left to look at the 3D model
NADPH is an abbreviation for nicotinamide adenine dinucleotide phosphate, a reducing agent required in many anabolic reactions, such as lipid and nucleic acid synthesis. The presence of the NADPH in the molecular structure of mammalian catalase was an unexpected and even surprising discovery. Interestingly, the NADPH binding residue is highly conserved (10). This evolutionary conservation of the NADPH binding site suggests that its function is vital for catalase activity. NADPH seems to play no role in the catalysis of hydrogen peroxide and there are no nucleotide binding sites in the catalase structure. However, it has been discovered that under certain conditions catalases can undergo undesired side reactions that can eventually inactivate the enzyme (9). For instance, at low hydrogen peroxide concentrations and in the presence of certain organic substrates, the Cmpd I intermediate, which forms during the hydrogen peroxide breakdown cycle, can undergo a one-electron reduction to form Compound II (Cmpd II) (7):
Catalase FeO3+ (Cmpd I) + AH → catalase FeOH3+ (Cmpd II) + A (9)
Cmpd II (with Fe4+) cannot be further reduced by 2-electron substrates like H2O2 (10), hence the reaction pathway of catalase is terminated. The biological role of NADP(H) thought to be the protection against this kind of enzyme inactivation during periods of slow catalytic turnover (8). It is believed that NADPH transfers electrons to the heme group to reduce Cmpd II back to Cmpd I and regenerate activity of the enzyme (10). Through experiments it has been proven that removal of the catalase-bound NADP did not abolish the ability of the enzyme to catalyze the conversion of H2O2 to oxygen and water. However, when the enzyme bounded to NADPH was oxidized to NADP+, the activity of the catalase fell to about one-third of the initial activity (4). It has been concluded that NADPH may protect catalase from oxidative damage by preventing the oxidation of the heme group (4).In addition to the role of NADPH of restoring catalase activity, experimental evidence suggests that binding of NADPH stabilizes the quaternary structure of the enzyme. It has been proposed that the NADPH binding causes slight structural changes that stabilize the native conformation (10).
Is catalase important for our well-being?
Deficiency of catalase was discovered by ear, nose and throat surgeon Professor Takahara at the Okayama University Medical School. By his usual routine Takahara irrigated the surgical wound of the patient with H2O2. Instead of seeing the usual foam of oxygen bubbles, he saw the tissue turn black. Assuming that he had mistakenly used silver nitrate, he repeated the application but the same effect occurred (2). Due to its strong oxidative properties, H2O2 is very damaging to tissues because of the presence of proteins that are prone to the rapid loss of their healthy behavior via oxidation throughout the body.
Nowadays, advertisements bombard us with information that oxidative stress is damaging for hair, skin and nails. Unfortunately, oxidative damage to a human body is not limited to oxidants in the environment. Reactive and highly damaging hydrogen peroxide molecules are produced in a healthy functioning human body every second due to constant usage of oxygen in metabolic reactions (11). Apart from assisting the production of ATP molecules, oxygen forms free floating oxidizing agents. Research indicates various harmful consequences of high concentrations of H2O2 to cells such as an oxidative DNA damage (12), damage of membrane proteins and lipids, reduced life span of the red blood cells (13). Absence or reduction of catalase activity is most likely to lead to various pathological changes and the development of detrimental diseases.
Aerobic respiration benefits an organism significantly by allowing a much more efficient energy production. However, this privilege comes with its own cost. Molecular oxygen, an essential contributor to aerobic respiration, undergoes multiple chemical modifications before it is converted to water at the end of the cycle. Oxygen forms an intermediate that is very damaging for the cells in the form of highly reactive hydrogen peroxide (12) (13). Since erythrocytes, or red blood cells, are exposed to a high concentration of molecular oxygen, some of the oxygen will certainly form a hydrogen peroxide intermediate. Acatalasemia, inherited near-total deficiency of catalase activity in red blood cells, will inevitably result in a variety of health problems for an individual. Hemoglobin is especially susceptible to oxidative damage. Degradation of the heme moiety takes place in conjunction with the reaction of hydrogen peroxide with hemoglobin (13). The heme group of hemoglobin with its ferrous ion is especially reactive with a strong oxidizing agent, hydrogen peroxide. In addition to heme loss, H2O2 induces other structural changes which include irreversible oxidation of sulfur-containing amino acids in the β-globin chain as well as loss of α-helical structure integrity (14). Catalase is the primary enzyme responsible for protecting the red cell from hydrogen peroxide (13).
Studies also has shown the high (18.5%) prevalence of type II diabetes in people with inherited catalase deficiency. It has been suggested that the manifestation of the disease may be due to the oxidative damage of oxidant sensitive, insulin producing pancreatic beta-cells. 97 of the 114 acatalasemics had other diseases related to oxidative stress and aging. (15)
Catalase is one of the oldest enzymes to be studied. The history of catalases goes back to the 19th century when they became one of the first sources of valuable information about the nature and behavior of enzymes (10). Understanding catalase structure and function was an important breakthrough in proteomics that allowed for the characterization of the structures of many other enzymes with a similar mechanism of action. The heme group used for the hydrogen peroxide breakdown is a structure that allows catalase to achieve a rate of the reaction almost as fast as the diffusion controlled rate making catalase one of the fastest enzymes currently known. An NADPH ligand with a high reduction potential is embedded into the structure of catalase to guard the enzyme from inactivation by its own highly oxidizing substrate. Catalase is a classic example of the efficiency and thoughtfulness of the evolutionary process. When aerobic respiration was undertaken as a more efficient way of energy production, catalase became an inseparable part of this evolutionary modification. The molecular structure of catalase is perfectly designed to give this enzyme the ability to perform its function with maximum efficiency.
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