Oxidative Stress in Health and Disease

*Glutathione System and Oxidative Stress in Health and Disease*

lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages. Journal of Periodontal Research. 2015;**50**(6):737-747. DOI:

Sofuoğlu A, Taner L, Koch M. Effect of the topical use of the antioxidant taurine on the two basement membrane proteins of regenerating oral gingival epithelium. Journal of Periodontology. 2012;**83**:127- 134. DOI: 10.1902/jop.2011.100568

[62] Sree SL, Sethupathy S. Evaluation of the efficacy of taurine as an antioxidant in the management of patients with chronic periodontitis. Dental Research Journal (Isfahan). 2014;**11**(2):228-233

[63] Bonnefont-Rousselot D, Collin F. Melatonin: Action as antioxidant and potential applications in human disease and aging. Toxicology. 2010;**278**(1): 55-67. DOI: 10.1016/j.tox.2010.04.008

[64] Özdem M, Kırzıoğlu FY, Yılmaz HR,

Vural H, Fentoğlu Ö, Uz E, et al. Antioxidant effects of melatonin in heart tissue after induction of experimental periodontitis in rats. Journal of Oral Science. 2017;**59**(1): 23-29. DOI: 10.2334/josnusd.16-0034

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[61] Gültekin SE, Sengüven B,

[53] Armitage GC. Bi-directional relationship between pregnancy and periodontal disease. Periodontology

[54] Agueda A, Ramon JM, Manau C, Guerrero A, Echeverra JJ. Periodontal disease as a risk factor for adverse pregnancy outcomes: A prospective cohort study. Journal of Clinical Periodontology. 2008;**35**:16-22. DOI: 10.1111/j.1600-051X.2007.01166.x

[55] Brown RS, Arany PR. Mechanism of drug-induced gingival overgrowth revisited: A unifying hypothesis. Oral Diseases. 2015;**21**(1):e51-e61. DOI:

Sendrowski K, Sobaniec S, Pietruska M. Antioxidant activity of blood serum and saliva in patients with periodontal

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[56] Sobaniec H, Sobaniec W,

disease treated due to epilepsy. Advances in Medical Sciences. 2007;**52**(Suppl 1):204-206

[57] Preshaw PM. Host response modulation in periodontics.

[59] Akyol S, Ginis Z, Armutcu F, Ozturk G, Yigitoglu MR, Akyol O. The potential usage of caffeic acid phenethyl ester (CAPE) against chemotherapyinduced and radiotherapy-induced toxicity. Cell Biochemistry and Function. 2012;**30**(5):438-443. DOI: 10.1002/

[60] Choi EY, Choe SH, Hyeon JY, Choi JI, Choi IS, Kim SJ. Effect of caffeic acid phenethyl ester on Prevotella intermedia

Periodontology 2000. 2008;**48**:92-110. DOI: 10.1111/j.1600-0757.2008.00252.x

[58] Yağan A, Kesim S, Liman N. Effect of low-dose doxycycline on serum oxidative status, gingival antioxidant levels, and alveolar bone loss in experimental periodontitis in rats. Journal of Periodontology. 2014;**85**(3):478-489. DOI: 10.1902/jop.2013.130138

2000. 2013;**61**:160-176. DOI: 10.1111/j.1600-0757.2011.00396.x

**96**

cbf.2817

**99**

**Chapter 6**

**Abstract**

the female and the male.

**1. Introduction**

Sperm Cells

*and Raúl Sánchez Sánchez*

Effect of Oxidative Stress on

*Gustavo Ruiz Lang, Rubén Huerta Crispín,* 

*Abel E. Villa Mancera, Pedro Sánchez Aparico* 

*Alejando Córdova Izquierdo, Adrian Emmanuel Iglesias Reyes,* 

*Alda Roció Ortiz Muñiz, María de Lourdes Juárez Mosqueda,* 

*Jesús Alberto Guevara González, Juan Eulogio Guerra Liera,* 

Free radicals are unstable molecules that have an unpaired electron in their last orbital, which makes them highly unstable agents. In medicine, it has been discovered that they play an important role in cell signaling and without them some cells such as leukocytes or sperm could not perform their biological functions. To protect itself from these oxidizing agents, the cell has a defense system based on antioxidants; however, when this balance is lost and oxidizing agents exceed the cellular antioxidant capacity, the cell enters oxidative stress, which affects cellular components such as proteins, nucleic acids, lipids, amino acids, and carbohydrates, among others. In the case of spermatozoa, due to their high metabolic rate, they produce large quantities of oxygen reactive species (ROS), decreasing sperm motility, alterations in cytoplasmic components, modifications in genetic material, or sperm death. In this chapter, a review is made of a brief history of how the toxicity of oxygen and free radicals was discovered, the oxidative stress in cells, and the effect of oxidative stress in the cytoplasmic sperm membrane, in the spermatic mitochondria, in the spermatic acrosome, in the sperm DNA, and in the fertility of

**Keywords:** spermatozoa, oxidative stress, free radicals, reproduction

Semen freezing is one of the most important procedures in the development of biotechnologies for assisted reproduction. Among the advantages that we can find in artificial insemination is as follows: to keep the biological material viable for an indefinite time, the establishment of gene banks and the exchange of genetic material over very long distances economically rationalize the ejaculate; improve the use of wild boar elite, an adequate available germinal material of economic interest for man; and perform the collection of semen only in the most favorable reproductive seasons. However, the composition of the plasma membrane of the pig sperm, the

**Chapter 6**

## Effect of Oxidative Stress on Sperm Cells

*Alejando Córdova Izquierdo, Adrian Emmanuel Iglesias Reyes, Alda Roció Ortiz Muñiz, María de Lourdes Juárez Mosqueda, Jesús Alberto Guevara González, Juan Eulogio Guerra Liera, Gustavo Ruiz Lang, Rubén Huerta Crispín, Abel E. Villa Mancera, Pedro Sánchez Aparico and Raúl Sánchez Sánchez*

#### **Abstract**

Free radicals are unstable molecules that have an unpaired electron in their last orbital, which makes them highly unstable agents. In medicine, it has been discovered that they play an important role in cell signaling and without them some cells such as leukocytes or sperm could not perform their biological functions. To protect itself from these oxidizing agents, the cell has a defense system based on antioxidants; however, when this balance is lost and oxidizing agents exceed the cellular antioxidant capacity, the cell enters oxidative stress, which affects cellular components such as proteins, nucleic acids, lipids, amino acids, and carbohydrates, among others. In the case of spermatozoa, due to their high metabolic rate, they produce large quantities of oxygen reactive species (ROS), decreasing sperm motility, alterations in cytoplasmic components, modifications in genetic material, or sperm death. In this chapter, a review is made of a brief history of how the toxicity of oxygen and free radicals was discovered, the oxidative stress in cells, and the effect of oxidative stress in the cytoplasmic sperm membrane, in the spermatic mitochondria, in the spermatic acrosome, in the sperm DNA, and in the fertility of the female and the male.

**Keywords:** spermatozoa, oxidative stress, free radicals, reproduction

#### **1. Introduction**

Semen freezing is one of the most important procedures in the development of biotechnologies for assisted reproduction. Among the advantages that we can find in artificial insemination is as follows: to keep the biological material viable for an indefinite time, the establishment of gene banks and the exchange of genetic material over very long distances economically rationalize the ejaculate; improve the use of wild boar elite, an adequate available germinal material of economic interest for man; and perform the collection of semen only in the most favorable reproductive seasons. However, the composition of the plasma membrane of the pig sperm, the

large phospholipid layer (the comparison of bull sperm, which has a smaller layer of skin), is the cause of the sperm cell. Free radical changes that occur during freezing, the occasion when the effects of sperm freezing occur in the wild boar, affect the integrity of the plasma membrane, the acrosome, the nucleus, as well as the mitochondrial functions and motility of spermatozoa [1–4]. The purpose of this review is to publicize the main causes of ROS generation in sperm cells, as well as a brief explanation of how ROS is a part of sperm parts.

#### **2. Background**

Air is a vital element for any living being and is a mixture of gases based on nitrogen (78%), oxygen (21%), water vapor (variable between 0 and 7%), ozone, carbon dioxide (CO2), hydrogen, and some noble gases such as krypton, neon, helium, and argon. Of these, oxygen (which appeared approximately 2500 million years ago) plays a vital role in the processes of aerobic life, being the second most abundant element in the atmosphere [5–7].

Antoine Lavoisier in the eighteenth century gives the name to "oxygen" which means "generator of acids," because despite having a therapeutic use, it was already known that it was a toxic substance, due to its great oxidizing power. In 1774, the toxic effects of the gas are demonstrated, and 6 years later (1780) experiments are made of the use of oxygen in newborns; in 1878, the toxic effect of oxygen in the brain is documented by Paul Bert, manifested by the presence of convulsive crises to more than three atmospheres, and in 1899, when trying to replicate the Bert effect, J. Lorrain Smith reports fatal pneumonia in rats exposed to 73% oxygen for 4 days. In 1940, it is reported that babies with periodic breathing pattern improved with the use of oxygen to 70%, beginning the routine use of oxygen in premature babies. Between 1951 and 1956, it is demonstrated that oxygen was safe when it occurred in concentrations lower than 40%. Harman in 1954 stated that the life expectancy increases decreasing the degree of oxidative phenomena. Thus, throughout history, it has been described that the higher the toxicity of O2 is, the higher is the metabolic rate of the species considered [6, 8].

In veterinary and human medicine, more and more agents that cause diseases in the body have been discovered; some of them are derived from metabolic processes of oxygen, among which are the production of energy, detoxification of harmful compounds, and defense against pathogens, among which are free radicals (RL), which are highly reactive oxidation agents, which act as short-lived chemical intermediates on lipids, amino acids, carbohydrates, and nucleic acids [5, 7].

The RL can be divided into the following: (i) reactive oxygen species (ROS), which are highly reactive molecules that constantly attack organisms through oxidation-reduction reactions, among which are molecular oxygen (O2), superoxide anion (O21) hydrogen peroxide (H2O2), hydroperoxyl (HO2), and hydroxyl radical (OH); (ii) the transition metals, which have unpaired electrons and can exist as RL; and (iii) reactive nitrogen species (ERN), which are capable of generating oxidative damage and cell death, among which are nitric oxide (NO), peroxynitrite anion (ONOO−), and nitric dioxide (NO2) [9–11].

The RL must be attenuated by different antioxidant defense systems, which involve enzymes and molecules. Antioxidants are divided into enzymatic, also called endogenous production, which are the first line of defense against the production of RL and are proteins with antioxidant capacity that are not consumed when reacting with the RL. Among the most important of this group are catalase, superoxide dismutase, and glutathione peroxidase. The nonenzymatic ones come mainly from the diet and are small liposoluble molecules, which, unlike the

**101**

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

processes [7, 14, 17, 18].

or regeneration of nonenzymatic antioxidants [9].

**3. Effect of oxidative stress on cells**

diseases such as heart failure [7].

enzymatic, are consumed during their antioxidant action, so they must be replaced; among the most important in this group are vitamins E and C, beta-carotenes, retinol, uric acid, pyruvate, albumin, carnitine, taurine, hypotaurine, transferrin, ceruloplasmin, polyphenoids, flavonoids, and trace elements [12–16]. These antioxidant defense systems are linked in a cellular buffer system, where they add up and collaborate with each other, to deal with any oxidative aggression in cells, for example, nonenzymatic antioxidants can have synergistic effects in combination with enzymatic antioxidants, regenerating enzymatic antioxidants through the donation of hydrogen, neutralizing molecular oxygen, and catalyzing the synthesis

When there is an imbalance and the amount of RL exceeds the balance between oxidant production and antioxidant capacity, a phenomenon known as oxidative stress (EO) is generated, which has negative consequences on multiple cellular

Due to aerobic conditions, cells maintain a high concentration of oxidant products in their metabolism, such as RL, which are generated as a result of cellular metabolism and in cellular physiological concentrations are related to cell signaling processes or to fulfill their functions biological, including leukocytes that are recruited to the sites of infection by chemotactic factors and are able to eliminate microorganisms through phagocytosis, exposing them to high concentrations of ROS (superoxide and hydrogen peroxide) and other microbicidal products contained in cell granules. However, when EO exists, ROS can mainly affect cellular

components such as proteins, nucleic acids, sugars, and lipids [7, 9, 17].

leads to the formation of peroxynitrites, which alters sperm motility [27].

It has been observed that in the ejaculate, the main sources of ROS are leukocytes and abnormal sperm cells, although it has been proposed that there are other

Most of the main diseases that cause the death of animals and people or deteriorate their quality of life are caused by the RL. Each cell of the body suffers about 10,000 impacts of free radicals per day. For this reason, the EO has been the target of intense research in recent years, mainly in the implications on how mitochondria produce ROS, since they are of vital importance to understand their relationship with the pathogenesis of several chronic diseases such as cancer, osteoporosis, Alzheimer's, type 2 diabetes, neurodegenerative diseases, and cardiovascular

The spermatozoon was the first cell type in which the presence of ROS could be identified, because until a few years ago, ROS were considered toxic elements for sperm; however, the RL are currently known (mainly O2.-) in low concentrations in semen, which play a fundamental role in their biological functions during sperm capacitation, sperm maturation, tyrosine phosphorylation, intergame interaction, and the acrosomal reaction that occurs for fertilization of the oocyte; these phenomena are controlled by the mechanism of defense of enzymatic and nonenzymatic antioxidants that when this balance is broken between the RL and the antioxidant defense system, damages are induced in the nucleic acids, proteins, and lipids present in the membrane of the sperm, causing loss of mobility, decrease in viability, and alterations in the intermediate piece, which finally produce a decrease in seminal quality or sperm death [2, 7, 14, 16, 19–26]. A clear example of this is nitric oxide (NO), which has an important function in the sperm pathophysiology, since in low concentrations it favors the processes of sperm capacitation, the acrosomal reaction, and the union to the zona pelucida; however, in high concentrations it

#### *Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

*Glutathione System and Oxidative Stress in Health and Disease*

explanation of how ROS is a part of sperm parts.

abundant element in the atmosphere [5–7].

higher is the metabolic rate of the species considered [6, 8].

(ONOO−), and nitric dioxide (NO2) [9–11].

mediates on lipids, amino acids, carbohydrates, and nucleic acids [5, 7].

**2. Background**

large phospholipid layer (the comparison of bull sperm, which has a smaller layer of skin), is the cause of the sperm cell. Free radical changes that occur during freezing, the occasion when the effects of sperm freezing occur in the wild boar, affect the integrity of the plasma membrane, the acrosome, the nucleus, as well as the mitochondrial functions and motility of spermatozoa [1–4]. The purpose of this review is to publicize the main causes of ROS generation in sperm cells, as well as a brief

Air is a vital element for any living being and is a mixture of gases based on nitrogen (78%), oxygen (21%), water vapor (variable between 0 and 7%), ozone, carbon dioxide (CO2), hydrogen, and some noble gases such as krypton, neon, helium, and argon. Of these, oxygen (which appeared approximately 2500 million years ago) plays a vital role in the processes of aerobic life, being the second most

Antoine Lavoisier in the eighteenth century gives the name to "oxygen" which means "generator of acids," because despite having a therapeutic use, it was already known that it was a toxic substance, due to its great oxidizing power. In 1774, the toxic effects of the gas are demonstrated, and 6 years later (1780) experiments are made of the use of oxygen in newborns; in 1878, the toxic effect of oxygen in the brain is documented by Paul Bert, manifested by the presence of convulsive crises to more than three atmospheres, and in 1899, when trying to replicate the Bert effect, J. Lorrain Smith reports fatal pneumonia in rats exposed to 73% oxygen for 4 days. In 1940, it is reported that babies with periodic breathing pattern improved with the use of oxygen to 70%, beginning the routine use of oxygen in premature babies. Between 1951 and 1956, it is demonstrated that oxygen was safe when it occurred in concentrations lower than 40%. Harman in 1954 stated that the life expectancy increases decreasing the degree of oxidative phenomena. Thus, throughout history, it has been described that the higher the toxicity of O2 is, the

In veterinary and human medicine, more and more agents that cause diseases in the body have been discovered; some of them are derived from metabolic processes of oxygen, among which are the production of energy, detoxification of harmful compounds, and defense against pathogens, among which are free radicals (RL), which are highly reactive oxidation agents, which act as short-lived chemical inter-

The RL can be divided into the following: (i) reactive oxygen species (ROS), which are highly reactive molecules that constantly attack organisms through oxidation-reduction reactions, among which are molecular oxygen (O2), superoxide anion (O21) hydrogen peroxide (H2O2), hydroperoxyl (HO2), and hydroxyl radical (OH); (ii) the transition metals, which have unpaired electrons and can exist as RL; and (iii) reactive nitrogen species (ERN), which are capable of generating oxidative damage and cell death, among which are nitric oxide (NO), peroxynitrite anion

The RL must be attenuated by different antioxidant defense systems, which involve enzymes and molecules. Antioxidants are divided into enzymatic, also called endogenous production, which are the first line of defense against the production of RL and are proteins with antioxidant capacity that are not consumed when reacting with the RL. Among the most important of this group are catalase, superoxide dismutase, and glutathione peroxidase. The nonenzymatic ones come mainly from the diet and are small liposoluble molecules, which, unlike the

**100**

enzymatic, are consumed during their antioxidant action, so they must be replaced; among the most important in this group are vitamins E and C, beta-carotenes, retinol, uric acid, pyruvate, albumin, carnitine, taurine, hypotaurine, transferrin, ceruloplasmin, polyphenoids, flavonoids, and trace elements [12–16]. These antioxidant defense systems are linked in a cellular buffer system, where they add up and collaborate with each other, to deal with any oxidative aggression in cells, for example, nonenzymatic antioxidants can have synergistic effects in combination with enzymatic antioxidants, regenerating enzymatic antioxidants through the donation of hydrogen, neutralizing molecular oxygen, and catalyzing the synthesis or regeneration of nonenzymatic antioxidants [9].

When there is an imbalance and the amount of RL exceeds the balance between oxidant production and antioxidant capacity, a phenomenon known as oxidative stress (EO) is generated, which has negative consequences on multiple cellular processes [7, 14, 17, 18].

#### **3. Effect of oxidative stress on cells**

Due to aerobic conditions, cells maintain a high concentration of oxidant products in their metabolism, such as RL, which are generated as a result of cellular metabolism and in cellular physiological concentrations are related to cell signaling processes or to fulfill their functions biological, including leukocytes that are recruited to the sites of infection by chemotactic factors and are able to eliminate microorganisms through phagocytosis, exposing them to high concentrations of ROS (superoxide and hydrogen peroxide) and other microbicidal products contained in cell granules. However, when EO exists, ROS can mainly affect cellular components such as proteins, nucleic acids, sugars, and lipids [7, 9, 17].

Most of the main diseases that cause the death of animals and people or deteriorate their quality of life are caused by the RL. Each cell of the body suffers about 10,000 impacts of free radicals per day. For this reason, the EO has been the target of intense research in recent years, mainly in the implications on how mitochondria produce ROS, since they are of vital importance to understand their relationship with the pathogenesis of several chronic diseases such as cancer, osteoporosis, Alzheimer's, type 2 diabetes, neurodegenerative diseases, and cardiovascular diseases such as heart failure [7].

The spermatozoon was the first cell type in which the presence of ROS could be identified, because until a few years ago, ROS were considered toxic elements for sperm; however, the RL are currently known (mainly O2.-) in low concentrations in semen, which play a fundamental role in their biological functions during sperm capacitation, sperm maturation, tyrosine phosphorylation, intergame interaction, and the acrosomal reaction that occurs for fertilization of the oocyte; these phenomena are controlled by the mechanism of defense of enzymatic and nonenzymatic antioxidants that when this balance is broken between the RL and the antioxidant defense system, damages are induced in the nucleic acids, proteins, and lipids present in the membrane of the sperm, causing loss of mobility, decrease in viability, and alterations in the intermediate piece, which finally produce a decrease in seminal quality or sperm death [2, 7, 14, 16, 19–26]. A clear example of this is nitric oxide (NO), which has an important function in the sperm pathophysiology, since in low concentrations it favors the processes of sperm capacitation, the acrosomal reaction, and the union to the zona pelucida; however, in high concentrations it leads to the formation of peroxynitrites, which alters sperm motility [27].

It has been observed that in the ejaculate, the main sources of ROS are leukocytes and abnormal sperm cells, although it has been proposed that there are other possibilities on the generation of intracellular ROS in the spermatozoon, such as the leakage of electrons from the mitochondrial transport chain, NADPH oxidase as a possible source of ROS, and the generation of RL by means of nitric oxide in the post-acrosomal and equatorial regions, which can generate a change in the basal state of the oxidizing agents and induce changes in sperm activity [7].

#### **4. Effect of oxidative stress on the cytoplasmic sperm membrane**

The spermatic membrane is asymmetric in its structure and functions. It is formed by an association of phospholipids, plasmalogens, and sphingomyelins in dynamic equilibrium with membrane proteins making it an easy target of oxidizing agents. Cholesterol and phospholipids are important in maintaining the structural integrity of membrane systems. In particular, the plasma membrane of the sperm possesses a large quantity of polyunsaturated fatty acids (PUFA), which are necessary for the acrosome reaction and the interaction with the oocyte membrane. On the other hand, the high content of polyunsaturated fatty acids in the plasma membranes of sperm makes them very susceptible to lipoperoxidation (LP), making it highly vulnerable to oxidative stress [7, 14, 20, 24].

The low concentrations of antioxidant enzymes (catalases, dismutases, peroxidases, and glutathione reductase) in the plasma membrane also convert sperm into cells susceptible to the attack of the RL (particularly the attack of hydroxyl radical (OH) and hydroperoxyl (HO2)), on all the post-acrosomal region, causing alterations in its permeability (since ROS induces LP of the phospholipids of the membrane, which causes the appearance of "orifices"), affecting the Na+ and Ca2+ pumps, causing these to enter cations into the sperm, altering the osmolarity, which causes the formation of few soluble calcium phosphates, depletion of ATP, and activation by means of Ca2+ of proteolytic and phosphoglycolytic enzymes. It also damages the enzymes lactate dehydrogenase, pyruvate kinase, glyceraldehyde 3 phosphate dehydrogenase, and ATPase, generating loss or reduction in mobility, protein and lipid damage, alterations in deoxyribonucleic acid (DNA), anomalies in its morphology, fertility problems, and cell death [9, 14, 20, 23, 24, 28, 29].

#### **5. Effect of oxidative stress on sperm mitochondria**

Mitochondria are considered one of the main cellular sources of ROS, which are responsible for regulating physiological processes such as transduction of intracellular signals, the response to oxidative stress, embryonic development, cell proliferation and adhesion, gene expression, and apoptosis [7].

In the sperm mitochondria provide the highest amount of ATP, through glycolysis and oxidative phosphorylation, contributing to the formation of RL during these processes [7, 30, 31]. However, when there is disruption of the mitochondrial respiratory chain (during freezing), these are responsible for the formation and release of ROS. This interruption causes oxygen to undergo complete reductions producing, instead of water molecules, intermediate molecules such as superoxide anion, hydroxyl radical, and hydrogen peroxide, triggering a phenomenon similar to apoptosis, responsible for both the death of sperm and the sublethal damages that decrease the half-life and fertilizing capacity of the cells (**Figure 1**) [32].

The freezing of semen also exerts an important damage in the mitochondria, since it has been demonstrated that the EO induces damage in the mitochondrial DNA, observing that the mutation spectrum of said DNA, in the spermatozoon, can

**103**

**Figure 1.**

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

be 10–100 times greater than to nuclear DNA. This can be explained by the crosslinking of DNA proteins that cause RL, exchange of sister chromatids, damage to the structure of deoxyribose phosphate, oxidation of nitrogenous bases, conversion of bases (the deamination of cytosine into uracil and of the 5-methylcytosine in thymidine), ring openings, base release, and chain breaking (one or two strands).

The acrosome is also affected by the action of the RL during the transport of the sperm through the epididymis, mainly by hydrogen peroxide, since it inhibits the induction of the acrosomal reaction and damages the integrity of the acrosome,

Much of the DNA damage in the sperm is generated by the EO. The damage that ROS exerts directly on sperm DNA can induce mutations, affecting the paternal genomics of the embryo, and can be an indication of male fertility [20, 24]. To demonstrate this, in studies where sperm were exposed to high concentrations of artificially produced ROS, a significant increase in DNA damage, decreased sperm motility, and induction in apoptotic processes could be observed [7]. These damages in the chromatic sperm depend on endogenous factors such as in the testicles or the epididymis (during sperm maturation), and exogenous factors as DNA peroxidative damage, infections, immunological factors, or various chemical agents. These may be related to failures in packaging, nuclear maturity, chromatin

This leads directly to a decrease in fertility [4, 7, 9, 24, 33].

*Lesions resulting from the freezing of pig semen (modified from [4]).*

**7. Effect of oxidative stress on sperm DNA**

**6. Effect of oxidative stress on the spermatic acrosome**

fragmentation, aneuploidies, or DNA integrity defects [7, 24].

producing a malfunction at the time of fertilization of the oocyte [34].

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

*Glutathione System and Oxidative Stress in Health and Disease*

ing it highly vulnerable to oxidative stress [7, 14, 20, 24].

possibilities on the generation of intracellular ROS in the spermatozoon, such as the leakage of electrons from the mitochondrial transport chain, NADPH oxidase as a possible source of ROS, and the generation of RL by means of nitric oxide in the post-acrosomal and equatorial regions, which can generate a change in the basal

state of the oxidizing agents and induce changes in sperm activity [7].

**4. Effect of oxidative stress on the cytoplasmic sperm membrane**

The spermatic membrane is asymmetric in its structure and functions. It is formed by an association of phospholipids, plasmalogens, and sphingomyelins in dynamic equilibrium with membrane proteins making it an easy target of oxidizing agents. Cholesterol and phospholipids are important in maintaining the structural integrity of membrane systems. In particular, the plasma membrane of the sperm possesses a large quantity of polyunsaturated fatty acids (PUFA), which are necessary for the acrosome reaction and the interaction with the oocyte membrane. On the other hand, the high content of polyunsaturated fatty acids in the plasma membranes of sperm makes them very susceptible to lipoperoxidation (LP), mak-

The low concentrations of antioxidant enzymes (catalases, dismutases, peroxidases, and glutathione reductase) in the plasma membrane also convert sperm into cells susceptible to the attack of the RL (particularly the attack of hydroxyl radical (OH) and hydroperoxyl (HO2)), on all the post-acrosomal region, causing alterations in its permeability (since ROS induces LP of the phospholipids of the membrane, which causes the appearance of "orifices"), affecting the Na+

Ca2+ pumps, causing these to enter cations into the sperm, altering the osmolarity, which causes the formation of few soluble calcium phosphates, depletion of ATP, and activation by means of Ca2+ of proteolytic and phosphoglycolytic enzymes. It also damages the enzymes lactate dehydrogenase, pyruvate kinase, glyceraldehyde 3 phosphate dehydrogenase, and ATPase, generating loss or reduction in mobility, protein and lipid damage, alterations in deoxyribonucleic acid (DNA), anomalies in

Mitochondria are considered one of the main cellular sources of ROS, which are responsible for regulating physiological processes such as transduction of intracellular signals, the response to oxidative stress, embryonic development, cell prolif-

In the sperm mitochondria provide the highest amount of ATP, through glycolysis and oxidative phosphorylation, contributing to the formation of RL during these processes [7, 30, 31]. However, when there is disruption of the mitochondrial respiratory chain (during freezing), these are responsible for the formation and release of ROS. This interruption causes oxygen to undergo complete reductions producing, instead of water molecules, intermediate molecules such as superoxide anion, hydroxyl radical, and hydrogen peroxide, triggering a phenomenon similar to apoptosis, responsible for both the death of sperm and the sublethal damages that decrease the half-life and fertilizing capacity of the cells (**Figure 1**) [32]. The freezing of semen also exerts an important damage in the mitochondria, since it has been demonstrated that the EO induces damage in the mitochondrial DNA, observing that the mutation spectrum of said DNA, in the spermatozoon, can

its morphology, fertility problems, and cell death [9, 14, 20, 23, 24, 28, 29].

**5. Effect of oxidative stress on sperm mitochondria**

eration and adhesion, gene expression, and apoptosis [7].

and

**102**

**Figure 1.** *Lesions resulting from the freezing of pig semen (modified from [4]).*

be 10–100 times greater than to nuclear DNA. This can be explained by the crosslinking of DNA proteins that cause RL, exchange of sister chromatids, damage to the structure of deoxyribose phosphate, oxidation of nitrogenous bases, conversion of bases (the deamination of cytosine into uracil and of the 5-methylcytosine in thymidine), ring openings, base release, and chain breaking (one or two strands). This leads directly to a decrease in fertility [4, 7, 9, 24, 33].

#### **6. Effect of oxidative stress on the spermatic acrosome**

The acrosome is also affected by the action of the RL during the transport of the sperm through the epididymis, mainly by hydrogen peroxide, since it inhibits the induction of the acrosomal reaction and damages the integrity of the acrosome, producing a malfunction at the time of fertilization of the oocyte [34].

### **7. Effect of oxidative stress on sperm DNA**

Much of the DNA damage in the sperm is generated by the EO. The damage that ROS exerts directly on sperm DNA can induce mutations, affecting the paternal genomics of the embryo, and can be an indication of male fertility [20, 24]. To demonstrate this, in studies where sperm were exposed to high concentrations of artificially produced ROS, a significant increase in DNA damage, decreased sperm motility, and induction in apoptotic processes could be observed [7]. These damages in the chromatic sperm depend on endogenous factors such as in the testicles or the epididymis (during sperm maturation), and exogenous factors as DNA peroxidative damage, infections, immunological factors, or various chemical agents. These may be related to failures in packaging, nuclear maturity, chromatin fragmentation, aneuploidies, or DNA integrity defects [7, 24].

In any part of the spermatogenesis, a damage to the spermatic DNA can be induced, which despite is being a multifactorial phenomenon and not being completely delimited; some of the factors that can produce irreversible damage is the generation of ROS, which come from the respiratory chain, since these oxidative molecules react with the nitrogenous bases and with deoxyribose, causing DNA fragmentation, problems in the compaction and winding of the DNA inside the chromatin, deletions, mutations, translocations, degradation of purine or pyrimidic bases, rupture of chains, and cross-links between proteins and DNA. The magnitude of damage induced by RL during sperm transit through the epididymis depends on the levels of these produced by immature sperm, the presence of epithelial cells or activated leukocytes in the epididymis, and the levels of antioxidant enzymes present in the epididymis lumen [2, 4, 21, 23, 24, 34–37].

It is important to note that there are mainly two RL that affect the DNA strand. The first is the OH radical, which results in the formation of 8-OH-guanine and 8-OH-2 deoxyguanosine at the first stage, attacking the purines as pyrimidines, causing fragmentation of double-stranded DNA, and the second is the radical O21, which generally produces only guanine adducts, especially 8-hydroxyguanine, which affect sperm motility [4, 7, 9, 24]. If a sperm with fragmentation of double-stranded DNA manages to fertilize an oocyte, it is incompatible and may affect the normal development of pregnancy [24].

#### **8. Effect of oxidative stress on female and male fertility**

Infertility is defined as the inability of a couple to conceive after a year of sexual intercourse without contraceptive measures [24]. There are multiple causes of male infertility, which may be congenital or acquired; of all of them, idiopathic infertility is caused by multiple factors such as endocrine alterations, oxidative stress, and genetic or epigenetic alterations [38].

In particular, the role of EO as one of the main causes of male infertility has been well established, since ROS can affect all cellular components, including the AGP of membranes, proteins, and nucleic acids, causing in males oligozoospermia, prostate carcinoma, cryptorchidism, varicocele, low seminal quality, low motility of spermatozoa, decreased sperm concentration, and acceleration in the process of apoptosis of geminous cells [24, 27].

In a study conducted by Pérez [27], it was observed that in asthenozoospermic patients have an overexpression of the enzyme inducible nitric oxide synthase (iNOS), compared with the normospermic, which results in a sperm dysfunction and in the decrease of the fecundate capacity of sperm. It has also been shown that in sperm of individuals whose partners have recurrent early embryonic death, there is a significant increase in aneuploidies, abnormal chromatin condensation, DNA fragmentation, apoptosis, and abnormal sperm morphology [19].

It is important to highlight the importance of antioxidants in semen, since it has been observed that the low levels or deficiency of antioxidants in the seminal plasma leaves the sperm unprotected to the EO [20]. So the use of antioxidants has been proposed as a tool to protect sperm from oxidative damage, and it has even been proven that the addition of antioxidants (vitamin C, E or glutathione), at the time of the seminal conservation, produces better results in the seminal evaluation at the time of insemination [4, 7, 29, 39].

In the case of females, it has been suggested that ROS can participate in the formation of adhesions associated with endometriosis, decreasing its fertility. There are also alterations of folliculogenesis caused by ROS, which can deteriorate the quality of the oocyte and have been proposed as a cause of subfertility associated

**105**

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

**9. Conclusions**

**Thanks**

number 624422.

**Implications**

the male.

importance at present.

**Declaration of conflict of interest**

The authors declare that there are no conflicts of interest.

affecting the growth rate of piglets [33, 40–42].

with endometriosis. The EO has also been associated with numerous pathologies among which we can mention mastitis, edema of the udder, higher incidence of diseases in the peripartum period, deficit in the synthesis of steroid horns in cows, and degenerative nutritional myopathy in sheep. In the case of sows, the EO can cause postweaning inflammatory states, modifying the status of selenium and vitamin E

The effect of EO on sperm cells significantly affects the fecundating capacity of sperm, causing infertility in males and/or low reproductive parameters in females so that the issue of EO in the fertilizing capacity of spermatozoa mammals is of utmost

To CONACYT, for the support to the second author as a fellow with registration

In this paper, a review is made of a brief history of how the toxicity of oxygen and free radicals was discovered, the oxidative stress in cells, and the effect of oxidative stress in the cytoplasmic sperm membrane, in the spermatic mitochondria, in the spermatic acrosome, in the sperm DNA, and in the fertility of the female and *Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

with endometriosis. The EO has also been associated with numerous pathologies among which we can mention mastitis, edema of the udder, higher incidence of diseases in the peripartum period, deficit in the synthesis of steroid horns in cows, and degenerative nutritional myopathy in sheep. In the case of sows, the EO can cause postweaning inflammatory states, modifying the status of selenium and vitamin E affecting the growth rate of piglets [33, 40–42].

#### **9. Conclusions**

*Glutathione System and Oxidative Stress in Health and Disease*

In any part of the spermatogenesis, a damage to the spermatic DNA can be induced, which despite is being a multifactorial phenomenon and not being completely delimited; some of the factors that can produce irreversible damage is the generation of ROS, which come from the respiratory chain, since these oxidative molecules react with the nitrogenous bases and with deoxyribose, causing DNA fragmentation, problems in the compaction and winding of the DNA inside the chromatin, deletions, mutations, translocations, degradation of purine or pyrimidic bases, rupture of chains, and cross-links between proteins and DNA. The magnitude of damage induced by RL during sperm transit through the epididymis depends on the levels of these produced by immature sperm, the presence of epithelial cells or activated leukocytes in the epididymis, and the levels of antioxidant

It is important to note that there are mainly two RL that affect the DNA strand. The first is the OH radical, which results in the formation of 8-OH-guanine and 8-OH-2 deoxyguanosine at the first stage, attacking the purines as pyrimidines, causing fragmentation of double-stranded DNA, and the second is the radical O21, which generally produces only guanine adducts, especially 8-hydroxyguanine, which affect sperm motility [4, 7, 9, 24]. If a sperm with fragmentation of double-stranded DNA manages to fertilize an oocyte, it is incompatible and may affect the normal

Infertility is defined as the inability of a couple to conceive after a year of sexual intercourse without contraceptive measures [24]. There are multiple causes of male infertility, which may be congenital or acquired; of all of them, idiopathic infertility is caused by multiple factors such as endocrine alterations, oxidative stress, and

In particular, the role of EO as one of the main causes of male infertility has been well established, since ROS can affect all cellular components, including the AGP of membranes, proteins, and nucleic acids, causing in males oligozoospermia, prostate carcinoma, cryptorchidism, varicocele, low seminal quality, low motility of spermatozoa, decreased sperm concentration, and acceleration in the process of

In a study conducted by Pérez [27], it was observed that in asthenozoospermic patients have an overexpression of the enzyme inducible nitric oxide synthase (iNOS), compared with the normospermic, which results in a sperm dysfunction and in the decrease of the fecundate capacity of sperm. It has also been shown that in sperm of individuals whose partners have recurrent early embryonic death, there is a significant increase in aneuploidies, abnormal chromatin condensation, DNA

It is important to highlight the importance of antioxidants in semen, since it has been observed that the low levels or deficiency of antioxidants in the seminal plasma leaves the sperm unprotected to the EO [20]. So the use of antioxidants has been proposed as a tool to protect sperm from oxidative damage, and it has even been proven that the addition of antioxidants (vitamin C, E or glutathione), at the time of the seminal conservation, produces better results in the seminal evaluation

In the case of females, it has been suggested that ROS can participate in the formation of adhesions associated with endometriosis, decreasing its fertility. There are also alterations of folliculogenesis caused by ROS, which can deteriorate the quality of the oocyte and have been proposed as a cause of subfertility associated

enzymes present in the epididymis lumen [2, 4, 21, 23, 24, 34–37].

**8. Effect of oxidative stress on female and male fertility**

fragmentation, apoptosis, and abnormal sperm morphology [19].

development of pregnancy [24].

genetic or epigenetic alterations [38].

apoptosis of geminous cells [24, 27].

at the time of insemination [4, 7, 29, 39].

**104**

The effect of EO on sperm cells significantly affects the fecundating capacity of sperm, causing infertility in males and/or low reproductive parameters in females so that the issue of EO in the fertilizing capacity of spermatozoa mammals is of utmost importance at present.

#### **Declaration of conflict of interest**

The authors declare that there are no conflicts of interest.

#### **Thanks**

To CONACYT, for the support to the second author as a fellow with registration number 624422.

#### **Implications**

In this paper, a review is made of a brief history of how the toxicity of oxygen and free radicals was discovered, the oxidative stress in cells, and the effect of oxidative stress in the cytoplasmic sperm membrane, in the spermatic mitochondria, in the spermatic acrosome, in the sperm DNA, and in the fertility of the female and the male.

### **Author details**

Alejando Córdova Izquierdo1 \*, Adrian Emmanuel Iglesias Reyes2 , Alda Roció Ortiz Muñiz3 , María de Lourdes Juárez Mosqueda4 , Jesús Alberto Guevara González1 , Juan Eulogio Guerra Liera5 , Gustavo Ruiz Lang1 , Rubén Huerta Crispín6 , Abel E. Villa Mancera6 , Pedro Sánchez Aparico7 and Raúl Sánchez Sánchez8

1 Department of Agricultural and Animal Production, University Autonomous Unit Xochimilco, Mexico City, Mexico

2 Master's Degree in Agricultural Sciences, University Autonomous Unit Xochimilco, Mexico City, Mexico

3 University Autonomous Metropolitan Unit Iztapalapa, Mexico City, Mexico

4 Department of Morphology, FMVZ-UNAM, Mexico City, Mexico

5 Faculty of Agronomy, Autonomous University of Sinaloa, Mexico

6 Faculty of Veterinary, Benemérita Autonomous University of Puebla, Mexico

7 Faculty of Veterinary, Autonomous Mexico State University, Mexico

8 Department of Reproduction, INIA, Madrid, Spain

\*Address all correspondence to: acordova@correo.xoc.uam.mx

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**107**

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

[1] Restrepo BG, Pizarro LE, Albero RB. Estrés oxidativo en el semen equino criopreservado. Revista Lasallista de Investivación. 2012;**9**(1):128-136

[9] Quintanar EMA, Calderón, Víctor SJ. La capacidad antioxidante total. Bases y Aplicaciones. REB.

[10] Berzosa S. Estudio del daño oxidativo, niveles de defensa

Fisiología; 2011. pp. 11-15

[11] García TBE, Saldaña BA, Saldaña GL. El estrés oxidativo y los antioxidantes en la prevención del cáncer. Revista Habanera de Ciencias

Médicas. 2012;**12**(2):187-196

[12] Villa NA, Sánchez LE, Ceballos A. Actividad de glutatión peroxidasa y superóxido dismutasa en plasma seminal y sangre en cerdos reproductores. Veterinaria e Zootecnia. 2009;**3**(1):9-51

[13] Gašparovic AC, Lovakovic T, Zarkovic N. Oxidative stress and antioxidants: Biological response modifiers of oxidative homeostasis in cancer. Periodicum Biologorum.

[14] Flores C, Márquez Y, Vilanova L, Mendoza C. Dienos conjugados y malondialdehído como indicadores de lipoperoxidación en semen de toros "Carora". Revista Veterinaria.

[15] Villalba MC. Implicaciones del estrés oxidativo en la infertilidad masculina: Análisis de marcadores bioquímicos en plasma seminal y su asociación con parámetros del seminograma y la capacitación espermática [Mediora presentada por Celia Villalba Martínez

Universitat d' Alacant. Departamento de

para optar al grado de Doctor].

Biotecnología; 2014. pp. 1-321

2010;**112**(4):433-439

2011;**22**(2):91-94

antioxidantes y efecto ergogénico de la melatonina en pruebas de esfuerzo físico agudo [Tesis doctoral para optar al grado de doctor europeo]. Universidad de Zaragoza. Facultad de Medicina. Departamento de Farmacología y

2009;**28**(3):89-101

[2] Williams S. Criopreservación de semen porcino: Desafíos y perspectivas. Revista Brasileira de Reprodução Animal. 2013;**37**(2):207-2012

[3] Gallardo BJO, Vargas SCA. Evaluacion de tres diluyentes para criopreservar semen bovino de toros cruce Sahiwal (Bos Taurus) En el trópico Húmedo [Tesis previa a la obtención del título de Ingeniero Agropecuario]. Universidad de las Fuerzas Armadas. Departamento de Ciencias de la Vida y la

Agricultura; 2015. pp. 1-89

2017;**2017**:1-12

2011;**20**(1):42-45

2015;**80**(6):486-492

Farmacia. 2005;**39**(3):1-11

[4] Yeste M, Rodríguez GJE, Bonet S. Artificial insemination with frozenthawed boar sperm. Molecular Reproduction and Development.

[5] Villa ANA, Ceballos MA. Radicales libres e infertilidad en el macho.

Veterinaria e Zootecnia. 2007;**1**(2):87-97

[6] Sánchez I, Torres V, Moreno O, Rodríguez A. Determinación del estrés oxidativo mediante peroxidación lipídica en cristalinos humanos con cataratas. Revista de Facultad de Medicina, Universidad de Los Andes.

[7] Mayorga TJM, Camargo M, Cadavid Ángela P, Cardona Maya Walter D. Estrés oxidativo: ¿un estado celular defectuoso para la función espermática? Revista Chilena de Obstetricia y Ginecología.

[8] Martínez SG. Especies reactivas de oxígeno y balance redox, parte 1: Aspectos básicos y principales especies reactivas de oxígeno. Revista Cubana de

**References**

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

#### **References**

*Glutathione System and Oxidative Stress in Health and Disease*

**106**

**Author details**

Alejando Córdova Izquierdo1

Jesús Alberto Guevara González1

Xochimilco, Mexico City, Mexico

Xochimilco, Mexico City, Mexico

Alda Roció Ortiz Muñiz3

Rubén Huerta Crispín6

and Raúl Sánchez Sánchez8

\*, Adrian Emmanuel Iglesias Reyes2

, Juan Eulogio Guerra Liera5

, María de Lourdes Juárez Mosqueda4

1 Department of Agricultural and Animal Production, University Autonomous Unit

, Abel E. Villa Mancera6

2 Master's Degree in Agricultural Sciences, University Autonomous Unit

4 Department of Morphology, FMVZ-UNAM, Mexico City, Mexico

5 Faculty of Agronomy, Autonomous University of Sinaloa, Mexico

7 Faculty of Veterinary, Autonomous Mexico State University, Mexico

\*Address all correspondence to: acordova@correo.xoc.uam.mx

8 Department of Reproduction, INIA, Madrid, Spain

provided the original work is properly cited.

3 University Autonomous Metropolitan Unit Iztapalapa, Mexico City, Mexico

6 Faculty of Veterinary, Benemérita Autonomous University of Puebla, Mexico

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

,

, Gustavo Ruiz Lang1

,

,

, Pedro Sánchez Aparico7

[1] Restrepo BG, Pizarro LE, Albero RB. Estrés oxidativo en el semen equino criopreservado. Revista Lasallista de Investivación. 2012;**9**(1):128-136

[2] Williams S. Criopreservación de semen porcino: Desafíos y perspectivas. Revista Brasileira de Reprodução Animal. 2013;**37**(2):207-2012

[3] Gallardo BJO, Vargas SCA. Evaluacion de tres diluyentes para criopreservar semen bovino de toros cruce Sahiwal (Bos Taurus) En el trópico Húmedo [Tesis previa a la obtención del título de Ingeniero Agropecuario]. Universidad de las Fuerzas Armadas. Departamento de Ciencias de la Vida y la Agricultura; 2015. pp. 1-89

[4] Yeste M, Rodríguez GJE, Bonet S. Artificial insemination with frozenthawed boar sperm. Molecular Reproduction and Development. 2017;**2017**:1-12

[5] Villa ANA, Ceballos MA. Radicales libres e infertilidad en el macho. Veterinaria e Zootecnia. 2007;**1**(2):87-97

[6] Sánchez I, Torres V, Moreno O, Rodríguez A. Determinación del estrés oxidativo mediante peroxidación lipídica en cristalinos humanos con cataratas. Revista de Facultad de Medicina, Universidad de Los Andes. 2011;**20**(1):42-45

[7] Mayorga TJM, Camargo M, Cadavid Ángela P, Cardona Maya Walter D. Estrés oxidativo: ¿un estado celular defectuoso para la función espermática? Revista Chilena de Obstetricia y Ginecología. 2015;**80**(6):486-492

[8] Martínez SG. Especies reactivas de oxígeno y balance redox, parte 1: Aspectos básicos y principales especies reactivas de oxígeno. Revista Cubana de Farmacia. 2005;**39**(3):1-11

[9] Quintanar EMA, Calderón, Víctor SJ. La capacidad antioxidante total. Bases y Aplicaciones. REB. 2009;**28**(3):89-101

[10] Berzosa S. Estudio del daño oxidativo, niveles de defensa antioxidantes y efecto ergogénico de la melatonina en pruebas de esfuerzo físico agudo [Tesis doctoral para optar al grado de doctor europeo]. Universidad de Zaragoza. Facultad de Medicina. Departamento de Farmacología y Fisiología; 2011. pp. 11-15

[11] García TBE, Saldaña BA, Saldaña GL. El estrés oxidativo y los antioxidantes en la prevención del cáncer. Revista Habanera de Ciencias Médicas. 2012;**12**(2):187-196

[12] Villa NA, Sánchez LE, Ceballos A. Actividad de glutatión peroxidasa y superóxido dismutasa en plasma seminal y sangre en cerdos reproductores. Veterinaria e Zootecnia. 2009;**3**(1):9-51

[13] Gašparovic AC, Lovakovic T, Zarkovic N. Oxidative stress and antioxidants: Biological response modifiers of oxidative homeostasis in cancer. Periodicum Biologorum. 2010;**112**(4):433-439

[14] Flores C, Márquez Y, Vilanova L, Mendoza C. Dienos conjugados y malondialdehído como indicadores de lipoperoxidación en semen de toros "Carora". Revista Veterinaria. 2011;**22**(2):91-94

[15] Villalba MC. Implicaciones del estrés oxidativo en la infertilidad masculina: Análisis de marcadores bioquímicos en plasma seminal y su asociación con parámetros del seminograma y la capacitación espermática [Mediora presentada por Celia Villalba Martínez para optar al grado de Doctor]. Universitat d' Alacant. Departamento de Biotecnología; 2014. pp. 1-321

[16] Gumbao B, David D. Efecto antioxidante del glutatión aplicado en el medio de descongelación seminal de tres especies con interés. Universidad de Murcia. Departamento de Fisiología; 2015. pp. 1-154

[17] Zepeda AB, Farías JB. Antioxidantes frente a estrés oxidativo inducido por hipoxia hipobária en testículo y epidídimo. Revista de Farmacología de Chile. 2013;**6**(1):31-36

[18] Cota MAI. Actividad de las enzimas antioxidantes: Superóxido dismutasa, catalasa y glutatión peroxidasa, en el espermatozoide y líquido seminal de conejo nueva Zelanda y su relación con el sobrepeso [tesis para obtener el grado de Maestra en Biología de la Reproducción Animal]. Universidad Autónoma Metropolitana Unidad Iztapalapa; 2014. pp. 1-71

[19] Rodríguez E, Gil VAM, Aguirre ADC, Cardona MW, Cadavid AP. Evaluación de parámetros seminales no convencionales en individuos cuyas parejas presentan muerte embrionaria temprana recurrente: En busca de un valor de referencia. Biomédica. 2011;**31**:100-107

[20] Villa NA, Castaño D, Duque PC, Ceballos A. Glutatione peroxidase and superoxide dismutase activities in blood and seminal plasma in colombian stallions. Revista Colombiana de Ciencias Pecuarias. 2012;**25**:64-70

[21] Santiani A. Uso de Antioxidantes para mejorar la calidad de semen criopreservado. Spermova. 2013;**3**(2):154-157

[22] Zhong RZ, Zhou DW. Oxidative stress and role of natural plant derived antioxidants in animal reproduction. Journal of Integrative Agriculture. 2013;**12**(10):1826-1838

[23] Orozco BMG, Navarrete MR, Murray NR, Curiel PE. Efecto de

la temperatura en el proceso de criopreservación, sobre la motilidad progresiva del espermatozoide de cerdo. Revista Educatecnociencia. 2014;**4**(59):53-64

[24] Paparella CV, Pavesi AB, Feldman RN, Bouvet BR. Importancia de la evaluación del estrés oxidativo en el semen humano. Archivio di Medicina Interna. 2015;**37**(1):7-14

[25] Álvarez Rodríguez M, Vicente Carrillo A, Rodríguez Martínez H. Exogenous individual lecthin-phospholipids (Phosphatidylchoine and Phosphatidylglycerol) cannot prevent the oxidative stress imposed by criopreservation of boar sperm. Journal of Veterinary Medicine and Surgery. 2017;**1**(2):1-11

[26] Domínguez CO, Toledano OÁ, Ávalos RA. Efecto del suplemento de astaxantina sobre la calidad seminal en *Moenkhausia sanctaefiloenae* (Teleostei: Characidae). Latin American Journal of Aquatic Research. 2015;**43**(1):215-221

[27] Pérez MS. Óxido nítrico sintetasa y nitración en tirosina en la astenozoospermia: Un estudio inmunológico. Revista Saegre. 2012;**19**(3):57.59

[28] Pulgar PEA. Sistemas transportadores de vitamina C en células espermatogénicas y espermatozoides [tesis de grado presentada como parte de los requisitos para optar al grado de Licenciado en bioquímica y al título profesional de bioquímico]. Facultad de Ciencias, Universidad Austral de Chile; 2009. pp. 1-192

[29] Thongrueang N, Chaibangyang N, Chanapiwat P, Kaeoket K. Effects of adding melatonin on the quality of frozen-thawed boar semen. Journal of Applied Animal Science. 2017;**10**(2):47-56

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2012;**5**(1):9-19

*Effect of Oxidative Stress on Sperm Cells DOI: http://dx.doi.org/10.5772/intechopen.88499*

*Glutathione System and Oxidative Stress in Health and Disease*

la temperatura en el proceso de criopreservación, sobre la motilidad progresiva del espermatozoide de cerdo. Revista Educatecnociencia.

[24] Paparella CV, Pavesi AB,

Interna. 2015;**37**(1):7-14

[25] Álvarez Rodríguez M, Vicente Carrillo A, Rodríguez Martínez H. Exogenous

(Phosphatidylchoine and

2017;**1**(2):1-11

2015;**43**(1):215-221

2012;**19**(3):57.59

2009. pp. 1-192

2017;**10**(2):47-56

[27] Pérez MS. Óxido nítrico sintetasa y nitración en tirosina en la astenozoospermia: Un estudio inmunológico. Revista Saegre.

[28] Pulgar PEA. Sistemas

individual lecthin-phospholipids

Phosphatidylglycerol) cannot prevent the oxidative stress imposed by

criopreservation of boar sperm. Journal of Veterinary Medicine and Surgery.

[26] Domínguez CO, Toledano OÁ, Ávalos RA. Efecto del suplemento de astaxantina sobre la calidad seminal en *Moenkhausia sanctaefiloenae* (Teleostei: Characidae). Latin

American Journal of Aquatic Research.

transportadores de vitamina C en células espermatogénicas y espermatozoides [tesis de grado presentada como parte de los requisitos para optar al grado de Licenciado en bioquímica y al título profesional de bioquímico]. Facultad de Ciencias, Universidad Austral de Chile;

[29] Thongrueang N, Chaibangyang N, Chanapiwat P, Kaeoket K. Effects of adding melatonin on the quality of frozen-thawed boar semen. Journal of Applied Animal Science.

Feldman RN, Bouvet BR. Importancia de la evaluación del estrés oxidativo en el semen humano. Archivio di Medicina

2014;**4**(59):53-64

[16] Gumbao B, David D. Efecto antioxidante del glutatión aplicado en el medio de descongelación seminal de tres especies con interés. Universidad de Murcia. Departamento de Fisiología;

[17] Zepeda AB, Farías JB. Antioxidantes frente a estrés oxidativo inducido por hipoxia hipobária en testículo y epidídimo. Revista de Farmacología

[18] Cota MAI. Actividad de las enzimas antioxidantes: Superóxido dismutasa, catalasa y glutatión peroxidasa, en el espermatozoide y líquido seminal de conejo nueva Zelanda y su relación con el sobrepeso [tesis para obtener el grado de Maestra en Biología de la Reproducción Animal]. Universidad Autónoma Metropolitana Unidad

2015. pp. 1-154

de Chile. 2013;**6**(1):31-36

Iztapalapa; 2014. pp. 1-71

[19] Rodríguez E, Gil VAM, Aguirre ADC, Cardona MW,

Cadavid AP. Evaluación de parámetros seminales no convencionales en individuos cuyas parejas presentan muerte embrionaria temprana recurrente: En busca de un valor de referencia. Biomédica. 2011;**31**:100-107

[20] Villa NA, Castaño D, Duque PC, Ceballos A. Glutatione peroxidase and superoxide dismutase activities in blood and seminal plasma in colombian stallions. Revista Colombiana de Ciencias Pecuarias. 2012;**25**:64-70

[21] Santiani A. Uso de Antioxidantes para mejorar la calidad de semen criopreservado. Spermova.

[22] Zhong RZ, Zhou DW. Oxidative stress and role of natural plant derived antioxidants in animal reproduction. Journal of Integrative Agriculture.

[23] Orozco BMG, Navarrete MR, Murray NR, Curiel PE. Efecto de

2013;**3**(2):154-157

2013;**12**(10):1826-1838

**108**

[30] Ramón MMO. Estudio de la actividad aminopeptidásica en espermatozoide astenozoospérmicos. Comparación clínica [tesis doctoral]. Universidad del País Vasco, Facultad de Medicina y Odontología, Departamento de Fisiología; 2014. pp. 1-249

[31] Flores C, Vilanova L. Metabolismo espermático. Gaceta de Ciencias Veterinarias. 2015;**20**(1):23-32

[32] Ortega FC. Factores implicados en la variabilidad individual en la respuesta a la congelación del eyaculado equino: Estructura de subpoblaciones, estrés oxidativo y cambios apoptóticos [tesis doctoral]. Universidad de Extremadura, Facultad de veterinaria, Departamento de Medicina Animal; 2011. pp. 1-176

[33] Gupta S, Goldberg Jeffrey M, Aziz N, Goldberg E, Krajcir N, Agarwal A. Mecanismos patogénicos de la infertilidad asociada con endometriosis. Revista Mexicana de Medicina de la Reproducción. 2010;**3**(2):83-97

[34] Córdova JCA. Control de la peroxidación lipídica del semen refrigerado y criopreservado de verraco mediante antioxidantes (α-Tocoferol/ Glutatión reducido) y su repercusión sobre la calidad espermática [tesis doctoral]. Universidad de León. Facultad de Veterinaria. Departamento de Sanidad Animal; 2010. pp. 1-290

[35] Birben E, Murat SU, Sackesen C, Erzyrum S, Kalayci O. Oxidative stress and antioxidant defense. The World Allergy Organization Journal. 2012;**5**(1):9-19

[36] Mayorga TJM, Peña B, Cadavid ÁP, Walter CM. La importancia clínica del ADN espermática en el análisis seminal cotidiano. Revista Chilena de Obstetricia y Ginecología. 2010;**80**(3):256-268

[37] Leyland F. Assessment of ageingdependent effects on sperm functions following semen cryopreservation. Veterinary Medicine—Open Journal. 2017;**2**(2):1-2

[38] Palma C, Vinay BJI. Infertilidad masculina. Revista Médica Clínica Las Condes. 2014;**25**(1):122-128

[39] Córdova IA, Iglesias RAE, Espinosa CR, Guerra LJE, Villa MAE, Huerta CR, et al. Effect of addition of antioxidants in the extender to freeze boar in two types of straws on sperm quality. International Journal of Recent Scientific Research. 2017;**8**(6):17466-17468

[40] Castro C, Márquez A. Uso de antioxidantes en animales domésticos. Gaceta de Ciencias Veterinarias. 2006;**12**(1):5-12

[41] Quiles A. Efecto del selenio en la producción porcina. Mundo Ganadero. 2008;**8**:42-44

[42] Reinoso V, Soto C. Importancia de la vitamina E y el selenio en vacas lecheras. In: Artigas, Uruguay. Sitio Argentino de Producción Animal, 1-3. 2009. Obtenido de: http://www.produccion-animal. com.ar/suplementacion\_mineral/104- Vit\_E\_y\_Se.pdf

**111**

**Chapter 7**

**Abstract**

Structure

ity of these enzymes.

**1. Introduction**

*Yonca Yuzugullu Karakus*

Typical Catalases: Function and

Catalase (EC 1.11.1.6) is a heme-containing enzyme ubiquitously present in most aerobic organisms. Although the full range of biological functions of catalase still remains unclear, its main function is the decomposition of hydrogen peroxide into water and oxygen. Catalases have been studied for over 100 years, with examples of the enzyme isolated, purified, and characterized from many different organisms. The crystal structures of 16 heme-containing catalases have now been solved, revealing a common, highly conserved core in all enzymes. The active center consists of a heme with a tyrosine ligand on the proximal side and a conserved histidine and an aspartate on the distal side. Although catalases have been studied for many years, additional functions of catalases have recently been recognized. For example, *Scytalidium thermophilum* catalase (CATPO) has been shown to oxidize o-diphenolic and some p-diphenolic compounds in the absence of hydrogen peroxide. This and other studies have led to the proposal that this secondary oxidative activity may be a general characteristic of catalases. The present chapter will focus on the function and structure of monofunctional heme catalases, emphasizing the information obtained in the last few years mainly in relation to the secondary activ-

**Keywords:** catalase, oxidase, heme, NADPH, channel, secondary activity

Catalases are one of the most studied groups of enzymes. The term catalase was first identified by Loew as hydrogen peroxide (H2O2) degrading enzyme in 1901, and the protein has been the focus of study for biochemists and molecular biologists ever since. The overall reaction for catalase can simply be described as the degradation of two molecules of hydrogen peroxide to water and oxygen (reaction 1). This catalytic reaction occurs in two distinct stages, but what each of the stages includes is mainly based on the kind of catalase [1]. The first stage involves oxidation of the heme using first hydrogen peroxide molecule to form an oxyferryl species in which one oxidation equivalent is taken off from the iron and one from the porphyrin ring to make a porphyrin cation radical (reaction 2). In the second stage, this radical intermediate, known as compound I, is reduced by a second hydrogen peroxide to regenerate the resting state enzyme, water and oxygen (reaction 3) [2, 3]. Catalases can also function as peroxidases, in which suitable organic compound is used as an electron donor. During peroxidase reaction, compound I is converted to compound II (reaction 4), which can be oxidized by another hydrogen peroxide to produce the inactive compound III (reaction 5). For NADPH-binding catalases, it has been

**Chapter 7**

## Typical Catalases: Function and Structure

*Yonca Yuzugullu Karakus*

#### **Abstract**

Catalase (EC 1.11.1.6) is a heme-containing enzyme ubiquitously present in most aerobic organisms. Although the full range of biological functions of catalase still remains unclear, its main function is the decomposition of hydrogen peroxide into water and oxygen. Catalases have been studied for over 100 years, with examples of the enzyme isolated, purified, and characterized from many different organisms. The crystal structures of 16 heme-containing catalases have now been solved, revealing a common, highly conserved core in all enzymes. The active center consists of a heme with a tyrosine ligand on the proximal side and a conserved histidine and an aspartate on the distal side. Although catalases have been studied for many years, additional functions of catalases have recently been recognized. For example, *Scytalidium thermophilum* catalase (CATPO) has been shown to oxidize o-diphenolic and some p-diphenolic compounds in the absence of hydrogen peroxide. This and other studies have led to the proposal that this secondary oxidative activity may be a general characteristic of catalases. The present chapter will focus on the function and structure of monofunctional heme catalases, emphasizing the information obtained in the last few years mainly in relation to the secondary activity of these enzymes.

**Keywords:** catalase, oxidase, heme, NADPH, channel, secondary activity

#### **1. Introduction**

Catalases are one of the most studied groups of enzymes. The term catalase was first identified by Loew as hydrogen peroxide (H2O2) degrading enzyme in 1901, and the protein has been the focus of study for biochemists and molecular biologists ever since. The overall reaction for catalase can simply be described as the degradation of two molecules of hydrogen peroxide to water and oxygen (reaction 1). This catalytic reaction occurs in two distinct stages, but what each of the stages includes is mainly based on the kind of catalase [1]. The first stage involves oxidation of the heme using first hydrogen peroxide molecule to form an oxyferryl species in which one oxidation equivalent is taken off from the iron and one from the porphyrin ring to make a porphyrin cation radical (reaction 2). In the second stage, this radical intermediate, known as compound I, is reduced by a second hydrogen peroxide to regenerate the resting state enzyme, water and oxygen (reaction 3) [2, 3]. Catalases can also function as peroxidases, in which suitable organic compound is used as an electron donor. During peroxidase reaction, compound I is converted to compound II (reaction 4), which can be oxidized by another hydrogen peroxide to produce the inactive compound III (reaction 5). For NADPH-binding catalases, it has been

suggested that enzyme inhibition through the appearance of compound III can be prevented by the NADPH blocking or releasing compound II generation [4–6].

$$2\text{H}\_2\text{O}\_2 \rightarrow 2\text{H}\_2\text{O} + \text{O}\_2 \tag{1}$$

$$\text{Enz} \left( \text{Por-Fe}^{\text{III}} \right) + \text{H}\_2\text{O}\_2 \rightarrow \text{Cpd I} \left( \text{Por\*}^{\star} - \text{Fe}^{\text{IV}} = \text{O} \right) + \text{H}\_2\text{O} \tag{2}$$

$$\text{Cpd I (Por }\star\text{-}\text{-Fe IV = O) + H}\_2\text{O}\_2 \rightarrow \text{Enz (Por -Fe}^{\text{III}}\text{) + H}\_2\text{O}\_2 + \text{O}\_2 \tag{3}$$

$$\text{Cpd I (Por }\star\text{-Fe IV = O) + AH}\_2 \rightarrow \text{Cpd II (Por -Fe}^{\text{IV}}\text{--OH) + AH}^{\text{"}} \quad \text{(4)}$$

$$\text{Cpd II (Por-Fe}^{\text{IV}}\text{-OH)} + \text{H}\_2\text{O}\_2 \rightarrow \text{Cpd III (Por-Fe}^{\text{III}}\text{-O2}^{\text{-}\text{'}}\text{)} + \text{H}\_2\text{O} \tag{5}$$

Catalases have been classified into three groups: monofunctional heme-containing catalases, heme-containing catalase-peroxidases, and manganese-containing catalases [7]. Among them, monofunctional catalases constitute the largest and most extensively studied group of catalases [1, 2]. They all possess two-step mechanism for dismutation of hydrogen peroxide. Members of this largest class of catalases can be biochemically subdivided based on having large (75–84 kDa) subunits with heme *d* associated or small (55–69 kDa) subunits with heme *b* associated. All small subunit enzymes so far characterized, unlike larger enzymes, have been found with NADP(H) bound [1, 8]. In turn, larger subunit enzymes have been shown to exhibit significantly enhanced stability against high temperatures and proteolysis [1, 9]. The catalase-peroxidases, less widespread class, exhibit significant peroxidatic activity in addition to catalytic activity [2]. They are found in bacteria, archaebacteria, and fungi. Catalase-peroxidases have a molecular mass in the range of 120–340 kDa [10, 11]. Manganese-containing catalases are not as widespread as the heme-containing catalases, and there are only three of them so far characterized, one from lactic acid bacteria (*Lactobacillus plantarum*) and two from thermophilic bacteria (*Thermus thermophilus* and *Thermoleophilum album*) [1, 2]. These enzymes are also called pseudo-catalases as their active site contains a manganese-rich reaction instead of heme group [12, 13]. Crystal structures of two manganese catalases, one from *T. thermophilus* and the other from *L. plantarum*, show the presence of dimanganese group in the catalytic center [1].

Although monofunctional catalases are described as such due to the prolongedagreed belief that their only role is hydrogen peroxide removal, this rather limited catalytic role has recently been questioned. Vetrano et al. expressed a novel oxidase activity in the absence of hydrogen peroxide [14]. Later, a catalase from *S. thermophilum* was shown to have an unselective phenolic oxidase activity in the absence of hydrogen peroxide [15–17]. It is thought that such bifunctional enzymes might be more common due to the evidence on the presence of oxidase/ peroxidase activity in catalase enzymes from different organisms such as *Bacillus pumilus* [18], *Thermobifida fusca* [19], and *Amaranthus cruentus* [20]. Such studies are likely to give evidence that translates from various sources to a great deal of catalases. Bifunctional enzymes can be advantageous in many industrial applications including the removal of toxic chemicals and/or chemoprotective agent activity especially when the oxidase activity is enhanced by directed evolution or engineering.

**113**

*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

rial catalase expression [9, 21].

polymerase [21].

induction seems to be σ<sup>S</sup>

**3. Catalase cofactors**

additional transcription factors [22, 23].

ences between *b*-type and *d-*type heme.

**2. Regulation of catalase gene expression**

The study of the bacterial response to oxidative stress has given insights into how catalase synthesis is controlled in different cells. Studies with *E. coli* and *Salmonella typhimurium* have shown that there are two regulatory pathways available in bacte-

*E. coli* produces two catalases or hydroperoxidases, the bifunctional catalaseperoxidase HPI and the monofunctional catalase HPII. These two types of catalases are induced independently; HPI synthesis is promoted by H2O2 added to a medium, and HPII synthesis is induced during growth into stationary phase [22]. The katG gene, encoding HPI, has been found to be regulated by the OxyR regulon which responds to oxidative stress [9, 21, 22]. OxyR protein is a member of LysR family of regulatory proteins that respond to oxidant levels in the cell [9]. OxyR protein undergoes a conformational change during its transition from the reduced (transcriptionally inactive) to the oxidized (transcriptional active) form. This protein directly senses the oxidative stress by becoming oxidized, and that oxidation results in conformational change by which it transduces oxidative stress to RNA

The regulatory mechanism of the katE gene, encoding HPII, is quite different and requires a functional katF gene as a positive effector [22]. HPII levels are expressed at high levels when cells enter stationary phase and are unaffected by hydrogen peroxide and/or anaerobiosis [9, 22]. The most important factor for HPII

The prosthetic group of horse liver catalase enzyme was first isolated by Stern in 1935 [24]. This non-covalently bound component was identified as protoheme (also

The heme prosthetic group has been found to be buried inside the protein, approximately 20 Å from the surface in almost all hem-containing catalases whose structures have been dissolved [25–28]. Despite the similarities in heme-binding pocket, catalases from different sources contain different prosthetic groups [29]. All small subunit size catalases have been shown to include a non-covalently bound iron protoporphyrin IX (heme *b*) as prosthetic group per subunit [29, 30]. Consecutively, an oxidized form of protoporphyrin IX, heme *d*, has been found in almost all large subunit size catalases [30]. The heme *d* group characterized in the active sites of crystal structures of two large subunit size catalases, *Penicillium vitale* catalase (PVC) and HPII from *E. coli*, has the structure of the cis-hydroxy γ-spirolactone and is rotated 180 degrees about the axis defined by the α-γ-meso carbon atoms, with regard to the orientation found for heme *b* in small subunit size catalases like bovine liver catalase (BVC) [29]. **Figure 1** shows the structural differ-

The γ-spirolactone ring and additional hydroxyl group make heme *d* more asymmetric with respect to heme *b*. The conversion of heme *b* to heme *d* has been studied in *E. coli* by many scientists, and it is proposed that the oxidation of heme in HPII may be catalyzed by HPII itself. Loewen and colleagues [32] also reported this conversion in the presence of hydrogen peroxide. However, the modification takes place on the proximal side of ring III opposite to the essential distal histidine [29, 33]. Díaz et al. proposed another possible change of protoheme to heme,

called hematin), consisting of an iron atom and a porphyrin ring.

, as concluded from studies related with the involvement of

*Glutathione System and Oxidative Stress in Health and Disease*

suggested that enzyme inhibition through the appearance of compound III can be prevented by the NADPH blocking or releasing compound II generation [4–6].

Enz (Por– FeIII) + H2 O2 → Cpd I (Por+•– FeIV = O) + H2O (2)

Cpd I (Por +• – Fe IV = O) + H2 O2 → Enz (Por– FeIII) + H2O + O2 (3)

Cpd I (Por +•– Fe IV = O) + AH2 <sup>→</sup> Cpd II (Por– FeIV –OH) + AH• (4)

Cpd II (Por– FeIV –OH) + H2 O2 <sup>→</sup> Cpd III (Por– FeIII– O2−•) + H2O (5)

Catalases have been classified into three groups: monofunctional heme-containing catalases, heme-containing catalase-peroxidases, and manganese-containing catalases [7]. Among them, monofunctional catalases constitute the largest and most extensively studied group of catalases [1, 2]. They all possess two-step mechanism for dismutation of hydrogen peroxide. Members of this largest class of catalases can be biochemically subdivided based on having large (75–84 kDa) subunits with heme *d* associated or small (55–69 kDa) subunits with heme *b* associated. All small subunit enzymes so far characterized, unlike larger enzymes, have been found with NADP(H) bound [1, 8]. In turn, larger subunit enzymes have been shown to exhibit significantly enhanced stability against high temperatures and proteolysis [1, 9]. The catalase-peroxidases, less widespread class, exhibit significant peroxidatic activity in addition to catalytic activity [2]. They are found in bacteria, archaebacteria, and fungi. Catalase-peroxidases have a molecular mass in the range of 120–340 kDa [10, 11]. Manganese-containing catalases are not as widespread as the heme-containing catalases, and there are only three of them so far characterized, one from lactic acid bacteria (*Lactobacillus plantarum*) and two from thermophilic bacteria (*Thermus thermophilus* and *Thermoleophilum album*) [1, 2]. These enzymes are also called pseudo-catalases as their active site contains a manganese-rich reaction instead of heme group [12, 13]. Crystal structures of two manganese catalases, one from *T. thermophilus* and the other from *L. plantarum*,

show the presence of dimanganese group in the catalytic center [1].

Although monofunctional catalases are described as such due to the prolongedagreed belief that their only role is hydrogen peroxide removal, this rather limited catalytic role has recently been questioned. Vetrano et al. expressed a novel oxidase activity in the absence of hydrogen peroxide [14]. Later, a catalase from *S. thermophilum* was shown to have an unselective phenolic oxidase activity in the absence of hydrogen peroxide [15–17]. It is thought that such bifunctional enzymes might be more common due to the evidence on the presence of oxidase/ peroxidase activity in catalase enzymes from different organisms such as *Bacillus pumilus* [18], *Thermobifida fusca* [19], and *Amaranthus cruentus* [20]. Such studies are likely to give evidence that translates from various sources to a great deal of catalases. Bifunctional enzymes can be advantageous in many industrial applications including the removal of toxic chemicals and/or chemoprotective agent activity especially when the oxidase activity is enhanced by directed evolution or

2H2 O2 → 2H2O + O2 (1)

**112**

engineering.

### **2. Regulation of catalase gene expression**

The study of the bacterial response to oxidative stress has given insights into how catalase synthesis is controlled in different cells. Studies with *E. coli* and *Salmonella typhimurium* have shown that there are two regulatory pathways available in bacterial catalase expression [9, 21].

*E. coli* produces two catalases or hydroperoxidases, the bifunctional catalaseperoxidase HPI and the monofunctional catalase HPII. These two types of catalases are induced independently; HPI synthesis is promoted by H2O2 added to a medium, and HPII synthesis is induced during growth into stationary phase [22]. The katG gene, encoding HPI, has been found to be regulated by the OxyR regulon which responds to oxidative stress [9, 21, 22]. OxyR protein is a member of LysR family of regulatory proteins that respond to oxidant levels in the cell [9]. OxyR protein undergoes a conformational change during its transition from the reduced (transcriptionally inactive) to the oxidized (transcriptional active) form. This protein directly senses the oxidative stress by becoming oxidized, and that oxidation results in conformational change by which it transduces oxidative stress to RNA polymerase [21].

The regulatory mechanism of the katE gene, encoding HPII, is quite different and requires a functional katF gene as a positive effector [22]. HPII levels are expressed at high levels when cells enter stationary phase and are unaffected by hydrogen peroxide and/or anaerobiosis [9, 22]. The most important factor for HPII induction seems to be σ<sup>S</sup> , as concluded from studies related with the involvement of additional transcription factors [22, 23].

#### **3. Catalase cofactors**

The prosthetic group of horse liver catalase enzyme was first isolated by Stern in 1935 [24]. This non-covalently bound component was identified as protoheme (also called hematin), consisting of an iron atom and a porphyrin ring.

The heme prosthetic group has been found to be buried inside the protein, approximately 20 Å from the surface in almost all hem-containing catalases whose structures have been dissolved [25–28]. Despite the similarities in heme-binding pocket, catalases from different sources contain different prosthetic groups [29]. All small subunit size catalases have been shown to include a non-covalently bound iron protoporphyrin IX (heme *b*) as prosthetic group per subunit [29, 30]. Consecutively, an oxidized form of protoporphyrin IX, heme *d*, has been found in almost all large subunit size catalases [30]. The heme *d* group characterized in the active sites of crystal structures of two large subunit size catalases, *Penicillium vitale* catalase (PVC) and HPII from *E. coli*, has the structure of the cis-hydroxy γ-spirolactone and is rotated 180 degrees about the axis defined by the α-γ-meso carbon atoms, with regard to the orientation found for heme *b* in small subunit size catalases like bovine liver catalase (BVC) [29]. **Figure 1** shows the structural differences between *b*-type and *d-*type heme.

The γ-spirolactone ring and additional hydroxyl group make heme *d* more asymmetric with respect to heme *b*. The conversion of heme *b* to heme *d* has been studied in *E. coli* by many scientists, and it is proposed that the oxidation of heme in HPII may be catalyzed by HPII itself. Loewen and colleagues [32] also reported this conversion in the presence of hydrogen peroxide. However, the modification takes place on the proximal side of ring III opposite to the essential distal histidine [29, 33]. Díaz et al. proposed another possible change of protoheme to heme,

#### **Figure 1.**

*Structures of heme b (a) and heme d (b), taken from the study reported by Yuzugullu et al. [31].*

where γ-spirolactone is formed either by a singlet oxygen or in a light-mediated mechanism [34].

The residues in a contact with heme in the active center are shown to be different for protoheme and heme d enzymes. Such residues for BLC include Met60, Ser216, Leu298, and Met349, whereas analogous residues for PVC involve Ile41, Val209, Pro291, and Leu342 and for HPII contain Ile114, Ile279, Pro356, and Leu407 [29, 35].

Small subunit size catalases have the ability to bind NADP(H) cofactor which is not essential for the activity of catalase [36], but it is believed to have a role in protecting the enzyme from the formation of catalytically inactive intermediate (cpd II) by promoting its reduction to resting state (Fe3+) during catalytic cycle [37, 38]. According to this hypothesis, large subunit enzymes, whose catalytic cycle lacks compound II formation, do not require to bind NADP(H) [38]. It has also been found that NADP(H) is essential for the dismutation of small peroxides, other than hydrogen peroxide [37]. Instead, large subunit size catalases possess the extra C-terminal domain with a flavodoxin-like topology [29, 30]. Despite this difference, residues defining the NADPH pocket in the bovine liver catalase appear to be well preserved in HPII. Only two residues that interact ionically with NADP(H) in the bovine catalase (Asp212 and His304) differ in HPII (Glu270 and Glu362), but it has been proven that their mutation to the bovine sequence does not promote nucleotide binding [4].

#### **4. Catalase catalytic cycle**

As described previously, catalytic reaction occurs in two steps [1–3]. The first phase of catalytic cycle involves reaction of ferric enzyme and hydrogen peroxide molecule to generate compound I and water. In the second stage, compound I combines with a second molecule of hydrogen peroxide molecule to regenerate the ferric enzyme, molecular oxygen, and water [2].

Paulos and Kraut firstly proposed the formation of compound I using crystal structure of cytochrome c peroxidase in 1980 [39]. According to this mechanism,

**115**

*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

oxidation phase of the catalytic cycles [40, 41].

54,000 and 833,000 reactions per second [3].

**6. Overall structure of catalases**

(**Figure 2**) [26, 30, 46, 47]:

c.Wrapping domain

d.α-helical domain

a.An amino-terminal arm

tion above 3 M hydrogen peroxide concentrations [1, 3].

b.An anti-parallel eight-stranded β-barrel domain

**5. Kinetics**

proton transfer takes places from hydrogen peroxide to distal imidazole group, and iron-oxygen bond is generated [40]. The studies of water release or rebinding to the coproduct formation site have shown that compound I intermediate might exist in two forms either in a wet form in which a water molecule is present at or near the site of coproduct water formation or dry form where the coproduct water formation site is dry. It is assumed that the presence of water may play a significant role in both substrate selectivity and the variety of redox pathways available in the donor

Compound I intermediate is also perceived in the presence of organic peroxides as substrate, and the reaction rate of compound I production decreases with an increase in the molecular size of the leaving group such as H▬ > CH3▬ > HOCH2▬ > CH3C H2▬ > CH3C(〓O) ▬ > CH3(CH2)2▬ > CH3(CH2)3OOH▬ [42]. At low hydrogen peroxide concentrations and in the presence of suitable organic electron donors, compound I can be reduced by one-electron addition leading to the formation of compound II (a formal Fe4+ state) which can cause enzyme inactivation. In this reaction, the porphyrin accepts one electron, therefore losing its radical character [43, 44].

The proposed catalytic mechanism supports that catalase enzyme is never saturated with its substrate, H2O2, and that turnover of enzyme increases indefinitely as substrate concentration increases [2]. Apparently, catalases have been recognized with a rapid turnover rate and the maximum observed velocities ranging between

The classical kinetic parameters, Vmax, kcat, and Km, cannot be directly applied to the observed data as catalases do not follow Michaelis-Menten kinetics except at very low substrate concentrations. However, at concentrations below 200 mM, all small subunit size catalases show Michaelis-Menten-like dependence of velocity. At concentrations above 300–500 mM, most small subunit size catalases suffer inactivation. Conversely, large subunit size catalases begin to suffer inhibi-

All catalases, whose structure have been dissolved, exhibit highly conserved

β-barrel core structure [45]. Their structure is composed of four domains

The amino-terminal domain is an extended arm and is quite variable in length ranging from 53 residues in *Proteus mirabilis* catalase (PMC) to 127 in HPII [30, 47]. This domain is shown to constitute expanded intersubunit interactions, and residues from this region confer us to describe the heme pocket of a

#### *Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

proton transfer takes places from hydrogen peroxide to distal imidazole group, and iron-oxygen bond is generated [40]. The studies of water release or rebinding to the coproduct formation site have shown that compound I intermediate might exist in two forms either in a wet form in which a water molecule is present at or near the site of coproduct water formation or dry form where the coproduct water formation site is dry. It is assumed that the presence of water may play a significant role in both substrate selectivity and the variety of redox pathways available in the donor oxidation phase of the catalytic cycles [40, 41].

Compound I intermediate is also perceived in the presence of organic peroxides as substrate, and the reaction rate of compound I production decreases with an increase in the molecular size of the leaving group such as H▬ > CH3▬ > HOCH2▬ > CH3C H2▬ > CH3C(〓O) ▬ > CH3(CH2)2▬ > CH3(CH2)3OOH▬ [42]. At low hydrogen peroxide concentrations and in the presence of suitable organic electron donors, compound I can be reduced by one-electron addition leading to the formation of compound II (a formal Fe4+ state) which can cause enzyme inactivation. In this reaction, the porphyrin accepts one electron, therefore losing its radical character [43, 44].

#### **5. Kinetics**

*Glutathione System and Oxidative Stress in Health and Disease*

where γ-spirolactone is formed either by a singlet oxygen or in a light-mediated

*Structures of heme b (a) and heme d (b), taken from the study reported by Yuzugullu et al. [31].*

The residues in a contact with heme in the active center are shown to be different for protoheme and heme d enzymes. Such residues for BLC include Met60, Ser216, Leu298, and Met349, whereas analogous residues for PVC involve Ile41, Val209, Pro291, and Leu342 and for HPII contain Ile114, Ile279, Pro356, and Leu407

Small subunit size catalases have the ability to bind NADP(H) cofactor which is not essential for the activity of catalase [36], but it is believed to have a role in protecting the enzyme from the formation of catalytically inactive intermediate (cpd II) by promoting its reduction to resting state (Fe3+) during catalytic cycle [37, 38]. According to this hypothesis, large subunit enzymes, whose catalytic cycle lacks compound II formation, do not require to bind NADP(H) [38]. It has also been found that NADP(H) is essential for the dismutation of small peroxides, other than hydrogen peroxide [37]. Instead, large subunit size catalases possess the extra C-terminal domain with a flavodoxin-like topology [29, 30]. Despite this difference, residues defining the NADPH pocket in the bovine liver catalase appear to be well preserved in HPII. Only two residues that interact ionically with NADP(H) in the bovine catalase (Asp212 and His304) differ in HPII (Glu270 and Glu362), but it has been proven that their mutation to the bovine sequence does not promote nucleo-

As described previously, catalytic reaction occurs in two steps [1–3]. The first phase of catalytic cycle involves reaction of ferric enzyme and hydrogen peroxide molecule to generate compound I and water. In the second stage, compound I combines with a second molecule of hydrogen peroxide molecule to regenerate the

Paulos and Kraut firstly proposed the formation of compound I using crystal structure of cytochrome c peroxidase in 1980 [39]. According to this mechanism,

**114**

mechanism [34].

tide binding [4].

**4. Catalase catalytic cycle**

ferric enzyme, molecular oxygen, and water [2].

[29, 35].

**Figure 1.**

The proposed catalytic mechanism supports that catalase enzyme is never saturated with its substrate, H2O2, and that turnover of enzyme increases indefinitely as substrate concentration increases [2]. Apparently, catalases have been recognized with a rapid turnover rate and the maximum observed velocities ranging between 54,000 and 833,000 reactions per second [3].

The classical kinetic parameters, Vmax, kcat, and Km, cannot be directly applied to the observed data as catalases do not follow Michaelis-Menten kinetics except at very low substrate concentrations. However, at concentrations below 200 mM, all small subunit size catalases show Michaelis-Menten-like dependence of velocity. At concentrations above 300–500 mM, most small subunit size catalases suffer inactivation. Conversely, large subunit size catalases begin to suffer inhibition above 3 M hydrogen peroxide concentrations [1, 3].

#### **6. Overall structure of catalases**

All catalases, whose structure have been dissolved, exhibit highly conserved β-barrel core structure [45]. Their structure is composed of four domains (**Figure 2**) [26, 30, 46, 47]:


The amino-terminal domain is an extended arm and is quite variable in length ranging from 53 residues in *Proteus mirabilis* catalase (PMC) to 127 in HPII [30, 47]. This domain is shown to constitute expanded intersubunit interactions, and residues from this region confer us to describe the heme pocket of a

symmetry-associated subunit. The frequency of intersubunit interactions increases with the length of the domain demonstrating catalases' molecular stability [30].

The second domain, referred to as β-barrel domain, is the central feature of catalase. Most of the residues involved in forming the cavity on the distal side of the heme are placed in the first half of the β-barrel. On the other hand, the second half corresponds to the NADP(H)-binding pocket in small subunit catalases. This domain also involves at least six helices situated in two long insertions between β-strands along the polypeptide chain [30, 47].

The wrapping loop is an extended region of almost 110 residues that link the β-barrel and α-helical parts. This region, residues from 366 to 420, does not have any secondary structure except the essential helix (α9) stating the proximal side of heme with tyrosine residue. This part of the polypeptide chain is involved in different interdomain and intersubunit interactions especially with residues from the amino-terminal arm region from another subunit [30, 47].

The α-helical region contains four anti-parallel helices that are close to some of the helices from the β-barrel domain [30, 47].

Unlike BLC, the structures of PVC and HPII present an extra carboxy-terminal domain including roughly 150 residues with a high content of secondary structure elements organized with a "flavodoxin-like" topology [30, 46, 47]. The possible role of this extra domain in PVC remains unknown [30]. In BLC, prior to the flavodoxin-like domain is occupied by an NADP(H) molecule [48].

Although PVC and HPII share common structural similarities, HPII differs in the existence of 60 residues at N-terminal end that increase the contact area between subunits [25].

#### **Figure 2.**

*Schematic drawing of the polypeptide chain and elements of secondary structure in a* S. thermophilum *catalase subunit. The heme is colored green, Tyr369 magenta, His82 gray, Asn155 purple blue, Val123 red, Phe160 lemon, Phe161 yellow, and Phe168 orange. This figure is taken from the report of Yuzugullu et al. [17].*

**117**

*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

In all catalases, the heme group is deeply buried in the core structure, and its distance from the nearest part of the molecular surface is about 20 Å [9, 30]. Three residues, tyrosine on the proximal side of the heme (Tyr415 in HPII) and histidine and asparagine on the distal side (His128 and Asn201 in HPII), are believed to be essential for catalysis [30]. The oxygen of phenolic hydroxyl group in tyrosine residue is the proximal ligand of heme iron and is probably deprotonated with negative charge, so that it can lead to the stabilization of iron's high oxidation states. The imidazole ring of distal histidine is placed almost parallel to the heme at a mean distance of about 3.5 Å above either pyrrole ring III in PMC or pyrrole ring IV in PVC and HPII [9]. The histidine and asparagine residues on the distal side of the heme make the environment strongly hydrophobic [30]. A conserved serine residue (Ser167 in HPII) is also found to be hydrogen

Despite possessing the same type of heme in active site, PVC and HPII differ in the presence of covalent bond between tyrosine and histidine residues. HPII

The limited accessibility to heme grouping catalases requires the presence of channels [30]. The heme of the enzyme is connected to the exterior surface by three channels, namely, the main channel, the lateral channel, and the central channel. Among them, the main channel is placed perpendicular to the surface of the heme. The lateral channel approaches horizontal to the heme and the central one heading

The main channel is considered to be the primary route for substrate movement to the active site [1, 3]. It is funnel-shaped with 30 Å long in small catalases [30, 48], while in large catalases that channel is replaced by an elongated, constricted, and possibly bifurcated channel that includes the C-terminal domain of

The conserved residues in the main channel are shown in **Figure 3** including the essential histidine, a valine, and an aspartate (His82, Val123, and Asp135 in CATPO) situated 4, 8, and 12 Å from the heme, respectively [17]. The histidine residue is essential for catalysis in HPII, and the side chain of valine residue makes the channel narrower to a diameter of about 3 Å that prevents any molecule larger than H2O and H2O2 from gaining access to the active site. The role of aspartate has not been investigated in any catalase, but the presence of negatively charged side chain has

The lateral or minor channel approaches heme above and below the essential asparagine and emerges in the molecular surface at location corresponding to the NADP(H)-binding pocket in catalases that bind a cofactor (**Figure 4**) [30, 50]. The function of this channel remains unknown [34]. Molecular dynamics analysis

The main channel is a preferred route for substrate entry, but it might be too long and narrow for the release of reaction products (water and molecular oxygen). As the central channel is mainly hydrophilic and leads to the central cavity that is contiguous to the bulk water, this could be a way out for O2. However, substitutions of amino acid residues extending the major channel in large catalases might allow the exit of oxygen through the main channel. In fact, oxygen preferentially exits

indicates that water can exit the protein through this channel [4].

contains a novel type of covalent bond joining the C<sup>β</sup>

of His392 but not in PVC [33, 44, 46, 49].

**6.2 Channels to the heme group**

from the distal side [34, 45].

adjacent subunit [3, 30].

been found to be critical for catalysis [45].

of the essential histidine and might facilitate the enzymatic

of the essential Tyr415 and the

**6.1 Heme pocket**

bonded to the N<sup>δ</sup>

mechanism [46].

Nδ

#### **6.1 Heme pocket**

*Glutathione System and Oxidative Stress in Health and Disease*

β-strands along the polypeptide chain [30, 47].

the helices from the β-barrel domain [30, 47].

between subunits [25].

amino-terminal arm region from another subunit [30, 47].

flavodoxin-like domain is occupied by an NADP(H) molecule [48].

symmetry-associated subunit. The frequency of intersubunit interactions increases with the length of the domain demonstrating catalases' molecular stability [30]. The second domain, referred to as β-barrel domain, is the central feature of catalase. Most of the residues involved in forming the cavity on the distal side of the heme are placed in the first half of the β-barrel. On the other hand, the second half corresponds to the NADP(H)-binding pocket in small subunit catalases. This domain also involves at least six helices situated in two long insertions between

The wrapping loop is an extended region of almost 110 residues that link the β-barrel and α-helical parts. This region, residues from 366 to 420, does not have any secondary structure except the essential helix (α9) stating the proximal side of heme with tyrosine residue. This part of the polypeptide chain is involved in different interdomain and intersubunit interactions especially with residues from the

The α-helical region contains four anti-parallel helices that are close to some of

Unlike BLC, the structures of PVC and HPII present an extra carboxy-terminal domain including roughly 150 residues with a high content of secondary structure elements organized with a "flavodoxin-like" topology [30, 46, 47]. The possible role of this extra domain in PVC remains unknown [30]. In BLC, prior to the

Although PVC and HPII share common structural similarities, HPII differs in the existence of 60 residues at N-terminal end that increase the contact area

*Schematic drawing of the polypeptide chain and elements of secondary structure in a* S. thermophilum *catalase subunit. The heme is colored green, Tyr369 magenta, His82 gray, Asn155 purple blue, Val123 red, Phe160 lemon, Phe161 yellow, and Phe168 orange. This figure is taken from the report of Yuzugullu et al. [17].*

**116**

**Figure 2.**

In all catalases, the heme group is deeply buried in the core structure, and its distance from the nearest part of the molecular surface is about 20 Å [9, 30]. Three residues, tyrosine on the proximal side of the heme (Tyr415 in HPII) and histidine and asparagine on the distal side (His128 and Asn201 in HPII), are believed to be essential for catalysis [30]. The oxygen of phenolic hydroxyl group in tyrosine residue is the proximal ligand of heme iron and is probably deprotonated with negative charge, so that it can lead to the stabilization of iron's high oxidation states. The imidazole ring of distal histidine is placed almost parallel to the heme at a mean distance of about 3.5 Å above either pyrrole ring III in PMC or pyrrole ring IV in PVC and HPII [9]. The histidine and asparagine residues on the distal side of the heme make the environment strongly hydrophobic [30]. A conserved serine residue (Ser167 in HPII) is also found to be hydrogen bonded to the N<sup>δ</sup> of the essential histidine and might facilitate the enzymatic mechanism [46].

Despite possessing the same type of heme in active site, PVC and HPII differ in the presence of covalent bond between tyrosine and histidine residues. HPII contains a novel type of covalent bond joining the C<sup>β</sup> of the essential Tyr415 and the Nδ of His392 but not in PVC [33, 44, 46, 49].

#### **6.2 Channels to the heme group**

The limited accessibility to heme grouping catalases requires the presence of channels [30]. The heme of the enzyme is connected to the exterior surface by three channels, namely, the main channel, the lateral channel, and the central channel. Among them, the main channel is placed perpendicular to the surface of the heme. The lateral channel approaches horizontal to the heme and the central one heading from the distal side [34, 45].

The main channel is considered to be the primary route for substrate movement to the active site [1, 3]. It is funnel-shaped with 30 Å long in small catalases [30, 48], while in large catalases that channel is replaced by an elongated, constricted, and possibly bifurcated channel that includes the C-terminal domain of adjacent subunit [3, 30].

The conserved residues in the main channel are shown in **Figure 3** including the essential histidine, a valine, and an aspartate (His82, Val123, and Asp135 in CATPO) situated 4, 8, and 12 Å from the heme, respectively [17]. The histidine residue is essential for catalysis in HPII, and the side chain of valine residue makes the channel narrower to a diameter of about 3 Å that prevents any molecule larger than H2O and H2O2 from gaining access to the active site. The role of aspartate has not been investigated in any catalase, but the presence of negatively charged side chain has been found to be critical for catalysis [45].

The lateral or minor channel approaches heme above and below the essential asparagine and emerges in the molecular surface at location corresponding to the NADP(H)-binding pocket in catalases that bind a cofactor (**Figure 4**) [30, 50]. The function of this channel remains unknown [34]. Molecular dynamics analysis indicates that water can exit the protein through this channel [4].

The main channel is a preferred route for substrate entry, but it might be too long and narrow for the release of reaction products (water and molecular oxygen). As the central channel is mainly hydrophilic and leads to the central cavity that is contiguous to the bulk water, this could be a way out for O2. However, substitutions of amino acid residues extending the major channel in large catalases might allow the exit of oxygen through the main channel. In fact, oxygen preferentially exits

**Figure 3.** *Channels in CATPO of* S. thermophilum*.*

#### **Figure 4.**

*View of chain A of CATPO complex with 3TR (PDB 5ZZ1, gray) superposed onto human catalase (PDB 1DGH7, blue). CATPO loop 533–537 lies across the top of the NADPH-binding pocket, clashing with the position of the NADPH in the human enzyme [50].*

through the main channel instead of central one in all catalases having b-type heme in the active site. Thus, the presence of minor channels might be an alternative

**119**

*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

a result of oxygen.

production of superoxide [14].

sumed to also occur at the heme active site.

in small catalases through the major channel [34, 51].

**6.3 Bifunctionality of catalase and phenol oxidase**

oxidase from sweet potatoes (*Ipomoea batatas*) [57].

mechanism for a fast release of products under the condition of high H2O2 stress. These results indicate that O2 can exit the enzyme through different channels although the main exit in large catalases might be through the central channel and

Many reports on catalase and phenol oxidase enzymes suggest that the activities may flap in some way that catalases exhibit additional oxidase activity and phenol oxidases present further catalase activity. This relationship can be explained by the release of H2O2 due to polyphenol oxidation [52]. Hydrogen peroxide generation by phenol oxidation was also reported by Aoshima and Ayebe [53]. They observed high concentrations of H2O2 in beverages like tea or coffee directly after opening caps as

Jolley et al. first developed mushroom tyrosinase with catalase activity in the presence of hydrogen peroxide [54]. Garcia-Molina et al*.* [55] and Yamazaki et al. [56] also studied this bifunctional behavior of tyrosinase. In addition to this novel tyrosinase, a catalase-like process was found to have one isozyme of catechol

In literature, the first report on catalase known as a monofunctional enzyme but possessing secondary activity (oxidase) was introduced for mammalian

catalase. This enzyme has been reported to present oxidase activity when hydrogen peroxide is absent or levels of H2O2 are low. As mentioned previously, the main function of catalase is the decomposition of hydrogen peroxide into water and oxygen (catalytic activity). Moreover, it is known that catalases can oxidize low molecular weight alcohols in the presence of low concentrations of H2O2 (peroxidatic activity). The catalytic mechanism of catalases is a two-step process in which catalase heme Fe3+ reduces one hydrogen peroxide molecule to water and generates a porphyrin cation radical called compound I, which is then oxidized by a second hydrogen peroxide to give molecular oxygen and water. The peroxidase activity stems from the oxidation of alcohols by compound I through single-electron transfer. Vetrano et al. expressed a novel oxidase activity in the absence of hydrogen peroxide. This oxidase reaction involves the interaction of catalase heme with a strong reducing agent like benzidine (HB) and molecular oxygen leading to the formation of a compound II-like intermediate. The subsequent electron transfer causes substrate oxidation and regeneration of resting enzyme. An incomplete reaction may result in the formation of radical centered intermediates and the

Later, catalase from the thermophilic fungus *S. thermophilum* has been reported to possess additional phenol oxidase activity [16]. This enzyme, named as CATPO, is the first bifunctional catalase-phenol oxidase in the literature that is characterized in detail. *S. thermophilum* CATPO is a homotetramer with a molecular mass of 320 kDa. Based on the amino acid sequence and preliminary three-dimensional structure [58], CATPO is classified as a large heme catalase with the highest structural homology (77%) to catalase of *Penicillium vitale* [16]. CATPO can oxidize *o*-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide, and the highest oxidase activity is observed against catechol. This enzymatic activity is oxygen-dependent and is inhibited by classic catalase inhibitors, including 3-amino-1,2,4-triazole (3TR). The peroxide-independent secondary activity has also been identified in other catalases [14, 19, 20] and has been pre-

There are a great number of reports available describing the structural and biochemical characterization of catalases. However, basic questions related to

*Glutathione System and Oxidative Stress in Health and Disease*

**118**

**Figure 4.**

*position of the NADPH in the human enzyme [50].*

**Figure 3.**

*Channels in CATPO of* S. thermophilum*.*

through the main channel instead of central one in all catalases having b-type heme in the active site. Thus, the presence of minor channels might be an alternative

*View of chain A of CATPO complex with 3TR (PDB 5ZZ1, gray) superposed onto human catalase (PDB 1DGH7, blue). CATPO loop 533–537 lies across the top of the NADPH-binding pocket, clashing with the* 

mechanism for a fast release of products under the condition of high H2O2 stress. These results indicate that O2 can exit the enzyme through different channels although the main exit in large catalases might be through the central channel and in small catalases through the major channel [34, 51].

#### **6.3 Bifunctionality of catalase and phenol oxidase**

Many reports on catalase and phenol oxidase enzymes suggest that the activities may flap in some way that catalases exhibit additional oxidase activity and phenol oxidases present further catalase activity. This relationship can be explained by the release of H2O2 due to polyphenol oxidation [52]. Hydrogen peroxide generation by phenol oxidation was also reported by Aoshima and Ayebe [53]. They observed high concentrations of H2O2 in beverages like tea or coffee directly after opening caps as a result of oxygen.

Jolley et al. first developed mushroom tyrosinase with catalase activity in the presence of hydrogen peroxide [54]. Garcia-Molina et al*.* [55] and Yamazaki et al. [56] also studied this bifunctional behavior of tyrosinase. In addition to this novel tyrosinase, a catalase-like process was found to have one isozyme of catechol oxidase from sweet potatoes (*Ipomoea batatas*) [57].

In literature, the first report on catalase known as a monofunctional enzyme but possessing secondary activity (oxidase) was introduced for mammalian catalase. This enzyme has been reported to present oxidase activity when hydrogen peroxide is absent or levels of H2O2 are low. As mentioned previously, the main function of catalase is the decomposition of hydrogen peroxide into water and oxygen (catalytic activity). Moreover, it is known that catalases can oxidize low molecular weight alcohols in the presence of low concentrations of H2O2 (peroxidatic activity). The catalytic mechanism of catalases is a two-step process in which catalase heme Fe3+ reduces one hydrogen peroxide molecule to water and generates a porphyrin cation radical called compound I, which is then oxidized by a second hydrogen peroxide to give molecular oxygen and water. The peroxidase activity stems from the oxidation of alcohols by compound I through single-electron transfer. Vetrano et al. expressed a novel oxidase activity in the absence of hydrogen peroxide. This oxidase reaction involves the interaction of catalase heme with a strong reducing agent like benzidine (HB) and molecular oxygen leading to the formation of a compound II-like intermediate. The subsequent electron transfer causes substrate oxidation and regeneration of resting enzyme. An incomplete reaction may result in the formation of radical centered intermediates and the production of superoxide [14].

Later, catalase from the thermophilic fungus *S. thermophilum* has been reported to possess additional phenol oxidase activity [16]. This enzyme, named as CATPO, is the first bifunctional catalase-phenol oxidase in the literature that is characterized in detail. *S. thermophilum* CATPO is a homotetramer with a molecular mass of 320 kDa. Based on the amino acid sequence and preliminary three-dimensional structure [58], CATPO is classified as a large heme catalase with the highest structural homology (77%) to catalase of *Penicillium vitale* [16]. CATPO can oxidize *o*-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide, and the highest oxidase activity is observed against catechol. This enzymatic activity is oxygen-dependent and is inhibited by classic catalase inhibitors, including 3-amino-1,2,4-triazole (3TR). The peroxide-independent secondary activity has also been identified in other catalases [14, 19, 20] and has been presumed to also occur at the heme active site.

There are a great number of reports available describing the structural and biochemical characterization of catalases. However, basic questions related to

substrate and product flow remain unanswered, particularly related to the oxidase activity. Therefore researchers have recently focused on the investigation of the region of CATPO that corresponds to the NADPH-binding region of bovine liver catalase (BLC) and the lateral channel. A number of mutations were introduced into this region, and the properties of these mutant variants, including their specific activities and sensitivities to various inhibitors, are interpreted in terms of a role for the lateral channel in CATPO. The structural, mutation, and kinetic evidences suggested that this pocket at the entrance to the lateral channel, captured by NADPH's nicotinamide moiety in mammalian catalases, should be the site of both oxidase substrate and 3TR binding. The promiscuous nature of CATPO oxidase is clarified by the presence of numerous ordered water molecules which facilitate substrate binding through hydrogen bond formation and can be transferred to accommodate various size and shaped substrates. Peroxide-independent phenolic substrate oxidation is then likely to happen in a similar manner to NADPH oxidation, by electron transfer from the substrate to a high-valent iron-oxo intermediate, apparently arisen through reaction with oxygen [50].

#### **7. Conclusions**

Catalases have been studied for over 100 years, with examples of the enzyme isolated, purified, and characterized from many different organisms. The crystal structures of 16 monofunctional catalases have been solved at high resolution. These structures show that they are tetramers, and each of the four active sites consists of a pentacoordinated-iron protoporphyrin IX prosthetic group with a tyrosinate axial ligand. Some also contain a NADPH cofactor tightly bound at the periphery of each subunit. Recently, it has been found that these enzymes exhibit an oxidase activity in addition to their H2O2 degrading activity. Although they are old enzymes, a peroxide-independent oxidase activity of catalases is new in the literature. Such studies have led to the proposal that this secondary oxidative activity may be a general characteristic of catalases.

#### **Acknowledgements**

The author is grateful to the Department of Biotechnology, Middle East Technical University, Turkey, for providing the necessary facilities during PhD program and many thanks to the Department of Biology, Kocaeli University and TUBİTAK (grant No. 113Z744) for financial support. The author is also grateful to the EU COST Action CM1306 "Movement and Mechanism in Molecular Machines" for STSM support.

**121**

**Author details**

Turkey

Yonca Yuzugullu Karakus

provided the original work is properly cited.

Department of Biology, Faculty of Arts and Sciences, Kocaeli University, Kocaeli,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: yonca.yuzugullu@kocaeli.edu.tr

*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

HPI hydroperoxidase I HPII hydroperoxidase II

HB benzidine

POR porphyrin

3TR 3-amino-1,2,4-triazole H2O2 hydrogen peroxide

L-DOPA L-3,4-dihydroxy-phenylalanine

PVC *Penicillium vitale* catalase PMC *Proteus mirabilis* catalase

NADP(H) nicotinamide adenine dinucleotide phosphate

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Appendices and nomenclature**


*Typical Catalases: Function and Structure DOI: http://dx.doi.org/10.5772/intechopen.90048*

*Glutathione System and Oxidative Stress in Health and Disease*

arisen through reaction with oxygen [50].

ity may be a general characteristic of catalases.

The authors declare no conflict of interest.

**Appendices and nomenclature**

BLC bovine liver catalase CATPO catalase-phenol oxidase

Cpd compound

**7. Conclusions**

**Acknowledgements**

for STSM support.

**Conflict of interest**

substrate and product flow remain unanswered, particularly related to the oxidase activity. Therefore researchers have recently focused on the investigation of the region of CATPO that corresponds to the NADPH-binding region of bovine liver catalase (BLC) and the lateral channel. A number of mutations were introduced into this region, and the properties of these mutant variants, including their specific activities and sensitivities to various inhibitors, are interpreted in terms of a role for the lateral channel in CATPO. The structural, mutation, and kinetic evidences suggested that this pocket at the entrance to the lateral channel, captured by NADPH's nicotinamide moiety in mammalian catalases, should be the site of both oxidase substrate and 3TR binding. The promiscuous nature of CATPO oxidase is clarified by the presence of numerous ordered water molecules which facilitate substrate binding through hydrogen bond formation and can be transferred to accommodate various size and shaped substrates. Peroxide-independent phenolic substrate oxidation is then likely to happen in a similar manner to NADPH oxidation, by electron transfer from the substrate to a high-valent iron-oxo intermediate, apparently

Catalases have been studied for over 100 years, with examples of the enzyme isolated, purified, and characterized from many different organisms. The crystal structures of 16 monofunctional catalases have been solved at high resolution. These structures show that they are tetramers, and each of the four active sites consists of a pentacoordinated-iron protoporphyrin IX prosthetic group with a tyrosinate axial ligand. Some also contain a NADPH cofactor tightly bound at the periphery of each subunit. Recently, it has been found that these enzymes exhibit an oxidase activity in addition to their H2O2 degrading activity. Although they are old enzymes, a peroxide-independent oxidase activity of catalases is new in the literature. Such studies have led to the proposal that this secondary oxidative activ-

The author is grateful to the Department of Biotechnology, Middle East Technical University, Turkey, for providing the necessary facilities during PhD program and many thanks to the Department of Biology, Kocaeli University and TUBİTAK (grant No. 113Z744) for financial support. The author is also grateful to the EU COST Action CM1306 "Movement and Mechanism in Molecular Machines"

**120**


### **Author details**

Yonca Yuzugullu Karakus Department of Biology, Faculty of Arts and Sciences, Kocaeli University, Kocaeli, Turkey

\*Address all correspondence to: yonca.yuzugullu@kocaeli.edu.tr

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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1980;**255**:8199-8205

M011413200

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DOI: 10.1021/bi061519w

[43] Alfonso-Prieto M, Borovik A, Carpena X, Murshudov G, Melik-Adamyan W, Fita I, et al. The structures

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A tetrameric enzyme with four tightly bound molecules of

[29] Murshudov GN, Grebenko AI, Barynin V, Dauter Z, Wilson KS, Vainshtein BK, et al. Structure of the heme d of *Penicillium vitale* and *Escherichia coli* catalases. The Journal of Biological Chemistry. 1996;**271**:8863- 8868. DOI: 10.1074/jbc.271.15.8863.

[30] Maté MJ, Murshudov G, Bravo J, Melik-Adamyan W, Loewen PC, Fita I. Heme catalases. In: Messerschmidt A, Huber R, Poulos T, Widghardt K, editors. Handbook of Metalloproteins. Chichester, UK: Wiley & Sons; 2001.

[31] Yuzugullu Y, Zengin M, Balci S, Goc G, Avci Duman Y. Role of active site residues on catalytic activity of catalase with oxidase activity from *Scytalidium thermophilum*. Procedia— Social and Behavioral Sciences. 2015;**195**:1728-1735. DOI: 10.1016/j.

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[33] Bravo J, Fita I, Ferrer JC, Ens W, Hillar A, Switala J, et al. Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of *Escherichia coli*. Protein Science.

1997;**6**:1016-1023. DOI: 10.1002/

[34] Díaz A, Muñoz-Clares RA, Rangel P, Valdés VJ, Hansberg W. Functional and structural analysis of catalase oxidized by singlet oxygen. Biochimie. 2005;**87**:205-214. DOI: 10.1016/j.

[35] Maj M, Loewen PC, Nicholls P. *E. coli* HPII catalase interaction with high spin ligands: Formate and fluoride as active site probes. Biochimica et

pro.5560060507

biochi.2004.10.014

pp. 486-502

sbspro.2015.06.289

[32] Loewen PC, Switala J,

DOI: 10.1021/bi00089a035

**124**

*pylori* and *Penicillium vitale* catalases determined by X-ray crystallography and QM/MM density functional theory calculations. Journal of American Chemical Society. 2007;**129**:4193-4205. DOI: 10.1021/ja063660y

[44] Maté MJ, Zamocky M, Nykyri LM, Herzog C, Alzari PM, Betzel C, et al. Structure of catalase A from *Saccharomyces cerevisiae*. Journal of Molecular Biology. 1999;**286**:135-139. DOI: 10.1006/jmbi.1998.2453.9

[45] Chelikani P, Carpena X, Fita I, Loewen PC. An electrical potential in the access channel of catalases enhances catalysis. The Journal of Biological Chemistry. 2003;**278**:31290-31296. DOI: 10.1074/jbc.M304076200

[46] Bravo J, Mate MJ, Schneider T, Switala J, Wilson K, Loewen PC, et al. Structure of catalase HPII from *Escherichia coli* at 1.9 Å resolution. Proteins. 1999;**34**:155-166. DOI: 10.1002/(SICI)1097-0134(19990201) 34:2<155:AID-PROT1>3.0.CO;2-P

[47] Melik-Adamyan WR, Barynin VV, Vagin AA, Borisov VV, Vainshtein BK, Fita I, et al. Comparison of beef liver and *Penicillium vitale* catalases. Journal of Molecular Biology. 1986;**188**:63-72. DOI: 10.1016/0022-2836(86)90480-8

[48] Fita I, Rossmann MG. The active center of catalase. Journal of Molecular Biology. 1985;**185**:21-37. DOI: 10.1016/0022-2836(85)90180-9

[49] Melik-Adamyan W, Bravo J, Carpena X, Switala J, Maté MJ, Fita I, et al. Substrate flowing catalases deduced from the crystal structures of active site variants of HPII from *Escherichia coli*. Proteins: Structure, Function, and Genetics. 2001;**44**:270- 281. DOI: 10.1002/prot.1092

[50] Yuzugullu Karakus Y, Goc G, Balci S, Yorke BA, Trinh CH, McPherson MJ,

et al. Identification of the site of oxidase substrate binding in the *Scytalidium thermophilum* catalase. Acta Crystallographica Section D. 2018;**74**:979- 985. DOI: 10.1107/S2059798318010628

[51] Kalko SG, Gelpí JL, Fita I, Orozco M. Theoretical study of the mechanisms of substrate recognition by catalase. Journal of American Chemical Society. 2001;**123**:9665-9672. DOI: 10.1021/ja010512t

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[53] Aoshima H, Ayabe S. Prevention of the deterioration of polyphenolrich beverages. Food Chemistry. 2007;**100**:350-355. DOI: 10.1016/j. foodchem.2005.09.052

[54] Jolley RL Jr, Evans LH, Makino N, Mason HS. Oxytyrosinase. The Journal of Biological Chemistry. 1974;**249**:335-345

[55] Garcia-Molina F, Hiner ANP, Fenoll LG, Rodriguez-Lopez JN, Garcia-Ruiz PA, Garcia-Canovas F, et al. Mushroom tyrosinase: Catalase activity, inhibition and suicide inactivation. Journal of Agricultural and Food Chemistry. 2005;**53**:3702-3709. DOI: 10.1021/jf048340h

[56] Yamazaki S, Morioka C, Itoh S. Kinetic evaluation of catalase and peroxygenase activities of tyrosinase. Biochemistry. 2004;**43**:11546-11553. DOI: 10.1021/bi048908f

[57] Gerdemann C, Eicken C, Magrini A, Meyer HE, Rompel A, Spener F, et al. Isoenzymes of Ipomoea batatas catechol oxidase differ in catalase-like

activity. Biochemica et Biophysica Acta. 2001;**1548**:94-105. DOI: 10.1016/ s0167-4838(01)00219-9

[58] Sutay Kocabas D, Pearson AR, Phillips SE, Bakir U, Ogel ZB, McPherson MJ, et al. Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from *Scytalidium thermophilum*. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 2009;**65**:486-488. DOI: 10.1107/S1744309109012007

*Glutathione System and Oxidative Stress in Health and Disease*

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s0167-4838(01)00219-9

**126**

### *Edited by Margarete Dulce Bagatini*

The imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses determines a state known as oxidative stress. Higher levels of pro-oxidants compared to antioxidant defenses may generate oxidative damage, which, in turn, may lead to modifications in cellular proteins, lipids, and DNA, reducing functional capacity and increasing the risk of diseases. Nevertheless, the clearance of harmful reactive chemical species is achieved by the antioxidant defense systems. These protection systems are referred to as the first and second lines of defense and comprise the classic antioxidants, enzymatic and nonenzymatic defenses, including glutathione. This book presents and discusses the advancement of research on health and diseases and their underlying mechanisms, exploring mainly aspects related to the glutathione antioxidant system.

Published in London, UK © 2020 IntechOpen © Igor Mishenev / iStock

Glutathione System and Oxidative Stress in Health and Disease

IntechOpen Book Series

Biochemistry, Volume 17

Glutathione System and

Oxidative Stress in Health

and Disease

*Edited by Margarete Dulce Bagatini*