Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity

Citation: Andrés, C.M.C.; Pérez de la Lastra, J.M.; Andrés Juan, C.; Plou, F.J.; Pérez-Lebeña, E. Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity. Int. J. Mol. Sci. 2023,24, 1841. https:// doi.org/10.3390/ijms24031841 Academic Editor: Claudio Santi Received: 27 December 2022 Revised: 9 January 2023 Accepted: 12 January 2023 Published: 17 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Review Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity Celia María Curieses Andrés1, JoséManuel Pérez de la Lastra 2,* , Celia Andrés Juan 3, Francisco J. Plou 4 and Eduardo Pérez-Lebeña 5 1Hospital Clínico Universitario of Valladolid, Avenida de Ramón y Cajal, 3, 47003 Valladolid, Spain 2Institute of Natural Products and Agrobiology, CSIC—Spanish Research Council, Avda. Astrofísico Fco. Sánchez, 3, 38206 La Laguna, Spain 3Cinquima Institute and Department of Organic Chemistry, Faculty of Sciences, Valladolid University, Paseo de Belén, 7, 47011 Valladolid, Spain 4Institute of Catalysis and Petrochemistry, CSIC—Spanish Research Council, 28049 Madrid, Spain 5Sistemas de Biotecnología y Recursos Naturales, 47625 Valladolid, Spain *Correspondence: jm.per[email protected] Abstract: Classically, superoxide anion O 2•− and reactive oxygen species ROS play a dual role. At the physiological balance level, they are a by-product of O 2 reduction, necessary for cell signalling, and at the pathological level they are considered harmful, as they can induce disease and apoptosis, necrosis, ferroptosis, pyroptosis and autophagic cell death. This revision focuses on understanding the main characteristics of the superoxide O 2•− , its generation pathways, the biomolecules it oxidizes and how it may contribute to their modification and toxicity. The role of superoxide dismutase, the enzyme responsible for the removal of most of the superoxide produced in living organisms, is studied. At the same time, the toxicity induced by superoxide and derived radicals is beneficial in the oxidative death of microbial pathogens, which are subsequently engulfed by specialized immune cells, such as neutrophils or macrophages, during the activation of innate immunity. Ultimately, this review describes in some depth the chemistry related to O 2•− and how it is harnessed by the innate immune system to produce lysis of microbial agents. Keywords: reactive species; ROS; reactive stress; superoxide anion; innate immunity 1. Introduction In medicine, a great interest in the study of cellular stress and free radicals has emerged in recent years, focused on deepening our knowledge of the mechanisms of cellular selfcontrol that allow us to improve the quality of human life and understand the origin of a large number of diseases [1]. Oxidative stress is a component of many diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer’s disease and cancer, among others [ 2 ]. Simultaneously, ROS are essential for a variety of biological functions, such as cell survival, growth, proliferation and differentiation, and immune response. However, one of the major obstacles to understanding the role of these species is the lack of adequate methods to detect ROS/RNS in vivo , mainly due to their very short lifetimes and the presence of several antioxidants in cells [ 3 ]. In fact, radicals are continuously generated by most organisms as a result of the use of O 2 as a terminal electron acceptor in the mitochondrial electron transport chains and in cytochrome P450 [4]. The term reactive species refers to two types of molecules: free radicals and nonradicals [ 5 ]. This set of molecules is formed as a result of cellular metabolism and is represented in biological systems by reactive oxygen species ROS and reactive nitrogen species RNS, which arise in both normal physiological and pathological processes. Not excluding that, there are also reactive species from other elements, such as chlorine RClS Int. J. Mol. Sci. 2023,24, 1841. https://doi.org/10.3390/ijms24031841 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 1841 2 of 44 and bromine RBrS, although ROS and RNS are the two major groups involved in redox biology [6]. The superoxide anion is a primary oxygen radical that is formed when an oxygen molecule acquires an electron. The initial formation of O 2•− triggers a cascade of ROS, some of which, such as H 2 O 2 , behave as key molecules in cell signalling, and others, such as HO, are damaging. Ultimately, the biological impact of these molecules will be determined by the amount of ROS, cellular defences and the capacity for cellular adaptation [7]. O 2•− is one of the most important reactive oxygen species ROS responsible for oxidative stress in bio-organisms and is generated as a by-product of the mitochondrial respiratory chain [8]. Because of its charge, superoxide has a low membrane permeability, it passes through anion channels, but this is inefficient, and superoxide reacts to a large extent in the physiological compartment where it is generated. Reactive oxygen species (ROS) are a group of highly reactive oxygen-containing chemicals produced exogenously or endogenously from the reduction of oxygen and include both radicals and non-radicals, one of which is superoxide. ROS present in the body are mostly of endogenous origin, although they can also be generated in response to external stimuli, such as ultraviolet light, ionising radiation, pollution, alcohol and tobacco consumption, drugs and toxic agents [9], Figure 1. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 45 body are mostly of endogenous origin, although they can also be generated in response to external stimuli, such as ultraviolet light, ionising radiation, pollution, alcohol and tobacco consumption, drugs and toxic agents [9], Figure 1. Figure 1. Nomenclature of reactive species and free radicals and other reactive oxygen, nitrogen and chlorine species. To control ROS, the body uses several antioxidant mechanisms, including enzymatic and non-enzymatic antioxidants [10]. Non-enzymatic low-molecular-weight antioxidant compounds include cellular glutathione, vitamins C and E, β-carotene, polyphenols and uric acid. Antioxidant enzymes include superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, among others. SOD catalyses the dismutation of superoxide to H2O2. Mammalian cells contain three forms of SOD: Mn-SOD, cytosolic Cu, Zn-SOD and extracellular Cu, Zn-SOD. MnSOD is most abundant in the mitochondria, whereas Cn, Zn-SOD predominates in the cytoplasm [11]. Catalase is an important antioxidant enzyme that catalyses the reduction of H2O2 to H2O. Glutathione peroxidase is another important enzyme for the decomposition of H2O2. Polyphenols, ingested regularly through the fruit and vegetable diet, are a large family of natural organic compounds characterized by multiple hydroxyl phenolic units, with a polyphenolic structure, (several hydroxyl groups on aromatic rings), including four main classes: phenolic acids, flavonoids, stilbenes and lignans [12]. Evidence and research to date supports the role of polyphenols in the prevention of cancer, cardiovascular and neurodegenerative diseases [13]. A significant part of their beneficial effects are based on the modulation of cell signalling pathways [14]. 2. Superoxide Radical Anion O2•− O2•− is a reduced form of molecular oxygen O2, consisting of two oxygen atoms with 17 electrons and a negative electrical charge, Figure 2. Superoxide is the first species produced in the respiratory chain by the reduction of oxygen by the transfer of an electron Figure 1. Nomenclature of reactive species and free radicals and other reactive oxygen, nitrogen and chlorine species. To control ROS, the body uses several antioxidant mechanisms, including enzymatic and non-enzymatic antioxidants [ 10 ]. Non-enzymatic low-molecular-weight antioxidant compounds include cellular glutathione, vitamins C and E, β -carotene, polyphenols and uric acid. Antioxidant enzymes include superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, among others. SOD catalyses the dismutation of superoxide to H 2 O 2 . Mammalian cells contain three forms of SOD: Mn-SOD, cytosolic Int. J. Mol. Sci. 2023,24, 1841 3 of 44 Cu, Zn-SOD and extracellular Cu, Zn-SOD. MnSOD is most abundant in the mitochondria, whereas Cn, Zn-SOD predominates in the cytoplasm [ 11 ]. Catalase is an important antioxidant enzyme that catalyses the reduction of H 2 O 2 to H 2 O. Glutathione peroxidase is another important enzyme for the decomposition of H 2 O 2 . Polyphenols, ingested regularly through the fruit and vegetable diet, are a large family of natural organic compounds characterized by multiple hydroxyl phenolic units, with a polyphenolic structure, (several hydroxyl groups on aromatic rings), including four main classes: phenolic acids, flavonoids, stilbenes and lignans [ 12 ]. Evidence and research to date supports the role of polyphenols in the prevention of cancer, cardiovascular and neurodegenerative diseases [ 13 ]. A significant part of their beneficial effects are based on the modulation of cell signalling pathways [ 14 ]. 2. Superoxide Radical Anion O2•− O 2•− is a reduced form of molecular oxygen O 2 , consisting of two oxygen atoms with 17 electrons and a negative electrical charge, Figure 2. Superoxide is the first species produced in the respiratory chain by the reduction of oxygen by the transfer of an electron and is one of the first species generated by various cellular systems. O 2•− is formed in all living aerobic organisms, and can act as a signalling agent, a toxic specie or a harmless intermediate that spontaneously decomposes. Its levels are limited in vivo by two different types of enzymes, superoxide reductase SOR and superoxide dismutase SOD. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 45 and is one of the first species generated by various cellular systems. O2•− is formed in all living aerobic organisms, and can act as a signalling agent, a toxic specie or a harmless intermediate that spontaneously decomposes. Its levels are limited in vivo by two different types of enzymes, superoxide reductase SOR and superoxide dismutase SOD. Figure 2. Molecular orbital diagram of O2 showing its biradical nature. Despite being a “free biradical”, oxygen has a low reactivity because the unpaired electrons of each oxygen atom have parallel spins, Figure 3. Figure 3. The molecular orbital of O2•– shows one unpaired electron and is delocalized between the π* orbitals of the two oxygen atoms. Superoxide is considered both a radical and a −1 charged anion. It is a relatively unstable molecule, with a half-life of milliseconds, a reasonably strong oxidant, in which case it is reduced to hydrogen peroxide, and can also act as a reductant and convert to oxygen. There are two standard redox potentials for O2•− showing that it can act as a reducing agent E′(O2/O2•−) = 0.33 V or as an oxidizing agent E′(O2•−/H2O2) = 0.93 V [15]), Figure 4. O2O2+ e Oxidation O2+ e + 2H H2O2 Reduction Figure 4. Oxidation and reduction of O2•− to form oxygen or hydrogen peroxide, respectively. O2•− is a relatively small anion, highly soluble in water, where it is solvated by four water molecules strongly bound by hydrogen bonds [16] and reacts with a proton or proton donor to form HO2•, Figure 5. Various organic and inorganic compounds can act as a source of a proton in a large number of reactions [17]. Figure 2. Molecular orbital diagram of O2showing its biradical nature. Despite being a “free biradical”, oxygen has a low reactivity because the unpaired electrons of each oxygen atom have parallel spins, Figure 3. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 45 and is one of the first species generated by various cellular systems. O2•− is formed in all living aerobic organisms, and can act as a signalling agent, a toxic specie or a harmless intermediate that spontaneously decomposes. Its levels are limited in vivo by two different types of enzymes, superoxide reductase SOR and superoxide dismutase SOD. Figure 2. Molecular orbital diagram of O2 showing its biradical nature. Despite being a “free biradical”, oxygen has a low reactivity because the unpaired electrons of each oxygen atom have parallel spins, Figure 3. Figure 3. The molecular orbital of O2•– shows one unpaired electron and is delocalized between the π* orbitals of the two oxygen atoms. Superoxide is considered both a radical and a −1 charged anion. It is a relatively unstable molecule, with a half-life of milliseconds, a reasonably strong oxidant, in which case it is reduced to hydrogen peroxide, and can also act as a reductant and convert to oxygen. There are two standard redox potentials for O2•− showing that it can act as a reducing agent E′(O2/O2•−) = 0.33 V or as an oxidizing agent E′(O2•−/H2O2) = 0.93 V [15]), Figure 4. O2O2+ e Oxidation O2+ e + 2H H2O2 Reduction Figure 4. Oxidation and reduction of O2•− to form oxygen or hydrogen peroxide, respectively. O2•− is a relatively small anion, highly soluble in water, where it is solvated by four water molecules strongly bound by hydrogen bonds [16] and reacts with a proton or proton donor to form HO2•, Figure 5. Various organic and inorganic compounds can act as a source of a proton in a large number of reactions [17]. Figure 3. The molecular orbital of O 2•− shows one unpaired electron and is delocalized between the π* orbitals of the two oxygen atoms. Superoxide is considered both a radical and a − 1 charged anion. It is a relatively unstable molecule, with a half-life of milliseconds, a reasonably strong oxidant, in which case it is reduced to hydrogen peroxide, and can also act as a reductant and convert to oxygen. There are two standard redox potentials for O 2•− showing that it can act as a Int. J. Mol. Sci. 2023,24, 1841 4 of 44 reducing agent E 0 (O 2 /O 2•− ) = 0.33 V or as an oxidizing agent E 0 (O 2•− /H 2 O 2 ) = 0.93 V [ 15 ]), Figure 4. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 45 and is one of the first species generated by various cellular systems. O2•− is formed in all living aerobic organisms, and can act as a signalling agent, a toxic specie or a harmless intermediate that spontaneously decomposes. Its levels are limited in vivo by two different types of enzymes, superoxide reductase SOR and superoxide dismutase SOD. Figure 2. Molecular orbital diagram of O2 showing its biradical nature. Despite being a “free biradical”, oxygen has a low reactivity because the unpaired electrons of each oxygen atom have parallel spins, Figure 3. Figure 3. The molecular orbital of O2•– shows one unpaired electron and is delocalized between the π* orbitals of the two oxygen atoms. Superoxide is considered both a radical and a −1 charged anion. It is a relatively unstable molecule, with a half-life of milliseconds, a reasonably strong oxidant, in which case it is reduced to hydrogen peroxide, and can also act as a reductant and convert to oxygen. There are two standard redox potentials for O2•− showing that it can act as a reducing agent E′(O2/O2•−) = 0.33 V or as an oxidizing agent E′(O2•−/H2O2) = 0.93 V [15]), Figure 4. O2O2+ e Oxidation O2+ e + 2H H2O2 Reduction Figure 4. Oxidation and reduction of O2•− to form oxygen or hydrogen peroxide, respectively. O2•− is a relatively small anion, highly soluble in water, where it is solvated by four water molecules strongly bound by hydrogen bonds [16] and reacts with a proton or proton donor to form HO2•, Figure 5. Various organic and inorganic compounds can act as a source of a proton in a large number of reactions [17]. Figure 4. Oxidation and reduction of O2•− to form oxygen or hydrogen peroxide, respectively. O 2•− is a relatively small anion, highly soluble in water, where it is solvated by four water molecules strongly bound by hydrogen bonds [ 16 ] and reacts with a proton or proton donor to form HO 2• , Figure 5. Various organic and inorganic compounds can act as a source of a proton in a large number of reactions [17]. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 45 Figure 5. Protonation of O2•− leads to the formation of HO2•. The superoxide radical is the conjugate base of a weak acid, the hydroperoxide radical HOO•, whose pKa is 4.88 [18]. The pH controls the distribution between HO2• and O2•. Near the membrane, where this radical is produced, the pH is much lower than in the cytoplasm, so the acid form or hydroperoxide radical will predominate. Due to its non-ionic nature, it can enter the cell membrane and trigger lipid peroxidation processes [19]. The hydroperoxide radical is much more reactive, more oxidising than the superoxide radical, but in aqueous solution at physiological pH the non-protonated form, i.e., the superoxide radical, predominates. Perhydroxyl constitutes less than 1% of superoxide at neutral pH so its impact is more limited. Superoxide absorbs light in the ultraviolet range with a maximum at 245 nm and an extinction coefficient of 2350 M−1 cm−1, whereas hydroperoxyl absorbs at 225 nm with an extinction coefficient of 1400 M−1 cm−1 [20]. O2•– is toxic, mainly because it damages proteins containing Fe-S centres, such as aconitase, succinate dehydrogenase and NADH-ubiquinone oxidoreductase, among others. However, it can also be the generator of other reactive species even more toxic than itself, the iron released from iron and sulphur proteins can give rise to secondary products, such as hydroxyl radicals, and these, plus peroxynitrite, are thought to be the main contributors to superoxide toxicity. Superoxide dismutase SOD is the enzyme responsible for transforming this reactive species into one of a lower toxicity, such as hydrogen peroxide H2O2, Figure 6. Figure 6. Generation of hydroxyl radical, peroxynitrite and hydrogen peroxide by the O2•– anion. O2•– reacts slowly with most molecular targets, although it has been shown to disrupt iron and sulphur group enzymes [21]. However, O2•– can rapidly react with other radicals to give other reactive species [22]. 3. Sources of Superoxide Anion 3.1. Biological Sources Oxygen is an element that has a dual physiological effect; it is essential for the development of aerobic life and has toxic effects inherent to its structure. Oxygen utilisation by aerobic organisms, under normal conditions, generates reactive oxygen metabolites that can lead to a state of oxidative stress if the pro-oxidant/antioxidant cell balance is disturbed. Superoxide is a primary radical formed when an oxygen molecule acquires an electron through enzymatic or non-enzymatic reactions [11], Figure 7. Figure 5. Protonation of O2•− leads to the formation of HO2•. The superoxide radical is the conjugate base of a weak acid, the hydroperoxide radical HOO•, whose pKa is 4.88 [18]. The pH controls the distribution between HO2•and O2•. Near the membrane, where this radical is produced, the pH is much lower than in the cytoplasm, so the acid form or hydroperoxide radical will predominate. Due to its non-ionic nature, it can enter the cell membrane and trigger lipid peroxidation processes [ 19 ]. The hydroperoxide radical is much more reactive, more oxidising than the superoxide radical, but in aqueous solution at physiological pH the non-protonated form, i.e., the superoxide radical, predominates. Perhydroxyl constitutes less than 1% of superoxide at neutral pH so its impact is more limited. Superoxide absorbs light in the ultraviolet range with a maximum at 245 nm and an extinction coefficient of 2350 M −1 cm −1 , whereas hydroperoxyl absorbs at 225 nm with an extinction coefficient of 1400 M−1cm−1[20]. O 2•− is toxic, mainly because it damages proteins containing Fe-S centres, such as aconitase, succinate dehydrogenase and NADH-ubiquinone oxidoreductase, among others. However, it can also be the generator of other reactive species even more toxic than itself, the iron released from iron and sulphur proteins can give rise to secondary products, such as hydroxyl radicals, and these, plus peroxynitrite, are thought to be the main contributors to superoxide toxicity. Superoxide dismutase SOD is the enzyme responsible for transforming this reactive species into one of a lower toxicity, such as hydrogen peroxide H 2 O 2 , Figure 6. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 45 Figure 5. Protonation of O2•− leads to the formation of HO2•. The superoxide radical is the conjugate base of a weak acid, the hydroperoxide radical HOO•, whose pKa is 4.88 [18]. The pH controls the distribution between HO2• and O2•. Near the membrane, where this radical is produced, the pH is much lower than in the cytoplasm, so the acid form or hydroperoxide radical will predominate. Due to its non-ionic nature, it can enter the cell membrane and trigger lipid peroxidation processes [19]. The hydroperoxide radical is much more reactive, more oxidising than the superoxide radical, but in aqueous solution at physiological pH the non-protonated form, i.e., the superoxide radical, predominates. Perhydroxyl constitutes less than 1% of superoxide at neutral pH so its impact is more limited. Superoxide absorbs light in the ultraviolet range with a maximum at 245 nm and an extinction coefficient of 2350 M−1 cm−1, whereas hydroperoxyl absorbs at 225 nm with an extinction coefficient of 1400 M−1 cm−1 [20]. O2•– is toxic, mainly because it damages proteins containing Fe-S centres, such as aconitase, succinate dehydrogenase and NADH-ubiquinone oxidoreductase, among others. However, it can also be the generator of other reactive species even more toxic than itself, the iron released from iron and sulphur proteins can give rise to secondary products, such as hydroxyl radicals, and these, plus peroxynitrite, are thought to be the main contributors to superoxide toxicity. Superoxide dismutase SOD is the enzyme responsible for transforming this reactive species into one of a lower toxicity, such as hydrogen peroxide H2O2, Figure 6. Figure 6. Generation of hydroxyl radical, peroxynitrite and hydrogen peroxide by the O2•– anion. O2•– reacts slowly with most molecular targets, although it has been shown to disrupt iron and sulphur group enzymes [21]. However, O2•– can rapidly react with other radicals to give other reactive species [22]. 3. Sources of Superoxide Anion 3.1. Biological Sources Oxygen is an element that has a dual physiological effect; it is essential for the development of aerobic life and has toxic effects inherent to its structure. Oxygen utilisation by aerobic organisms, under normal conditions, generates reactive oxygen metabolites that can lead to a state of oxidative stress if the pro-oxidant/antioxidant cell balance is disturbed. Superoxide is a primary radical formed when an oxygen molecule acquires an electron through enzymatic or non-enzymatic reactions [11], Figure 7. Figure 6. Generation of hydroxyl radical, peroxynitrite and hydrogen peroxide by the O2•− anion. O 2•− reacts slowly with most molecular targets, although it has been shown to disrupt iron and sulphur group enzymes [ 21 ]. However, O 2•− can rapidly react with other radicals to give other reactive species [22]. Int. J. Mol. Sci. 2023,24, 1841 5 of 44 3. Sources of Superoxide Anion 3.1. Biological Sources Oxygen is an element that has a dual physiological effect; it is essential for the development of aerobic life and has toxic effects inherent to its structure. Oxygen utilisation by aerobic organisms, under normal conditions, generates reactive oxygen metabolites that can lead to a state of oxidative stress if the pro-oxidant/antioxidant cell balance is disturbed. Superoxide is a primary radical formed when an oxygen molecule acquires an electron through enzymatic or non-enzymatic reactions [11], Figure 7. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 45 Figure 7. Oxygen reduction to O2•−. Under basal conditions, human cells produce about 2 trillion O2•− and H2O2 per cell per day, the major source of which is the mitochondria [23]. These organelles consume 8090% of cellular oxygen, in which they reduce water to obtain energy in the form of ATP. Although mitochondrial respiration is highly efficient, approximately 2% of the O2 consumed is partially reduced to O2•− and H2O2. Metabolic reactions that consume oxygen molecules are the main source of superoxide. Biologically, O2•− can be generated from the mitochondrial electron transport chain (ETC), which is the main source of O2•− , and many enzymes, such as NADPH oxidase NOX, xanthine oxidase XO, lipoxygenase, cyclooxygenase, and cytochrome P450 CYP/cytochrome P450 reductase POR, and electron transport chains found in the endoplasmic reticulum, peroxisomes, nuclear membrane and cytoplasmic membrane, convert O2 to superoxide [24], Figure 8. Superoxide can also be produced nonenzymatically. Figure 8. Enzymatic sources of superoxide anion and non-enzymatic production of superoxide [25]. 3.2. Mitochondrial Respiratory Chain The mitochondrion is the main producer of reactive oxygen species during the normal oxidative processes of metabolism, mainly through oxidation–reduction reactions occurring in electron transfer complexes with oxygen as the ultimate electron acceptor [26], Figure 9. Complex I is the first multi-enzyme complex of the respiratory chain, with a central role in cellular energy production, being, in turn, one of the sites of generation of O2•−. The electron flow through the enzyme complexes in the inner membrane generates an electrochemical proton gradient and, therefore, produces energy. An undesired effect of the redox reactions occurring in mitochondria is the generation of reactive oxygen species [27]. Figure 7. Oxygen reduction to O2•−. Under basal conditions, human cells produce about 2 trillion O 2•− and H 2 O 2 per cell per day, the major source of which is the mitochondria [ 23 ]. These organelles consume 80-90% of cellular oxygen, in which they reduce water to obtain energy in the form of ATP. Although mitochondrial respiration is highly efficient, approximately 2% of the O 2 consumed is partially reduced to O2•− and H2O2. Metabolic reactions that consume oxygen molecules are the main source of superoxide. Biologically, O 2•− can be generated from the mitochondrial electron transport chain (ETC), which is the main source of O 2•− , and many enzymes, such as NADPH oxidase NOX, xanthine oxidase XO, lipoxygenase, cyclooxygenase, and cytochrome P450 CYP/cytochrome P450 reductase POR, and electron transport chains found in the endoplasmic reticulum, peroxisomes, nuclear membrane and cytoplasmic membrane, convert O 2 to superoxide [ 24 ], Figure 8. Superoxide can also be produced non-enzymatically. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 45 Figure 7. Oxygen reduction to O2•−. Under basal conditions, human cells produce about 2 trillion O2•− and H2O2 per cell per day, the major source of which is the mitochondria [23]. These organelles consume 8090% of cellular oxygen, in which they reduce water to obtain energy in the form of ATP. Although mitochondrial respiration is highly efficient, approximately 2% of the O2 consumed is partially reduced to O2•− and H2O2. Metabolic reactions that consume oxygen molecules are the main source of superoxide. Biologically, O2•− can be generated from the mitochondrial electron transport chain (ETC), which is the main source of O2•− , and many enzymes, such as NADPH oxidase NOX, xanthine oxidase XO, lipoxygenase, cyclooxygenase, and cytochrome P450 CYP/cytochrome P450 reductase POR, and electron transport chains found in the endoplasmic reticulum, peroxisomes, nuclear membrane and cytoplasmic membrane, convert O2 to superoxide [24], Figure 8. Superoxide can also be produced nonenzymatically. Figure 8. Enzymatic sources of superoxide anion and non-enzymatic production of superoxide [25]. 3.2. Mitochondrial Respiratory Chain The mitochondrion is the main producer of reactive oxygen species during the normal oxidative processes of metabolism, mainly through oxidation–reduction reactions occurring in electron transfer complexes with oxygen as the ultimate electron acceptor [26], Figure 9. Complex I is the first multi-enzyme complex of the respiratory chain, with a central role in cellular energy production, being, in turn, one of the sites of generation of O2•−. The electron flow through the enzyme complexes in the inner membrane generates an electrochemical proton gradient and, therefore, produces energy. An undesired effect of the redox reactions occurring in mitochondria is the generation of reactive oxygen species [27]. Figure 8. Enzymatic sources of superoxide anion and non-enzymatic production of superoxide [ 25 ]. 3.2. Mitochondrial Respiratory Chain The mitochondrion is the main producer of reactive oxygen species during the normal oxidative processes of metabolism, mainly through oxidation–reduction reactions occurring in electron transfer complexes with oxygen as the ultimate electron acceptor [ 26 ], Figure 9. Complex I is the first multi-enzyme complex of the respiratory chain, with a central role in cellular energy production, being, in turn, one of the sites of generation of O 2•− . The electron flow through the enzyme complexes in the inner membrane generates an electrochemical proton gradient and, therefore, produces energy. An undesired effect of the redox reactions occurring in mitochondria is the generation of reactive oxygen species [ 27 ]. Int. J. Mol. Sci. 2023,24, 1841 6 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 45 Figure 9. Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen. Mitochondria, present in all aerobic cells, are the most important biological source of superoxide carried out by two components of the mitochondrial respiratory chain, ubisemiquinone and the flavin semiquinone of NADH dehydrogenase. The superoxide radical is not able to cross the inner mitochondrial membrane so it is confined to the matrix where it reacts rapidly with the enzyme manganese-superoxide dismutase Mn-SOD and nitric oxide to form hydrogen peroxide and peroxynitrite, respectively [28], Figure 10. Figure 10. The mitochondrial production of superoxide radicals is carried out through two fundamental reactions: the oxidation of ubiquinol UQ and the autoxidation of flavin by FMNH dehydrogenase. 3.3. NADPH Oxidases NADPH oxidase in phagocytic cells produces large amounts of O2•− in defence against pathogens and other aggressors [29]. The pentose phosphate pathway generates NADPH during the oxidative phase in which two NADP+ molecules are reduced to NADPH by utilising glucose-6-phosphate in ribulose 5-phosphate, Figure 11. Figure 11. Formation of NADPH molecule in the transformation of glucose-6-phosphate into ribulose 5-phosphate. NADPH subsequently reduces O2 to O2•− via the NADPH oxidase pathway. In the rest of the non-phagocytic cells, NADP oxidase is represented by NOX (non-phagocytic NADPH oxidase), enzymes producing small constitutive pulses of O2•−, which are key players in cell signalling [30], Figure 12. Figure 9. Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen. Mitochondria, present in all aerobic cells, are the most important biological source of superoxide carried out by two components of the mitochondrial respiratory chain, ubisemiquinone and the flavin semiquinone of NADH dehydrogenase. The superoxide radical is not able to cross the inner mitochondrial membrane so it is confined to the matrix where it reacts rapidly with the enzyme manganese-superoxide dismutase Mn-SOD and nitric oxide to form hydrogen peroxide and peroxynitrite, respectively [28], Figure 10. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 45 Figure 9. Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen. Mitochondria, present in all aerobic cells, are the most important biological source of superoxide carried out by two components of the mitochondrial respiratory chain, ubisemiquinone and the flavin semiquinone of NADH dehydrogenase. The superoxide radical is not able to cross the inner mitochondrial membrane so it is confined to the matrix where it reacts rapidly with the enzyme manganese-superoxide dismutase Mn-SOD and nitric oxide to form hydrogen peroxide and peroxynitrite, respectively [28], Figure 10. Figure 10. The mitochondrial production of superoxide radicals is carried out through two fundamental reactions: the oxidation of ubiquinol UQ and the autoxidation of flavin by FMNH dehydrogenase. 3.3. NADPH Oxidases NADPH oxidase in phagocytic cells produces large amounts of O2•− in defence against pathogens and other aggressors [29]. The pentose phosphate pathway generates NADPH during the oxidative phase in which two NADP+ molecules are reduced to NADPH by utilising glucose-6-phosphate in ribulose 5-phosphate, Figure 11. Figure 11. Formation of NADPH molecule in the transformation of glucose-6-phosphate into ribulose 5-phosphate. NADPH subsequently reduces O2 to O2•− via the NADPH oxidase pathway. In the rest of the non-phagocytic cells, NADP oxidase is represented by NOX (non-phagocytic NADPH oxidase), enzymes producing small constitutive pulses of O2•−, which are key players in cell signalling [30], Figure 12. Figure 10. The mitochondrial production of superoxide radicals is carried out through two fundamental reactions: the oxidation of ubiquinol UQ and the autoxidation of flavin by FMNH dehydrogenase. 3.3. NADPH Oxidases NADPH oxidase in phagocytic cells produces large amounts of O 2•− in defence against pathogens and other aggressors [29]. The pentose phosphate pathway generates NADPH during the oxidative phase in which two NADP+ molecules are reduced to NADPH by utilising glucose-6-phosphate in ribulose 5-phosphate, Figure 11. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 45 Figure 9. Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen. Mitochondria, present in all aerobic cells, are the most important biological source of superoxide carried out by two components of the mitochondrial respiratory chain, ubisemiquinone and the flavin semiquinone of NADH dehydrogenase. The superoxide radical is not able to cross the inner mitochondrial membrane so it is confined to the matrix where it reacts rapidly with the enzyme manganese-superoxide dismutase Mn-SOD and nitric oxide to form hydrogen peroxide and peroxynitrite, respectively [28], Figure 10. Figure 10. The mitochondrial production of superoxide radicals is carried out through two fundamental reactions: the oxidation of ubiquinol UQ and the autoxidation of flavin by FMNH dehydrogenase. 3.3. NADPH Oxidases NADPH oxidase in phagocytic cells produces large amounts of O2•− in defence against pathogens and other aggressors [29]. The pentose phosphate pathway generates NADPH during the oxidative phase in which two NADP+ molecules are reduced to NADPH by utilising glucose-6-phosphate in ribulose 5-phosphate, Figure 11. Figure 11. Formation of NADPH molecule in the transformation of glucose-6-phosphate into ribulose 5-phosphate. NADPH subsequently reduces O2 to O2•− via the NADPH oxidase pathway. In the rest of the non-phagocytic cells, NADP oxidase is represented by NOX (non-phagocytic NADPH oxidase), enzymes producing small constitutive pulses of O2•−, which are key players in cell signalling [30], Figure 12. Figure 11. Formation of NADPH molecule in the transformation of glucose-6-phosphate into ribulose 5-phosphate. NADPH subsequently reduces O 2 to O 2•− via the NADPH oxidase pathway. In the rest of the non-phagocytic cells, NADP oxidase is represented by NOX (non-phagocytic NADPH oxidase), enzymes producing small constitutive pulses of O 2•− , which are key players in cell signalling [30], Figure 12. Int. J. Mol. Sci. 2023,24, 1841 7 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 45 Figure 9. Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen. Mitochondria, present in all aerobic cells, are the most important biological source of superoxide carried out by two components of the mitochondrial respiratory chain, ubisemiquinone and the flavin semiquinone of NADH dehydrogenase. The superoxide radical is not able to cross the inner mitochondrial membrane so it is confined to the matrix where it reacts rapidly with the enzyme manganese-superoxide dismutase Mn-SOD and nitric oxide to form hydrogen peroxide and peroxynitrite, respectively [28], Figure 10. Figure 10. The mitochondrial production of superoxide radicals is carried out through two fundamental reactions: the oxidation of ubiquinol UQ and the autoxidation of flavin by FMNH dehydrogenase. 3.3. NADPH Oxidases NADPH oxidase in phagocytic cells produces large amounts of O2•− in defence against pathogens and other aggressors [29]. The pentose phosphate pathway generates NADPH during the oxidative phase in which two NADP+ molecules are reduced to NADPH by utilising glucose-6-phosphate in ribulose 5-phosphate, Figure 11. Figure 11. Formation of NADPH molecule in the transformation of glucose-6-phosphate into ribulose 5-phosphate. NADPH subsequently reduces O2 to O2•− via the NADPH oxidase pathway. In the rest of the non-phagocytic cells, NADP oxidase is represented by NOX (non-phagocytic NADPH oxidase), enzymes producing small constitutive pulses of O2•−, which are key players in cell signalling [30], Figure 12. Figure 12. Reduction of O2to O2•− by NADPH. NOX are mainly located in the plasma membrane [ 31 , 32 ]. NOX2 proteins are constitutively present and, upon inflammatory stimuli, activated NOX2 converts molecular oxygen to superoxide using electrons from NADPH and releases superoxide. NOX2 is predominantly expressed in phagocytes and produces a relatively large amount of superoxide in order to kill bacteria within phagosomes in an inflamed area [33,34], Figure 13. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 45 Figure 12. Reduction of O2 to O2•– by NADPH. NOX are mainly located in the plasma membrane [31,32]. NOX2 proteins are constitutively present and, upon inflammatory stimuli, activated NOX2 converts molecular oxygen to superoxide using electrons from NADPH and releases superoxide. NOX2 is predominantly expressed in phagocytes and produces a relatively large amount of superoxide in order to kill bacteria within phagosomes in an inflamed area [33,34], Figure 13. Figure 13. The NOX family of O2•– generating NADPH oxidases. Superoxide radicals, hydrogen peroxide, singlet oxygen and hypochlorous acid HOCl are generated in the cell membrane through the action of the enzymes NADPH oxidase, myeloperoxidase and xanthine oxidase [35,36]. Other enzymes, such as lipooxygenase and cyclooxygenase also generate ROS during the synthesis of leukotrienes, thromboxanes and prostaglandins [35]. 3.4. Cytochrome P450 CYP/Cytochrome P450 Reductase POR System In the endoplasmic reticulum, superoxide radicals and hydrogen peroxide are produced by the auto-oxidation of the flavoprotein NADPH, cytochrome P450 reductase and cytochrome P450. In addition, mixed function monooxygenases provide another important source of superoxide. The CYP reaction together with POR releases superoxide as a by-product of the oxidase reaction [36], Figure 14. e POR/CYP O2O2 XX X XOH Figure 14. The enzymes cytochrome P450 (CYP)/cytochrome P450 reductase POR, convert molecular oxygen to superoxide either as a main product or as a by-product during oxidation of a variety of compounds X. 3.5. Xanthine Oxidoreductase Xanthine oxidase XO uses oxygen molecules, instead of NAD+, as an electron acceptor and produces superoxide or hydrogen peroxide. It is a cytosolic metalloflavoprotein that can be in two interconvertible and distinct forms called xanthine dehydrogenase XDH and xanthine oxidase XO. XO belongs to a family of molybdoflavoenzymes and is released by a calcium-activated protease during hypoxia. XO is Figure 13. The NOX family of O2•− generating NADPH oxidases. Superoxide radicals, hydrogen peroxide, singlet oxygen and hypochlorous acid HOCl are generated in the cell membrane through the action of the enzymes NADPH oxidase, myeloperoxidase and xanthine oxidase [ 35 , 36 ]. Other enzymes, such as lipooxygenase and cyclooxygenase also generate ROS during the synthesis of leukotrienes, thromboxanes and prostaglandins [35]. 3.4. Cytochrome P450 CYP/Cytochrome P450 Reductase POR System In the endoplasmic reticulum, superoxide radicals and hydrogen peroxide are produced by the auto-oxidation of the flavoprotein NADPH, cytochrome P450 reductase and cytochrome P450. In addition, mixed function monooxygenases provide another important source of superoxide. The CYP reaction together with POR releases superoxide as a by-product of the oxidase reaction [36], Figure 14. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 45 Figure 12. Reduction of O2 to O2•– by NADPH. NOX are mainly located in the plasma membrane [31,32]. NOX2 proteins are constitutively present and, upon inflammatory stimuli, activated NOX2 converts molecular oxygen to superoxide using electrons from NADPH and releases superoxide. NOX2 is predominantly expressed in phagocytes and produces a relatively large amount of superoxide in order to kill bacteria within phagosomes in an inflamed area [33,34], Figure 13. Figure 13. The NOX family of O2•– generating NADPH oxidases. Superoxide radicals, hydrogen peroxide, singlet oxygen and hypochlorous acid HOCl are generated in the cell membrane through the action of the enzymes NADPH oxidase, myeloperoxidase and xanthine oxidase [35,36]. Other enzymes, such as lipooxygenase and cyclooxygenase also generate ROS during the synthesis of leukotrienes, thromboxanes and prostaglandins [35]. 3.4. Cytochrome P450 CYP/Cytochrome P450 Reductase POR System In the endoplasmic reticulum, superoxide radicals and hydrogen peroxide are produced by the auto-oxidation of the flavoprotein NADPH, cytochrome P450 reductase and cytochrome P450. In addition, mixed function monooxygenases provide another important source of superoxide. The CYP reaction together with POR releases superoxide as a by-product of the oxidase reaction [36], Figure 14. e POR/CYP O2O2 XX X XOH Figure 14. The enzymes cytochrome P450 (CYP)/cytochrome P450 reductase POR, convert molecular oxygen to superoxide either as a main product or as a by-product during oxidation of a variety of compounds X. 3.5. Xanthine Oxidoreductase Xanthine oxidase XO uses oxygen molecules, instead of NAD+, as an electron acceptor and produces superoxide or hydrogen peroxide. It is a cytosolic metalloflavoprotein that can be in two interconvertible and distinct forms called xanthine dehydrogenase XDH and xanthine oxidase XO. XO belongs to a family of molybdoflavoenzymes and is released by a calcium-activated protease during hypoxia. XO is Figure 14. The enzymes cytochrome P450 (CYP)/cytochrome P450 reductase POR, convert molecular oxygen to superoxide either as a main product or as a by-product during oxidation of a variety of compounds X. Int. J. Mol. Sci. 2023,24, 1841 8 of 44 3.5. Xanthine Oxidoreductase Xanthine oxidase XO uses oxygen molecules, instead of NAD+, as an electron acceptor and produces superoxide or hydrogen peroxide. It is a cytosolic metalloflavoprotein that can be in two interconvertible and distinct forms called xanthine dehydrogenase XDH and xanthine oxidase XO. XO belongs to a family of molybdo-flavoenzymes and is released by a calcium-activated protease during hypoxia. XO is unique in generating superoxide (28%) and H 2 O 2 (72%) by oxidation of hypoxanthine to xanthine, and xanthine to uric acid, Figure 15. XO activity is increased in inflammatory airway disorders, ischaemic reperfusion injury, atherosclerosis, diabetes and in autoimmune disorders [37]. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 45 unique in generating superoxide (28%) and H2O2 (72%) by oxidation of hypoxanthine to xanthine, and xanthine to uric acid, Figure 15. XO activity is increased in inflammatory airway disorders, ischaemic reperfusion injury, atherosclerosis, diabetes and in autoimmune disorders [37]. Figure 15. Reactions catalysed by xanthine oxidase. The first two processes involve the reduction of two electrons from O2 to form H2O2, then the remaining two electrons are each used to reduce O2 to O2•−. The total ROS produced is therefore two H2O2 and two O2•− molecules. 3.6. Non-Enzymatic Production of Superoxide Superoxide can also be produced by a number of non-enzymatic reactions [25]. Above all, non-enzymatic glycosylation, referred to as glycation, occurs under conditions of hyperglycaemia and produces a variety of compounds [38,39], Figure 16. Figure 16. Catalysed reactions by Xanthine Oxidase. 3.7. Non-Biochemical Sources Several techniques to produce superoxide have been used to study its reactions. Chemically, the main ways to produce superoxide are reactions involving ionising radiation or UV, and O2 reduction by transition metals or reducing radicals, Figure 17. Figure 17. Non-Biochemical sources of superoxide [25]. 3.8. Photolysis Figure 15. Reactions catalysed by xanthine oxidase. The first two processes involve the reduction of two electrons from O2to form H2O2, then the remaining two electrons are each used to reduce O 2 to O 2•− . The total ROS produced is therefore two H2O2and two O2•− molecules. 3.6. Non-Enzymatic Production of Superoxide Superoxide can also be produced by a number of non-enzymatic reactions [ 25 ]. Above all, non-enzymatic glycosylation, referred to as glycation, occurs under conditions of hyperglycaemia and produces a variety of compounds [38,39], Figure 16. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 45 unique in generating superoxide (28%) and H2O2 (72%) by oxidation of hypoxanthine to xanthine, and xanthine to uric acid, Figure 15. XO activity is increased in inflammatory airway disorders, ischaemic reperfusion injury, atherosclerosis, diabetes and in autoimmune disorders [37]. Figure 15. Reactions catalysed by xanthine oxidase. The first two processes involve the reduction of two electrons from O2 to form H2O2, then the remaining two electrons are each used to reduce O2 to O2•−. The total ROS produced is therefore two H2O2 and two O2•− molecules. 3.6. Non-Enzymatic Production of Superoxide Superoxide can also be produced by a number of non-enzymatic reactions [25]. Above all, non-enzymatic glycosylation, referred to as glycation, occurs under conditions of hyperglycaemia and produces a variety of compounds [38,39], Figure 16. Figure 16. Catalysed reactions by Xanthine Oxidase. 3.7. Non-Biochemical Sources Several techniques to produce superoxide have been used to study its reactions. Chemically, the main ways to produce superoxide are reactions involving ionising radiation or UV, and O2 reduction by transition metals or reducing radicals, Figure 17. Figure 17. Non-Biochemical sources of superoxide [25]. 3.8. Photolysis Figure 16. Catalysed reactions by Xanthine Oxidase. 3.7. Non-Biochemical Sources Several techniques to produce superoxide have been used to study its reactions. Chemically, the main ways to produce superoxide are reactions involving ionising radiation or UV, and O2reduction by transition metals or reducing radicals, Figure 17. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 45 unique in generating superoxide (28%) and H2O2 (72%) by oxidation of hypoxanthine to xanthine, and xanthine to uric acid, Figure 15. XO activity is increased in inflammatory airway disorders, ischaemic reperfusion injury, atherosclerosis, diabetes and in autoimmune disorders [37]. Figure 15. Reactions catalysed by xanthine oxidase. The first two processes involve the reduction of two electrons from O2 to form H2O2, then the remaining two electrons are each used to reduce O2 to O2•−. The total ROS produced is therefore two H2O2 and two O2•− molecules. 3.6. Non-Enzymatic Production of Superoxide Superoxide can also be produced by a number of non-enzymatic reactions [25]. Above all, non-enzymatic glycosylation, referred to as glycation, occurs under conditions of hyperglycaemia and produces a variety of compounds [38,39], Figure 16. Figure 16. Catalysed reactions by Xanthine Oxidase. 3.7. Non-Biochemical Sources Several techniques to produce superoxide have been used to study its reactions. Chemically, the main ways to produce superoxide are reactions involving ionising radiation or UV, and O2 reduction by transition metals or reducing radicals, Figure 17. Figure 17. Non-Biochemical sources of superoxide [25]. 3.8. Photolysis Figure 17. Non-Biochemical sources of superoxide [25]. Int. J. Mol. Sci. 2023,24, 1841 9 of 44 3.8. Photolysis For the generation of O 2•− during the ionising irradiation of air-saturated sodium formate, an in-line stopped-flow radiolysis apparatus with a Van de Graaff electron generator has been used to generate O 2•− during the ionising irradiation of sodium formate saturated with air at 2 MeV [40], Figure 18. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 45 For the generation of O2•– during the ionising irradiation of air-saturated sodium formate, an in-line stopped-flow radiolysis apparatus with a Van de Graaff electron generator has been used to generate O2•– during the ionising irradiation of sodium formate saturated with air at 2 MeV [40], Figure 18. Figure 18. The radiolysis of water is performed under high-energy electrons and if the water contains molecular oxygen and sodium formate, the primary radicals become O2•–/HO2•. Superoxide can also be formed in an O2-saturated aqueous formate solution after brief UV irradiation with a Xe or Ar lamp. The main process involves the photochemical decomposition of water through its dissociation into HO• y H•, Figure 19. Figure 19. Photochemical decomposition of water through its dissociation into HO• and H•. In the presence of O2, electron transfer occurs to produce O2•–. In addition, formate HCOO– can react with H• or HO• to form a common product CO2•, where CO2• can further reduce O2 to O2•–, Figures 20 and 21. Figure 20. Reaction of H• with oxygen to form O2•–. Figure 21. The CO2•− formed reduces the O2 to O2•–. Photolysis of an H2O2 solution can also convert HO• and H• to HO2• through the reactions shown in Figure 22. Figure 22. UV photolysis of H2O2 forms HO2•. Figure 18. The radiolysis of water is performed under high-energy electrons and if the water contains molecular oxygen and sodium formate, the primary radicals become O2•−/HO2•. Superoxide can also be formed in an O 2 -saturated aqueous formate solution after brief UV irradiation with a Xe or Ar lamp. The main process involves the photochemical decomposition of water through its dissociation into HO•y H•, Figure 19. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 45 For the generation of O2•– during the ionising irradiation of air-saturated sodium formate, an in-line stopped-flow radiolysis apparatus with a Van de Graaff electron generator has been used to generate O2•– during the ionising irradiation of sodium formate saturated with air at 2 MeV [40], Figure 18. Figure 18. The radiolysis of water is performed under high-energy electrons and if the water contains molecular oxygen and sodium formate, the primary radicals become O2•–/HO2•. Superoxide can also be formed in an O2-saturated aqueous formate solution after brief UV irradiation with a Xe or Ar lamp. The main process involves the photochemical decomposition of water through its dissociation into HO• y H•, Figure 19. Figure 19. Photochemical decomposition of water through its dissociation into HO• and H•. In the presence of O2, electron transfer occurs to produce O2•–. In addition, formate HCOO– can react with H• or HO• to form a common product CO2•, where CO2• can further reduce O2 to O2•–, Figures 20 and 21. Figure 20. Reaction of H• with oxygen to form O2•–. Figure 21. The CO2•− formed reduces the O2 to O2•–. Photolysis of an H2O2 solution can also convert HO• and H• to HO2• through the reactions shown in Figure 22. Figure 22. UV photolysis of H2O2 forms HO2•. Figure 19. Photochemical decomposition of water through its dissociation into HO•and H•. In the presence of O 2 , electron transfer occurs to produce O 2•− . In addition, formate HCOO – can react with H • or HO • to form a common product CO 2• , where CO 2• can further reduce O2to O2•− , Figures 20 and 21. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 45 For the generation of O2•– during the ionising irradiation of air-saturated sodium formate, an in-line stopped-flow radiolysis apparatus with a Van de Graaff electron generator has been used to generate O2•– during the ionising irradiation of sodium formate saturated with air at 2 MeV [40], Figure 18. Figure 18. The radiolysis of water is performed under high-energy electrons and if the water contains molecular oxygen and sodium formate, the primary radicals become O2•–/HO2•. Superoxide can also be formed in an O2-saturated aqueous formate solution after brief UV irradiation with a Xe or Ar lamp. The main process involves the photochemical decomposition of water through its dissociation into HO• y H•, Figure 19. Figure 19. Photochemical decomposition of water through its dissociation into HO• and H•. In the presence of O2, electron transfer occurs to produce O2•–. In addition, formate HCOO– can react with H• or HO• to form a common product CO2•, where CO2• can further reduce O2 to O2•–, Figures 20 and 21. Figure 20. Reaction of H• with oxygen to form O2•–. Figure 21. The CO2•− formed reduces the O2 to O2•–. Photolysis of an H2O2 solution can also convert HO• and H• to HO2• through the reactions shown in Figure 22. Figure 22. UV photolysis of H2O2 forms HO2•. Figure 20. Reaction of H•with oxygen to form O2•− . Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 45 For the generation of O2•– during the ionising irradiation of air-saturated sodium formate, an in-line stopped-flow radiolysis apparatus with a Van de Graaff electron generator has been used to generate O2•– during the ionising irradiation of sodium formate saturated with air at 2 MeV [40], Figure 18. Figure 18. The radiolysis of water is performed under high-energy electrons and if the water contains molecular oxygen and sodium formate, the primary radicals become O2•–/HO2•. Superoxide can also be formed in an O2-saturated aqueous formate solution after brief UV irradiation with a Xe or Ar lamp. The main process involves the photochemical decomposition of water through its dissociation into HO• y H•, Figure 19. Figure 19. Photochemical decomposition of water through its dissociation into HO• and H•. In the presence of O2, electron transfer occurs to produce O2•–. In addition, formate HCOO– can react with H• or HO• to form a common product CO2•, where CO2• can further reduce O2 to O2•–, Figures 20 and 21. Figure 20. Reaction of H• with oxygen to form O2•–. Figure 21. The CO2•− formed reduces the O2 to O2•–. Photolysis of an H2O2 solution can also convert HO• and H• to HO2• through the reactions shown in Figure 22. Figure 22. UV photolysis of H2O2 forms HO2•. Figure 21. The CO2•− formed reduces the O2to O2•− . Photolysis of an H 2 O 2 solution can also convert HO • and H • to HO 2• through the reactions shown in Figure 22. Int. J. Mol. Sci. 2023,24, 1841 16 of 44 another with the conserved active site ligand, glutamine 69. A ping-pong mechanism in the dismutation of O2•− by Fe-SOD is proposed [88], Figure 37. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 17 of 45 Figure 36. The mechanism includes the formation of both Mn3+SOD and the complex (Mn3+ SOD(H2O)-O22−). 4.1.5. Iron Superoxide Dismutase Fe-SOD It is a prokaryotic enzyme, discovered in some bacterial cells and in the cytosol of plants [86]. Most of the structural and mechanistic studies have been performed on FeSOD obtained from E. coli. The structures of Fe-SOD are dimers. The active site contains a single iron atom bound to three histidines, an aspartate and a water molecule [87]. The coordinated water molecule involves a hydrogen bond with an aspartate ligand and another with the conserved active site ligand, glutamine 69. A ping-pong mechanism in the dismutation of O2•– by Fe-SOD is proposed [88], Figure 37. Figure 37. A ping-pong mechanism is displayed in the dismutation of O2•– by FeSOD. 4.1.6. Iron Superoxide Reductase Fe-SOR SORs are small enzymes, with about 110–180 amino acids in their sequences. Based on the number of metal centres, there are two types of Fe-SORs: neelaredoxins (1Fe-SORs) and desulfoferrodoxins (2Fe-SOR) [89]. In Fe-SOR, the iron is close to the molecular surfaces and is exposed to the solvent. The metal centres in Fe-SODs are located inside the protein. Desulfoferrodoxins are a homodimeric non-heme iron protein found in some sulphate-reducing bacteria and archaea [90,91]. Neelaredoxins, from Archaeoglobus fulgidus, have biofunctional properties as SOD and ROS. Desulfoferrodoxins and neelaredoxins have been isolated mainly in the Fe2+ and Fe3+ forms, respectively. UVvisible spectroscopy has been applied to study the catalytic activity of SOR [92–99]. 4.1.7. Analytical Determination of Superoxide Dismutase Activity O2 can act as either an oxidant or a monovalent reductant. This dual reactivity has been exploited in the design of assays to analyse SOD activity. Thus, in some assays, Oz reduces tetranitro methane [11], cytochrome c or nitro tetrazolium blue [100], and in others it oxidises epinephrine, tiron pyrogallol or 6-hydroxydopamine [101,102], Figure 38. Figure 37. A ping-pong mechanism is displayed in the dismutation of O2•− by FeSOD. 4.1.6. Iron Superoxide Reductase Fe-SOR SORs are small enzymes, with about 110–180 amino acids in their sequences. Based on the number of metal centres, there are two types of Fe-SORs: neelaredoxins (1Fe-SORs) and desulfoferrodoxins (2Fe-SOR) [ 89 ]. In Fe-SOR, the iron is close to the molecular surfaces and is exposed to the solvent. The metal centres in Fe-SODs are located inside the protein. Desulfoferrodoxins are a homodimeric non-heme iron protein found in some sulphatereducing bacteria and archaea [ 90 , 91 ]. Neelaredoxins, from Archaeoglobus fulgidus, have biofunctional properties as SOD and ROS. Desulfoferrodoxins and neelaredoxins have been isolated mainly in the Fe 2+ and Fe 3+ forms, respectively. UV-visible spectroscopy has been applied to study the catalytic activity of SOR [92–99]. 4.1.7. Analytical Determination of Superoxide Dismutase Activity O 2 can act as either an oxidant or a monovalent reductant. This dual reactivity has been exploited in the design of assays to analyse SOD activity. Thus, in some assays, Oz reduces tetranitro methane [ 11 ], cytochrome c or nitro tetrazolium blue [ 100 ], and in others it oxidises epinephrine, tiron pyrogallol or 6-hydroxydopamine [101,102], Figure 38. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 18 of 45 Figure 38. O2 as a monovalent oxidant or reductant. The measurement of SOD activity presents a major problem due to the instability of the superoxide radical in aqueous media. Direct determination of SOD activity is carried out by observing the disappearance of the free superoxide radical catalysed by this enzyme, generated by a pulse of electrons on a millisecond scale. This assay is not affordable for most laboratories as it requires the presence of a linear electron accelerator, so that determinations of SOD activity have had to rely on variations in constant concentrations of superoxide radicals catalysed by the enzyme. The most used methods require two components: (a) a superoxide radical generator and (b) a detector of the superoxide radical [103]. The generator produces the superoxide radical at a constant controlled rate. In the absence of SOD, the superoxide radical accumulates to such a concentration that the rate of reaction with the detector is equal to the rate of production, and this equilibrium state is reached within one second. If SOD is present, it competes with the detector for the superoxide radical, resulting in a decrease in the superoxide radical taken up by the detector, with an inhibition of the detection level. In the method described by Minami and Yoshikawa, 1979, the generation of the superoxide radical is produced by the chemical autoxidation of pyrogallol at pH = 8.2 [104], Figure 39. Figure 39. Generation of the superoxide radical by the chemical autoxidation of pyrogallol. The detector for this radical is a dye, NBT, a yellow compound which, in the presence of superoxide radical, is reduced, giving an intensely blue compound, formazan blue, Figure 40. Figure 38. O2as a monovalent oxidant or reductant. The measurement of SOD activity presents a major problem due to the instability of the superoxide radical in aqueous media. Direct determination of SOD activity is carried out by observing the disappearance of the free superoxide radical catalysed by this enzyme, generated by a pulse of electrons on a millisecond scale. This assay is not affordable for most laboratories as it requires the presence of a linear electron accelerator, so that determinations of SOD activity have had to rely on variations in constant concentrations of superoxide Int. J. Mol. Sci. 2023,24, 1841 17 of 44 radicals catalysed by the enzyme. The most used methods require two components: (a) a superoxide radical generator and (b) a detector of the superoxide radical [103]. The generator produces the superoxide radical at a constant controlled rate. In the absence of SOD, the superoxide radical accumulates to such a concentration that the rate of reaction with the detector is equal to the rate of production, and this equilibrium state is reached within one second. If SOD is present, it competes with the detector for the superoxide radical, resulting in a decrease in the superoxide radical taken up by the detector, with an inhibition of the detection level. In the method described by Minami and Yoshikawa, 1979, the generation of the superoxide radical is produced by the chemical autoxidation of pyrogallol at pH = 8.2 [104], Figure 39. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 18 of 45 Figure 38. O2 as a monovalent oxidant or reductant. The measurement of SOD activity presents a major problem due to the instability of the superoxide radical in aqueous media. Direct determination of SOD activity is carried out by observing the disappearance of the free superoxide radical catalysed by this enzyme, generated by a pulse of electrons on a millisecond scale. This assay is not affordable for most laboratories as it requires the presence of a linear electron accelerator, so that determinations of SOD activity have had to rely on variations in constant concentrations of superoxide radicals catalysed by the enzyme. The most used methods require two components: (a) a superoxide radical generator and (b) a detector of the superoxide radical [103]. The generator produces the superoxide radical at a constant controlled rate. In the absence of SOD, the superoxide radical accumulates to such a concentration that the rate of reaction with the detector is equal to the rate of production, and this equilibrium state is reached within one second. If SOD is present, it competes with the detector for the superoxide radical, resulting in a decrease in the superoxide radical taken up by the detector, with an inhibition of the detection level. In the method described by Minami and Yoshikawa, 1979, the generation of the superoxide radical is produced by the chemical autoxidation of pyrogallol at pH = 8.2 [104], Figure 39. Figure 39. Generation of the superoxide radical by the chemical autoxidation of pyrogallol. The detector for this radical is a dye, NBT, a yellow compound which, in the presence of superoxide radical, is reduced, giving an intensely blue compound, formazan blue, Figure 40. Figure 39. Generation of the superoxide radical by the chemical autoxidation of pyrogallol. The detector for this radical is a dye, NBT, a yellow compound which, in the presence of superoxide radical, is reduced, giving an intensely blue compound, formazan blue, Figure 40. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 19 of 45 Figure 40. The reduction of NBT gives a compound with an intense blue colour, formazan blue. In this method, therefore, the inhibition of NBT reduction is measured, which is measurable spectrophotometrically. This inhibition is measured against a control in which there is no SOD. A unit of SOD activity is considered to be the activity of this enzyme that would produce 50% of the maximum inhibition caused by this enzyme on the reduction of NBT, according to the definition of McCord and Frldovich [105]. The NBT method has several disadvantages, such as the poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase. The replacement of NBT by Dojindo’s highly water-soluble tetrazolium salt WST-1 (2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt) overcomes these problems and makes it a very convenient SOD assay, yielding a stable water-soluble formazan dye with a high absorbance at 450 nm after reduction with a superoxide anion. The rate of reduction with O22is linearly related to the activity of xanthine oxidase (XO) and is inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a colorimetric method. Superoxide ions are generated from the conversion of xanthine and O2 to uric acid and H2O2 by xanthine oxidase (XO). The superoxide anion then converts the tetrazolium salt WST-1 to the coloured product WST-1 formazan [106], Figure 41. Figure 41. SOD inhibition assay mechanism. The absorbance is then measured at 450 nm using a standard microplate reader. The addition of SOD to this reaction reduces the levels of superoxide ions, which reduces the rate of WST-1 formazan formation, Figure 42. The SOD activity in the experimental sample is measured as the percentage inhibition of the rate of WST-1 formazan formation. Figure 40. The reduction of NBT gives a compound with an intense blue colour, formazan blue. In this method, therefore, the inhibition of NBT reduction is measured, which is measurable spectrophotometrically. This inhibition is measured against a control in which there is no SOD. A unit of SOD activity is considered to be the activity of this enzyme that would produce 50% of the maximum inhibition caused by this enzyme on the reduction of NBT, according to the definition of McCord and Frldovich [105]. The NBT method has several disadvantages, such as the poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase. The replacement of NBT by Dojindo’s highly water-soluble tetrazolium salt WST-1 (2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt) overcomes these problems and makes it a very convenient SOD assay, yielding a stable water-soluble formazan dye with a high absorbance at 450 nm after reduction with a superoxide anion. The rate of reduction with O 22is linearly related to the activity of xanthine oxidase (XO) and is inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a colorimetric method. Superoxide ions are generated from the conversion of xanthine and O 2 to uric acid and H2O2 by xanthine oxidase (XO). The superoxide anion then converts the tetrazolium salt WST-1 to the coloured product WST-1 formazan [106], Figure 41. Int. J. Mol. Sci. 2023,24, 1841 18 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 19 of 45 Figure 40. The reduction of NBT gives a compound with an intense blue colour, formazan blue. In this method, therefore, the inhibition of NBT reduction is measured, which is measurable spectrophotometrically. This inhibition is measured against a control in which there is no SOD. A unit of SOD activity is considered to be the activity of this enzyme that would produce 50% of the maximum inhibition caused by this enzyme on the reduction of NBT, according to the definition of McCord and Frldovich [105]. The NBT method has several disadvantages, such as the poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase. The replacement of NBT by Dojindo’s highly water-soluble tetrazolium salt WST-1 (2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt) overcomes these problems and makes it a very convenient SOD assay, yielding a stable water-soluble formazan dye with a high absorbance at 450 nm after reduction with a superoxide anion. The rate of reduction with O22is linearly related to the activity of xanthine oxidase (XO) and is inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a colorimetric method. Superoxide ions are generated from the conversion of xanthine and O2 to uric acid and H2O2 by xanthine oxidase (XO). The superoxide anion then converts the tetrazolium salt WST-1 to the coloured product WST-1 formazan [106], Figure 41. Figure 41. SOD inhibition assay mechanism. The absorbance is then measured at 450 nm using a standard microplate reader. The addition of SOD to this reaction reduces the levels of superoxide ions, which reduces the rate of WST-1 formazan formation, Figure 42. The SOD activity in the experimental sample is measured as the percentage inhibition of the rate of WST-1 formazan formation. Figure 41. SOD inhibition assay mechanism. The absorbance is then measured at 450 nm using a standard microplate reader. The addition of SOD to this reaction reduces the levels of superoxide ions, which reduces the rate of WST-1 formazan formation, Figure 42. The SOD activity in the experimental sample is measured as the percentage inhibition of the rate of WST-1 formazan formation. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 20 of 45 Figure 42. Structure of WST-1 (4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzene-1,3-disulfonate). 4.1.8. Reaction with Iron–Sulphur [Fe–S] Cluster The best-established detrimental effect of superoxide is the inactivation of iron/sulphur protein groups, such as aconitase, and bacterial dehydratases required for branched-chain amino acid synthesis. Mammalian aconitases (mitochondrial and cytosolic isoenzymes) are unique iron/sulphur-group-containing proteins in which the metal centre participates in the catalysis of a non-redox reaction. The [4Fe-4S] group exists in a cubic structure, with iron and sulphur atoms found in alternating corner positions. These groups are found in bacterial ferredoxins and within mitochondrial respiratory complexes. The detrimental effect of superoxide is the inactivation of iron/sulphurcontaining protein groups. These reactions have rate constants in excess of 106 M−1 s−1 and are highly selective for the superoxide [107]. Aconitase is an essential enzyme, particularly sensitive to oxidative damage and is preferentially modified and inactivated by mitochondrial oxidants during ageing and in pathologies involving mitochondrial dysfunction. It is one of the main targets of superoxide. Aconitase activity is also sensitive to nitric oxide, peroxynitrite and the carbonate radical [108]. Aconitase deficiency is associated with myopathies and low exercise endurance [109]. Certain variants of aconitase have been found to cause infantile cerebellar-retinal degeneration syndrome, which is characterised by various neurological and muscular symptoms [110]. Within the Cuban group of iron–sulphur aconitases, only three of the four iron ions have cysteine thiolate ligands; the fourth iron ion (Feα) is exposed to the solvent within the active site pocket and binds to the oxygen atoms of the water or substrates to be dehydrated. An example of binding of the ferro-sulphurised centre of aconitase with citrate is shown in Figure 43. Fe S S S Fe Fe Fe S S S Cys SCys Cys O H C C O O CH2 COO C OOC H H O H H Iron-sulfored center of Aconitase Citrate Figure 43. Iron–sulphur centre of aconitase and union with citrate and a water molecule. Mitochondrial aconitase is a ferrosulphoprotein located in the mitochondrial matrix that catalyses the reversible isomerisation of citrate to isocitrate via cis-aconitate in the Krebs cycle [111], Figure 44. The reaction mechanism can be divided into three phases, a dehydration phase, followed by a rotation and finally a rehydration phase. In dehydration, the OH group attached to the Fe-S centre is protonated and eliminated, forming cis-aconitate. In order to rehydrate, the molecule needs to rotate 180º, which takes Figure 42. Structure of WST-1 (4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzene1,3-disulfonate). 4.1.8. Reaction with Iron–Sulphur [Fe–S] Cluster The best-established detrimental effect of superoxide is the inactivation of iron/sulphur protein groups, such as aconitase, and bacterial dehydratases required for branched-chain amino acid synthesis. Mammalian aconitases (mitochondrial and cytosolic isoenzymes) are unique iron/sulphur-group-containing proteins in which the metal centre participates in the catalysis of a non-redox reaction. The [4Fe-4S] group exists in a cubic structure, with iron and sulphur atoms found in alternating corner positions. These groups are found in bacterial ferredoxins and within mitochondrial respiratory complexes. The detrimental effect of superoxide is the inactivation of iron/sulphur-containing protein groups. These reactions have rate constants in excess of 10 6 M −1 s −1 and are highly selective for the superoxide [ 107 ]. Aconitase is an essential enzyme, particularly sensitive to oxidative damage and is preferentially modified and inactivated by mitochondrial oxidants during ageing and in pathologies involving mitochondrial dysfunction. It is one of the main targets of superoxide. Aconitase activity is also sensitive to nitric oxide, peroxynitrite and the carbonate radical [108]. Aconitase deficiency is associated with myopathies and low exercise endurance [ 109 ]. Certain variants of aconitase have been found to cause infantile cerebellar-retinal degen- Int. J. Mol. Sci. 2023,24, 1841 19 of 44 eration syndrome, which is characterised by various neurological and muscular symptoms [110]. Within the Cuban group of iron–sulphur aconitases, only three of the four iron ions have cysteine thiolate ligands; the fourth iron ion (Fe α ) is exposed to the solvent within the active site pocket and binds to the oxygen atoms of the water or substrates to be dehydrated. An example of binding of the ferro-sulphurised centre of aconitase with citrate is shown in Figure 43. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 20 of 45 Figure 42. Structure of WST-1 (4-(3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzene-1,3-disulfonate). 4.1.8. Reaction with Iron–Sulphur [Fe–S] Cluster The best-established detrimental effect of superoxide is the inactivation of iron/sulphur protein groups, such as aconitase, and bacterial dehydratases required for branched-chain amino acid synthesis. Mammalian aconitases (mitochondrial and cytosolic isoenzymes) are unique iron/sulphur-group-containing proteins in which the metal centre participates in the catalysis of a non-redox reaction. The [4Fe-4S] group exists in a cubic structure, with iron and sulphur atoms found in alternating corner positions. These groups are found in bacterial ferredoxins and within mitochondrial respiratory complexes. The detrimental effect of superoxide is the inactivation of iron/sulphurcontaining protein groups. These reactions have rate constants in excess of 106 M−1 s−1 and are highly selective for the superoxide [107]. Aconitase is an essential enzyme, particularly sensitive to oxidative damage and is preferentially modified and inactivated by mitochondrial oxidants during ageing and in pathologies involving mitochondrial dysfunction. It is one of the main targets of superoxide. Aconitase activity is also sensitive to nitric oxide, peroxynitrite and the carbonate radical [108]. Aconitase deficiency is associated with myopathies and low exercise endurance [109]. Certain variants of aconitase have been found to cause infantile cerebellar-retinal degeneration syndrome, which is characterised by various neurological and muscular symptoms [110]. Within the Cuban group of iron–sulphur aconitases, only three of the four iron ions have cysteine thiolate ligands; the fourth iron ion (Feα) is exposed to the solvent within the active site pocket and binds to the oxygen atoms of the water or substrates to be dehydrated. An example of binding of the ferro-sulphurised centre of aconitase with citrate is shown in Figure 43. Fe S S S Fe Fe Fe S S S Cys SCys Cys O H C C O O CH2 COO C OOC H H O H H Iron-sulfored center of Aconitase Citrate Figure 43. Iron–sulphur centre of aconitase and union with citrate and a water molecule. Mitochondrial aconitase is a ferrosulphoprotein located in the mitochondrial matrix that catalyses the reversible isomerisation of citrate to isocitrate via cis-aconitate in the Krebs cycle [111], Figure 44. The reaction mechanism can be divided into three phases, a dehydration phase, followed by a rotation and finally a rehydration phase. In dehydration, the OH group attached to the Fe-S centre is protonated and eliminated, forming cis-aconitate. In order to rehydrate, the molecule needs to rotate 180º, which takes Figure 43. Iron–sulphur centre of aconitase and union with citrate and a water molecule. Mitochondrial aconitase is a ferrosulphoprotein located in the mitochondrial matrix that catalyses the reversible isomerisation of citrate to isocitrate via cis-aconitate in the Krebs cycle [ 111 ], Figure 44. The reaction mechanism can be divided into three phases, a dehydration phase, followed by a rotation and finally a rehydration phase. In dehydration, the OH group attached to the Fe-S centre is protonated and eliminated, forming cis-aconitate. In order to rehydrate, the molecule needs to rotate 180 º , which takes place in more than one step, a cis-aconitate molecule displaces the one it is attached to, and once it is properly attached, it is rehydrated, forming the final product. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 21 of 45 place in more than one step, a cis-aconitate molecule displaces the one it is attached to, and once it is properly attached, it is rehydrated, forming the final product. Figure 44. Mechanism of isomerisation of citrate to isocitrate by the enzyme aconitase. The aconitase differentiates between the two carboxylic groups of the citrate uniting it by three points. This aconitase-catalysed reaction is stereospecific. Isocitrate has two asymmetric carbons, so that from citrate the formation of four possible stereoisomers would be possible, but the reaction produces only one of these, D-isocitrate. The enzyme has an asymmetric binding site for citrate, allowing the substrate to bind only in a specific orientation, so the transfer of the OH occurs only if the appropriate orientation conditions are met [112]. The high reactivity of aconitase with superoxide ion is due to the presence of the [4Fe4S] centre with labile Fe in its active site [113]. The mechanism involves (i) electrostatic attraction of the superoxide to the solvent-exposed group, (ii) protonation to become a strong univalent oxidant, and (iii) single electron abstraction. The reactivity is attributed to the electrophilic character of Fe, the large nucleophilicity of O2•–, the increased oxidative potential of O2•– in its iron-bound state and the sensitivity of Fe-S bonds to oxidation [113]. The oxidised group is unstable, the iron atom coordinating the substrate dissociates, and the enzyme becomes inactive. Iron and hydrogen peroxide are released and may undergo other damaging reactions [114]. Consequently, inactivation of m-aconitase by superoxide can increase the formation of hydroxyl •OH radicals via the Fenton reaction in mitochondria, Figure 45. Figure 44. Mechanism of isomerisation of citrate to isocitrate by the enzyme aconitase. The aconitase differentiates between the two carboxylic groups of the citrate uniting it by three points. This aconitase-catalysed reaction is stereospecific. Isocitrate has two asymmetric carbons, so that from citrate the formation of four possible stereoisomers would be possible, but the reaction produces only one of these, D-isocitrate. The enzyme has an asymmetric Int. J. Mol. Sci. 2023,24, 1841 20 of 44 binding site for citrate, allowing the substrate to bind only in a specific orientation, so the transfer of the OH occurs only if the appropriate orientation conditions are met [112]. The high reactivity of aconitase with superoxide ion is due to the presence of the [4Fe-4S] centre with labile Fe in its active site [ 113 ]. The mechanism involves (i) electrostatic attraction of the superoxide to the solvent-exposed group, (ii) protonation to become a strong univalent oxidant, and (iii) single electron abstraction. The reactivity is attributed to the electrophilic character of Fe, the large nucleophilicity of O 2•− , the increased oxidative potential of O 2•− in its iron-bound state and the sensitivity of Fe-S bonds to oxidation [ 113 ]. The oxidised group is unstable, the iron atom coordinating the substrate dissociates, and the enzyme becomes inactive. Iron and hydrogen peroxide are released and may undergo other damaging reactions [ 114 ]. Consequently, inactivation of m-aconitase by superoxide can increase the formation of hydroxyl • OH radicals via the Fenton reaction in mitochondria, Figure 45. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 21 of 45 place in more than one step, a cis-aconitate molecule displaces the one it is attached to, and once it is properly attached, it is rehydrated, forming the final product. Figure 44. Mechanism of isomerisation of citrate to isocitrate by the enzyme aconitase. The aconitase differentiates between the two carboxylic groups of the citrate uniting it by three points. This aconitase-catalysed reaction is stereospecific. Isocitrate has two asymmetric carbons, so that from citrate the formation of four possible stereoisomers would be possible, but the reaction produces only one of these, D-isocitrate. The enzyme has an asymmetric binding site for citrate, allowing the substrate to bind only in a specific orientation, so the transfer of the OH occurs only if the appropriate orientation conditions are met [112]. The high reactivity of aconitase with superoxide ion is due to the presence of the [4Fe4S] centre with labile Fe in its active site [113]. The mechanism involves (i) electrostatic attraction of the superoxide to the solvent-exposed group, (ii) protonation to become a strong univalent oxidant, and (iii) single electron abstraction. The reactivity is attributed to the electrophilic character of Fe, the large nucleophilicity of O2•–, the increased oxidative potential of O2•– in its iron-bound state and the sensitivity of Fe-S bonds to oxidation [113]. The oxidised group is unstable, the iron atom coordinating the substrate dissociates, and the enzyme becomes inactive. Iron and hydrogen peroxide are released and may undergo other damaging reactions [114]. Consequently, inactivation of m-aconitase by superoxide can increase the formation of hydroxyl •OH radicals via the Fenton reaction in mitochondria, Figure 45. Figure 45. Reaction of the [4Fe-4S] cluster with O 2•− . Loss of iron labile from the Fe-S centre with the consequent formation of an inactive [3Fe-4S] centre and the release of Fe+2. Two aconitase isoenzymes are present in mammalian cells: the mitochondrial enzyme (m-aconitase) and the bifunctional cytosolic enzyme (c-aconitase/IRP1). The assembly and disassembly of Fe-S groups is a key process, not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron-sensing and RNA-binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). Iron deficiency can decrease aconitase protein levels and limit the assembly of Fe-S groups required for its enzymatic activities. As a result, iron deficiency can potentially affect the citric acid cycle, lipid biosynthesis, carbohydrate metabolism and many other biological pathways involving citrate. 4.1.9. Conversion of Nitric Oxide to Peroxynitrite The conversion of nitric oxide to peroxynitrite is a type of reaction between two radicals. O 2•− and • NO react and produce, within the mitochondrial matrix, ONOO – , a potent oxidant that is normally reduced by the action of mitochondrial reductants, such as NADH2, ubiquinol UQH2 and glutathione GSH. When produced in excess, because its control is lost (e.g., in ischaemia/reperfusion or inflammation), it leads to tyrosine nitration and mitochondrial dysfunction. Its cumulative effect would contribute to tissue ageing. Another radical formed extensively by both enzymatic and non-enzymatic processes is nitric oxide •NO, which serves as an intraand intercellular signalling molecule [115,116]. • NO and superoxide react in a diffusion-limited manner. This reaction terminates the chain reaction initiated by superoxide, although peroxynitrite is commonly considered a harmful molecule [117], Figure 46. Int. J. Mol. Sci. 2023,24, 1841 21 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 22 of 45 Figure 45. Reaction of the [4Fe-4S] cluster with O2•–. Loss of iron labile from the Fe-S centre with the consequent formation of an inactive [3Fe-4S] centre and the release of Fe+2. Two aconitase isoenzymes are present in mammalian cells: the mitochondrial enzyme (m-aconitase) and the bifunctional cytosolic enzyme (c-aconitase/IRP1). The assembly and disassembly of Fe-S groups is a key process, not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron-sensing and RNA-binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). Iron deficiency can decrease aconitase protein levels and limit the assembly of Fe-S groups required for its enzymatic activities. As a result, iron deficiency can potentially affect the citric acid cycle, lipid biosynthesis, carbohydrate metabolism and many other biological pathways involving citrate. 4.1.9. Conversion of Nitric Oxide to Peroxynitrite The conversion of nitric oxide to peroxynitrite is a type of reaction between two radicals. O2•– and •NO react and produce, within the mitochondrial matrix, ONOO–, a potent oxidant that is normally reduced by the action of mitochondrial reductants, such as NADH2, ubiquinol UQH2 and glutathione GSH. When produced in excess, because its control is lost (e.g., in ischaemia/reperfusion or inflammation), it leads to tyrosine nitration and mitochondrial dysfunction. Its cumulative effect would contribute to tissue ageing. Another radical formed extensively by both enzymatic and non-enzymatic processes is nitric oxide •NO, which serves as an intraand intercellular signalling molecule [115,116]. •NO and superoxide react in a diffusion-limited manner. This reaction terminates the chain reaction initiated by superoxide, although peroxynitrite is commonly considered a harmful molecule [117], Figure 46. Figure 46. Conversion of nitric oxide to peroxynitrite and rate of reaction. This reaction has a very high rate constant and is fast enough to compete with SODcatalysed dismutation [22,118]. Peroxynitrite is a highly reactive oxidant and is capable of undergoing a wide range of oxidative processes [119]. These include direct oxidations and secondary reactions due to nitrogen dioxide, hydroxyl and carbonate radicals [120,121]. Peroxynitrite is a short-lived and highly reactive oxidant and is therefore another reaction that confers indirect toxicity to O2•–, in particular to DNA, proteins and lipids [122]. In addition, peroxynitrite is capable of nitrating tyrosine or tryptophan residues, or oxidising methionine residues [123–128]. 4.1.10. Nucleophilic Substitution Reaction O2•– is a nucleophile in the reaction with alkyl halides and alkyl tosylates in DMSO and leads to the formation of alkylperoxy radicals and subsequently to peroxy anions via one-electron reduction [56,129], Figure 47. Figure 47. Formation of alkylperoxy radicals and peroxy anions. Figure 46. Conversion of nitric oxide to peroxynitrite and rate of reaction. This reaction has a very high rate constant and is fast enough to compete with SODcatalysed dismutation [ 22 , 118 ]. Peroxynitrite is a highly reactive oxidant and is capable of undergoing a wide range of oxidative processes [ 119 ]. These include direct oxidations and secondary reactions due to nitrogen dioxide, hydroxyl and carbonate radicals [120,121]. Peroxynitrite is a short-lived and highly reactive oxidant and is therefore another reaction that confers indirect toxicity to O 2•− , in particular to DNA, proteins and lipids [ 122 ]. In addition, peroxynitrite is capable of nitrating tyrosine or tryptophan residues, or oxidising methionine residues [123–128]. 4.1.10. Nucleophilic Substitution Reaction O 2•− is a nucleophile in the reaction with alkyl halides and alkyl tosylates in DMSO and leads to the formation of alkylperoxy radicals and subsequently to peroxy anions via one-electron reduction [56,129], Figure 47. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 22 of 45 Figure 45. Reaction of the [4Fe-4S] cluster with O2•–. Loss of iron labile from the Fe-S centre with the consequent formation of an inactive [3Fe-4S] centre and the release of Fe+2. Two aconitase isoenzymes are present in mammalian cells: the mitochondrial enzyme (m-aconitase) and the bifunctional cytosolic enzyme (c-aconitase/IRP1). The assembly and disassembly of Fe-S groups is a key process, not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron-sensing and RNA-binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). Iron deficiency can decrease aconitase protein levels and limit the assembly of Fe-S groups required for its enzymatic activities. As a result, iron deficiency can potentially affect the citric acid cycle, lipid biosynthesis, carbohydrate metabolism and many other biological pathways involving citrate. 4.1.9. Conversion of Nitric Oxide to Peroxynitrite The conversion of nitric oxide to peroxynitrite is a type of reaction between two radicals. O2•– and •NO react and produce, within the mitochondrial matrix, ONOO–, a potent oxidant that is normally reduced by the action of mitochondrial reductants, such as NADH2, ubiquinol UQH2 and glutathione GSH. When produced in excess, because its control is lost (e.g., in ischaemia/reperfusion or inflammation), it leads to tyrosine nitration and mitochondrial dysfunction. Its cumulative effect would contribute to tissue ageing. Another radical formed extensively by both enzymatic and non-enzymatic processes is nitric oxide •NO, which serves as an intraand intercellular signalling molecule [115,116]. •NO and superoxide react in a diffusion-limited manner. This reaction terminates the chain reaction initiated by superoxide, although peroxynitrite is commonly considered a harmful molecule [117], Figure 46. Figure 46. Conversion of nitric oxide to peroxynitrite and rate of reaction. This reaction has a very high rate constant and is fast enough to compete with SODcatalysed dismutation [22,118]. Peroxynitrite is a highly reactive oxidant and is capable of undergoing a wide range of oxidative processes [119]. These include direct oxidations and secondary reactions due to nitrogen dioxide, hydroxyl and carbonate radicals [120,121]. Peroxynitrite is a short-lived and highly reactive oxidant and is therefore another reaction that confers indirect toxicity to O2•–, in particular to DNA, proteins and lipids [122]. In addition, peroxynitrite is capable of nitrating tyrosine or tryptophan residues, or oxidising methionine residues [123–128]. 4.1.10. Nucleophilic Substitution Reaction O2•– is a nucleophile in the reaction with alkyl halides and alkyl tosylates in DMSO and leads to the formation of alkylperoxy radicals and subsequently to peroxy anions via one-electron reduction [56,129], Figure 47. Figure 47. Formation of alkylperoxy radicals and peroxy anions. Figure 47. Formation of alkylperoxy radicals and peroxy anions. The high stereoselectivity observed in this reaction is inconsistent with the intermediation of free alkyl radicals. Furthermore, it has been observed that the structure of the alkyl group attached to the halogen, the nature of the leaving group, and the polarity of the solvent exert influence on the course of the reaction in the carbon–oxygen bond formation step that is consistent with a mechanism involving an SN2 displacement on the carbon; therefore, it follows that the initial reaction of the superoxide with an alkyl halide produces an alkylperoxy radical [130], Figure 48. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 23 of 45 The high stereoselectivity observed in this reaction is inconsistent with the intermediation of free alkyl radicals. Furthermore, it has been observed that the structure of the alkyl group attached to the halogen, the nature of the leaving group, and the polarity of the solvent exert influence on the course of the reaction in the carbon–oxygen bond formation step that is consistent with a mechanism involving an SN2 displacement on the carbon; therefore, it follows that the initial reaction of the superoxide with an alkyl halide produces an alkylperoxy radical [130], Figure 48. Figure 48. Formation of the C-O bond by a mechanism involving a displacement of SN2 on carbon. 4.1.11. Reactions of Superoxide with Amino Acids Second order rate constant values for HO2• reactions for aliphatic amino acids are reported in the literature in the range of 1 × 10 M−1s−1 and a value of about 6 × 102 M−1s−1 for Cys, these values are even lower for O2•− reactions of these amino acids, with values ranging from 1.0 × 10−1 M−1s−1 to about 2 × 10 M−1s−1 [131]. The difference in reactivity of the two superoxide species may be due to the fact that HO2• acts as a weak oxidising agent, while O2•− behaves as a weak oxidising and reducing agent, and these values may also depend on whether the amino acids are protonated or not. Cystine and Met do not react with superoxide, but N-acetylcysteine and glutathione GSH do [132–134], Figure 49. Figure 49. Rate constants for the reaction of N-acetylcysteine and glutathione with O2•−. Despite the large body of experimental work dealing with the oxidation of thiols by superoxide, the mechanism of this reaction remains controversial, Figure 50. According to some authors [135–137], the superoxide radical reacts with RSH thiols by abstraction of the hydrogen atom giving the thiyl radical and hydrogen peroxide; other authors consider that the first step of the reaction is the formation of a three-electron-bond radical, which breaks into a sulfinyl radical and a hydroxide anion [132–134]. Figure 50. Two pathways for the reaction of O2•– with thiols. Figure 48. Formation of the C-O bond by a mechanism involving a displacement of SN2 on carbon. 4.1.11. Reactions of Superoxide with Amino Acids Second order rate constant values for HO 2• reactions for aliphatic amino acids are reported in the literature in the range of 1 × 10 M −1 s −1 and a value of about 6 × 10 2 M −1 s −1 for Cys, these values are even lower for O 2•− reactions of these amino acids, with values ranging from 1.0 × 10 −1 M −1 s −1 to about 2 × 10 M −1 s −1 [ 131 ]. The difference in reactivity of the two superoxide species may be due to the fact that HO 2• acts as a weak oxidising agent, while O 2•− behaves as a weak oxidising and reducing agent, and these values may also depend on whether the amino acids are protonated or not. Cystine and Met do not react with superoxide, but N-acetylcysteine and glutathione GSH do [132–134], Figure 49. Int. J. Mol. Sci. 2023,24, 1841 22 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 23 of 45 The high stereoselectivity observed in this reaction is inconsistent with the intermediation of free alkyl radicals. Furthermore, it has been observed that the structure of the alkyl group attached to the halogen, the nature of the leaving group, and the polarity of the solvent exert influence on the course of the reaction in the carbon–oxygen bond formation step that is consistent with a mechanism involving an SN2 displacement on the carbon; therefore, it follows that the initial reaction of the superoxide with an alkyl halide produces an alkylperoxy radical [130], Figure 48. Figure 48. Formation of the C-O bond by a mechanism involving a displacement of SN2 on carbon. 4.1.11. Reactions of Superoxide with Amino Acids Second order rate constant values for HO2• reactions for aliphatic amino acids are reported in the literature in the range of 1 × 10 M−1s−1 and a value of about 6 × 102 M−1s−1 for Cys, these values are even lower for O2•− reactions of these amino acids, with values ranging from 1.0 × 10−1 M−1s−1 to about 2 × 10 M−1s−1 [131]. The difference in reactivity of the two superoxide species may be due to the fact that HO2• acts as a weak oxidising agent, while O2•− behaves as a weak oxidising and reducing agent, and these values may also depend on whether the amino acids are protonated or not. Cystine and Met do not react with superoxide, but N-acetylcysteine and glutathione GSH do [132–134], Figure 49. Figure 49. Rate constants for the reaction of N-acetylcysteine and glutathione with O2•−. Despite the large body of experimental work dealing with the oxidation of thiols by superoxide, the mechanism of this reaction remains controversial, Figure 50. According to some authors [135–137], the superoxide radical reacts with RSH thiols by abstraction of the hydrogen atom giving the thiyl radical and hydrogen peroxide; other authors consider that the first step of the reaction is the formation of a three-electron-bond radical, which breaks into a sulfinyl radical and a hydroxide anion [132–134]. Figure 50. Two pathways for the reaction of O2•– with thiols. Figure 49. Rate constants for the reaction of N-acetylcysteine and glutathione with O2•− . Despite the large body of experimental work dealing with the oxidation of thiols by superoxide, the mechanism of this reaction remains controversial, Figure 50. According to some authors [ 135 – 137 ], the superoxide radical reacts with RSH thiols by abstraction of the hydrogen atom giving the thiyl radical and hydrogen peroxide; other authors consider that the first step of the reaction is the formation of a three-electron-bond radical, which breaks into a sulfinyl radical and a hydroxide anion [132–134]. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 23 of 45 The high stereoselectivity observed in this reaction is inconsistent with the intermediation of free alkyl radicals. Furthermore, it has been observed that the structure of the alkyl group attached to the halogen, the nature of the leaving group, and the polarity of the solvent exert influence on the course of the reaction in the carbon–oxygen bond formation step that is consistent with a mechanism involving an SN2 displacement on the carbon; therefore, it follows that the initial reaction of the superoxide with an alkyl halide produces an alkylperoxy radical [130], Figure 48. Figure 48. Formation of the C-O bond by a mechanism involving a displacement of SN2 on carbon. 4.1.11. Reactions of Superoxide with Amino Acids Second order rate constant values for HO2• reactions for aliphatic amino acids are reported in the literature in the range of 1 × 10 M−1s−1 and a value of about 6 × 102 M−1s−1 for Cys, these values are even lower for O2•− reactions of these amino acids, with values ranging from 1.0 × 10−1 M−1s−1 to about 2 × 10 M−1s−1 [131]. The difference in reactivity of the two superoxide species may be due to the fact that HO2• acts as a weak oxidising agent, while O2•− behaves as a weak oxidising and reducing agent, and these values may also depend on whether the amino acids are protonated or not. Cystine and Met do not react with superoxide, but N-acetylcysteine and glutathione GSH do [132–134], Figure 49. Figure 49. Rate constants for the reaction of N-acetylcysteine and glutathione with O2•−. Despite the large body of experimental work dealing with the oxidation of thiols by superoxide, the mechanism of this reaction remains controversial, Figure 50. According to some authors [135–137], the superoxide radical reacts with RSH thiols by abstraction of the hydrogen atom giving the thiyl radical and hydrogen peroxide; other authors consider that the first step of the reaction is the formation of a three-electron-bond radical, which breaks into a sulfinyl radical and a hydroxide anion [132–134]. Figure 50. Two pathways for the reaction of O2•– with thiols. Figure 50. Two pathways for the reaction of O2•− with thiols. In both reactions it is assumed that the intermediate RS or RSO will further interact with the thiolate to give the final disulphide RSSR. 4.1.12. Radical–Radical Reactions of Superoxide Phenoxyl radicals are produced by one-electron oxidation of phenols. The most common phenol in biological systems is tyrosine, either in its free form or in peptides and proteins. The phenoxyl radicals in Tyr initially react with superoxide by addition, and the intermediate formed either releases oxygen to regenerate the parent compound or is converted to a hydroperoxide. The hydroperoxide, due to the resonant forms of Tyr, can be formed in the ortho or para position with respect to the phenoxyl radical [ 138 – 140 ], Figures 51 and 52. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 24 of 45 In both reactions it is assumed that the intermediate RS or RSO will further interact with the thiolate to give the final disulphide RSSR. 4.1.12. Radical–Radical Reactions of Superoxide Phenoxyl radicals are produced by one-electron oxidation of phenols. The most common phenol in biological systems is tyrosine, either in its free form or in peptides and proteins. The phenoxyl radicals in Tyr initially react with superoxide by addition, and the intermediate formed either releases oxygen to regenerate the parent compound or is converted to a hydroperoxide. The hydroperoxide, due to the resonant forms of Tyr, can be formed in the ortho or para position with respect to the phenoxyl radical [138–140], Figures 51 and 52. Figure 51. The reaction of tyrosyl radicals with O2•– provides tyrosine and tyrosine hydroperoxides. Electron transfer repairs the Tyr radical and addition results in the formation of Tyr-hydroperoxide. Figure 52. Resonant shapes of the Tyr•. As a consequence of electron delocalisation by the benzene ring, the Tyr• undergo rapid dimerization reactions to give Tyr-Tyr cross-linked species producing isomers with different cross-linked bonds, C-C cross-linked and C-O cross-linked species, Figure 53. Figure 51. The reaction of tyrosyl radicals with O 2•− provides tyrosine and tyrosine hydroperoxides. Electron transfer repairs the Tyr radical and addition results in the formation of Tyr-hydroperoxide. Int. J. Mol. Sci. 2023,24, 1841 23 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 24 of 45 In both reactions it is assumed that the intermediate RS or RSO will further interact with the thiolate to give the final disulphide RSSR. 4.1.12. Radical–Radical Reactions of Superoxide Phenoxyl radicals are produced by one-electron oxidation of phenols. The most common phenol in biological systems is tyrosine, either in its free form or in peptides and proteins. The phenoxyl radicals in Tyr initially react with superoxide by addition, and the intermediate formed either releases oxygen to regenerate the parent compound or is converted to a hydroperoxide. The hydroperoxide, due to the resonant forms of Tyr, can be formed in the ortho or para position with respect to the phenoxyl radical [138–140], Figures 51 and 52. Figure 51. The reaction of tyrosyl radicals with O2•– provides tyrosine and tyrosine hydroperoxides. Electron transfer repairs the Tyr radical and addition results in the formation of Tyr-hydroperoxide. Figure 52. Resonant shapes of the Tyr•. As a consequence of electron delocalisation by the benzene ring, the Tyr• undergo rapid dimerization reactions to give Tyr-Tyr cross-linked species producing isomers with different cross-linked bonds, C-C cross-linked and C-O cross-linked species, Figure 53. Figure 52. Resonant shapes of the Tyr•. As a consequence of electron delocalisation by the benzene ring, the Tyr • undergo rapid dimerization reactions to give Tyr-Tyr cross-linked species producing isomers with different cross-linked bonds, C-C cross-linked and C-O cross-linked species, Figure 53. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 25 of 45 Figure 53. Formation and reactions of Tyr phenoxyl radicals Tyr•. Tyr• self-react to produce di-Tyr (o,o′-di-Tyr, blue; iso-di-Tyr, black). Reactions of superoxide with tyrosyl radicals on the original amino acid or on small peptides are fast enough to compete with both superoxide dismutation and tyrosyl radical combination to form dityrosine derivatives. The production of di-Tyr crosslinks is limited by these alternative reactions, such as the short-lived peroxide formation reaction. For tyrosyl radicals, the reaction with superoxide to form the addition product (tyrosine hydroperoxide) is three times faster than dimerization [139,141]. Tyrosine and aminophenols are anomalous in that they undergo many more additions, the amino group attaches to the b-position of the unsaturated a,b.ketone to give a heterocyclic product. When tyrosine is N-terminal, the main products are hydroperoxides that have been cyclised via 1,4-conjugated addition (intramolecular Michael addition) of the terminal amine, Figure 54. When tyrosine is not N-terminal, electron transfer from O to the peptide radical prevails. Superoxide and tyrosyl radicals are among the most frequent radicals generated biologically during oxidative stress. The reaction between the two is very favourable. O2•− can react rapidly with low molecular mass Tyrradicals, with a rate constant of ~1.5 × 109 M−1 s−1, which is about three times faster than dimerization, Figure 54. Figure 54. When tyrosine is N-terminal, the major products are hydroperoxides that have cyclized via intramolecular Michael addition of the terminal amine. Figure 53. Formation and reactions of Tyr phenoxyl radicals Tyr • . Tyr • self-react to produce di-Tyr (o,o0-di-Tyr, blue; iso-di-Tyr, black). Reactions of superoxide with tyrosyl radicals on the original amino acid or on small peptides are fast enough to compete with both superoxide dismutation and tyrosyl radical combination to form dityrosine derivatives. The production of di-Tyr crosslinks is limited by these alternative reactions, such as the short-lived peroxide formation reaction. For tyrosyl radicals, the reaction with superoxide to form the addition product (tyrosine hydroperoxide) is three times faster than dimerization [139,141]. Tyrosine and aminophenols are anomalous in that they undergo many more additions, the amino group attaches to the b-position of the unsaturated a,b.ketone to give a heterocyclic product. When tyrosine is N-terminal, the main products are hydroperoxides that have been cyclised via 1,4-conjugated addition (intramolecular Michael addition) of the terminal amine, Figure 54. When tyrosine is not N-terminal, electron transfer from O to the peptide radical prevails. Superoxide and tyrosyl radicals are among the most frequent radicals generated biologically during oxidative stress. The reaction between the two is very favourable. O 2•− can react rapidly with low molecular mass Tyrradicals, with a rate constant of ~1.5 × 10 9 M −1 s −1 , which is about three times faster than dimerization, Figure 54. Int. J. Mol. Sci. 2023,24, 1841 24 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 25 of 45 Figure 53. Formation and reactions of Tyr phenoxyl radicals Tyr•. Tyr• self-react to produce di-Tyr (o,o′-di-Tyr, blue; iso-di-Tyr, black). Reactions of superoxide with tyrosyl radicals on the original amino acid or on small peptides are fast enough to compete with both superoxide dismutation and tyrosyl radical combination to form dityrosine derivatives. The production of di-Tyr crosslinks is limited by these alternative reactions, such as the short-lived peroxide formation reaction. For tyrosyl radicals, the reaction with superoxide to form the addition product (tyrosine hydroperoxide) is three times faster than dimerization [139,141]. Tyrosine and aminophenols are anomalous in that they undergo many more additions, the amino group attaches to the b-position of the unsaturated a,b.ketone to give a heterocyclic product. When tyrosine is N-terminal, the main products are hydroperoxides that have been cyclised via 1,4-conjugated addition (intramolecular Michael addition) of the terminal amine, Figure 54. When tyrosine is not N-terminal, electron transfer from O to the peptide radical prevails. Superoxide and tyrosyl radicals are among the most frequent radicals generated biologically during oxidative stress. The reaction between the two is very favourable. O2•− can react rapidly with low molecular mass Tyrradicals, with a rate constant of ~1.5 × 109 M−1 s−1, which is about three times faster than dimerization, Figure 54. Figure 54. When tyrosine is N-terminal, the major products are hydroperoxides that have cyclized via intramolecular Michael addition of the terminal amine. Figure 54. When tyrosine is N-terminal, the major products are hydroperoxides that have cyclized via intramolecular Michael addition of the terminal amine. An indole radical (tryptophanyl radical Trp-) is readily formed on the indole ring of tryptophan Trp. These radicals undergo multiple reactions, including ring opening and dimerization. As a consequence of electron delocalisation on the indole ring, Trp • radicals undergo rapid dimerization reactions to give Trp-Trp cross-linked species that produce isomers with different cross-linkages, C-C cross-linked and C-C cross-linked species. C-N [142,143], Figure 55. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 26 of 45 An indole radical (tryptophanyl radical Trp-) is readily formed on the indole ring of tryptophan Trp. These radicals undergo multiple reactions, including ring opening and dimerization. As a consequence of electron delocalisation on the indole ring, Trp• radicals undergo rapid dimerization reactions to give Trp-Trp cross-linked species that produce isomers with different cross-linkages, C-C cross-linked and C-C cross-linked species. C-N [142,143], Figure 55. Figure 55. Proposed structures of isomeric Trp-Trp crosslinks. As a result of delocalization of electron density around the indole ring, Trp• can undergo dimerization reactions that produce isomers with different C-C and C-N bonds. The structures indicate R stereochemistry around the formed bonds. Trp• can undergo rapid dimerization to form a series of isomeric cross-linked TrpTrp species. These reactions have second-order rate constants in the range of k = 2–6 × 108 M−1 s−1 at pH 7.4 and k = 7.3 × 108 M−1 s−1 at pH 10. These high values suggest that dimerization can compete with other Trpreactions in complex systems. Trp• dimerization takes place under conditions where O2•– is absent or present in low concentrations [144]. The reaction of Trp• radical with O2•– gives a hydroperoxide. Trp• reacts very rapidly with O2•– with second order rate constants, k, in the range 0.7–2.2 × 109 M−1 s−1, the main initial products being hydroperoxides, in almost quantitative yields [145–151], Figure 56. Figure 56. Trp• reacts with O2•−, the major products being hydroperoxides. Subsequent decomposition of these species gives rise to N-formylquinurenine, quinurenine, alcohols and diols. These data indicate that the O2•− with Trp• reaction should be considered as an important pathway for the degradation of Trp in peptides and proteins subjected to oxidative damage, Figure 57. Figure 55. Proposed structures of isomeric Trp-Trp crosslinks. As a result of delocalization of electron density around the indole ring, Trp•can undergo dimerization reactions that produce isomers with different C-C and C-N bonds. The structures indicate R stereochemistry around the formed bonds. Trp • can undergo rapid dimerization to form a series of isomeric cross-linked Trp-Trp species. These reactions have second-order rate constants in the range of k = 2–6 ×108M−1s−1 at pH 7.4 and k = 7.3 × 108 M −1 s −1 at pH 10. These high values suggest that dimerization can compete with other Trpreactions in complex systems. Trp • dimerization takes place under conditions where O2•− is absent or present in low concentrations [144]. The reaction of Trp • radical with O 2•− gives a hydroperoxide. Trp • reacts very rapidly with O 2•− with second order rate constants, k, in the range 0.7–2.2 × 10 9 M −1 s −1 , the main initial products being hydroperoxides, in almost quantitative yields [145–151], Figure 56. Int. J. Mol. Sci. 2023,24, 1841 25 of 44 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 26 of 45 An indole radical (tryptophanyl radical Trp-) is readily formed on the indole ring of tryptophan Trp. These radicals undergo multiple reactions, including ring opening and dimerization. As a consequence of electron delocalisation on the indole ring, Trp• radicals undergo rapid dimerization reactions to give Trp-Trp cross-linked species that produce isomers with different cross-linkages, C-C cross-linked and C-C cross-linked species. C-N [142,143], Figure 55. Figure 55. Proposed structures of isomeric Trp-Trp crosslinks. As a result of delocalization of electron density around the indole ring, Trp• can undergo dimerization reactions that produce isomers with different C-C and C-N bonds. The structures indicate R stereochemistry around the formed bonds. Trp• can undergo rapid dimerization to form a series of isomeric cross-linked TrpTrp species. These reactions have second-order rate constants in the range of k = 2–6 × 108 M−1 s−1 at pH 7.4 and k = 7.3 × 108 M−1 s−1 at pH 10. These high values suggest that dimerization can compete with other Trpreactions in complex systems. Trp• dimerization takes place under conditions where O2•– is absent or present in low concentrations [144]. The reaction of Trp• radical with O2•– gives a hydroperoxide. Trp• reacts very rapidly with O2•– with second order rate constants, k, in the range 0.7–2.2 × 109 M−1 s−1, the main initial products being hydroperoxides, in almost quantitative yields [145–151], Figure 56. Figure 56. Trp• reacts with O2•−, the major products being hydroperoxides. Subsequent decomposition of these species gives rise to N-formylquinurenine, quinurenine, alcohols and diols. These data indicate that the O2•− with Trp• reaction should be considered as an important pathway for the degradation of Trp in peptides and proteins subjected to oxidative damage, Figure 57. Figure 56. Trp•reacts with O2•− , the major products being hydroperoxides. Subsequent decomposition of these species gives rise to N-formylquinurenine, quinurenine, alcohols and diols. These data indicate that the O 2•− with Trp • reaction should be considered as an important pathway for the degradation of Trp in peptides and proteins subjected to oxidative damage, Figure 57. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 27 of 45 Figure 57. The formation and subsequent decomposition of dioxetanes gives as the main product N-formylkynurenine NFK which is hydrolysed to kynurenine Kyn. 4.1.13. Proton–Radical Transfer Because the pKa of the conjugate acid of O2•– is 4.8, O2•– is considered a weak base. However, proton and radical transfer pathways have been proposed to demonstrate the antioxidant property of phenols and polyphenols against O2•–. Results obtained by cyclic voltammetry show that proton transfer and radical transfer pathways are present for both monophenols and polyphenols, and the relative contributions of the two pathways depend on the structure of the phenol. Polyphenols containing an o-diphenol ring (as flavonoids) have the highest reactivities. Proton transfer is the main mechanism for the reaction between monophenols and O2•– in aprotic solvents, such as DMF or DMSO. The mechanism involves a first proton transfer between O2•– acting as a weak base and the phenolic compound PhOH acting as a Bronsted acid according to Figure, in which the formation of phenoxide PhO– and HO2•, although thermodynamically unfavourable, can be completed by the electron transfer reaction between HO2• and O2•– to form HO2• (a very strong base) and O2. In this, the former can extract more protons from the phenol to form the phenoxide PhO– according to Figure, in which the former can extract more protons from the phenol to form the phenoxide PhO– according to Figure 58. Figure 58. Reaction of phenol with superoxide anion. Polyphenols, however, undergo a radical (or H-atom) transfer reaction with O2•– to form the phenoxyl radical PhO– and HO2•. Similar to monophenols, HO2• can also extract protons from PhOH to form phenoxide PhO–, Figure 59. Figure 57. The formation and subsequent decomposition of dioxetanes gives as the main product N-formylkynurenine NFK which is hydrolysed to kynurenine Kyn. 4.1.13. Proton–Radical Transfer Because the pKa of the conjugate acid of O 2•− is 4.8, O 2•− is considered a weak base. However, proton and radical transfer pathways have been proposed to demonstrate the antioxidant property of phenols and polyphenols against O 2•− . Results obtained by cyclic voltammetry show that proton transfer and radical transfer pathways are present for both monophenols and polyphenols, and the relative contributions of the two pathways depend on the structure of the phenol. Polyphenols containing an o-diphenol ring (as flavonoids) have the highest reactivities. Proton transfer is the main mechanism for the reaction between monophenols and O 2•− in aprotic solvents, such as DMF or DMSO. The mechanism involves a first proton transfer between O 2•− acting as a weak base and the phenolic compound PhOH acting as a Bronsted acid according to Figure, in which the formation of phenoxide PhO – and HO 2• , although thermodynamically unfavourable, can be completed by the electron transfer reaction between HO 2• and O 2•− to form HO 2• (a very strong base) and O 2 . In this, the former can extract more protons from the phenol to form the phenoxide PhO – according to Figure, in which the former can extract more protons from the phenol to form the phenoxide PhO–according to Figure 58. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 27 of 45 Figure 57. The formation and subsequent decomposition of dioxetanes gives as the main product N-formylkynurenine NFK which is hydrolysed to kynurenine Kyn. 4.1.13. Proton–Radical Transfer Because the pKa of the conjugate acid of O2•– is 4.8, O2•– is considered a weak base. However, proton and radical transfer pathways have been proposed to demonstrate the antioxidant property of phenols and polyphenols against O2•–. Results obtained by cyclic voltammetry show that proton transfer and radical transfer pathways are present for both monophenols and polyphenols, and the relative contributions of the two pathways depend on the structure of the phenol. Polyphenols containing an o-diphenol ring (as flavonoids) have the highest reactivities. Proton transfer is the main mechanism for the reaction between monophenols and O2•– in aprotic solvents, such as DMF or DMSO. The mechanism involves a first proton transfer between O2•– acting as a weak base and the phenolic compound PhOH acting as a Bronsted acid according to Figure, in which the formation of phenoxide PhO– and HO2•, although thermodynamically unfavourable, can be completed by the electron transfer reaction between HO2• and O2•– to form HO2• (a very strong base) and O2. In this, the former can extract more protons from the phenol to form the phenoxide PhO– according to Figure, in which the former can extract more protons from the phenol to form the phenoxide PhO– according to Figure 58. Figure 58. Reaction of phenol with superoxide anion. Polyphenols, however, undergo a radical (or H-atom) transfer reaction with O2•– to form the phenoxyl radical PhO– and HO2•. Similar to monophenols, HO2• can also extract protons from PhOH to form phenoxide PhO–, Figure 59. Figure 58. Reaction of phenol with superoxide anion. Int. J. Mol. Sci. 2023,24, 1841 32 of 44 In the phagocytosis area, O 2•− production by the phagocyte NOX has been associated with pathogen killing for the last decades. In contrast, at much lower concentrations, O 2•− and ROS are necessary for cell signalling. O 2•− produced by NOX acts as a signalling molecule by modifying the redox state of proteins or lipids, and one of its possible targets is even NOX itself. Superoxide and H 2 O 2 added to NOX subunits have been shown to decrease O 2•− production, but only when added prior to subunit assembly [ 172 ]. Additionally, O 2•− derived from different NOx influences distinct downstream signalling pathways, which may be the reason for the co-expression of more than one isoform of NOX in specific cell types [173]. Phagocytes can release O 2•− both into the phagosome and the extracellular space due to expression of NOX2 on both the phagosome and the plasma membrane [ 174 ]. The activation of the mammalian phagocyte NOX2 is tightly regulated and predominantly depends on the engagement of surface receptors by dedicated ligands. Some cell surface receptors (Toll-like receptors (TLR), G-protein-coupled receptors (GPCR) and TNF receptors (TNFR)), can prime the NOX2 activation [ 175 ]. Stimulation of other receptors, including Fc and integrin, result in direct activation of NOX2. As NOx is assembled, electrons pass from cytosolic NADPH to FAD and membraneintegrated heme groups and begin the reduction of molecular oxygen O 2 to O 2•− . Superoxide O 2•− is a highly reactive “non-diffusible” specie that is enzymatically transformed to hydrogen peroxide. In granulocytes, H 2 O 2 is quickly converted into hypochlorous acid HOCl [ 176 ] by the action of the enzyme myeloperoxidase (MPO) contained within them. HOCl has superior bactericidal characteristics to superoxide [177]. The role of superoxide in phagocytes differs depending on NOX2 expression and activity. After activation, neutrophils produce more O 2•− compared to monocytes and macrophages [ 178 ]. Dendritic cells (DC) express little NOX2 and consequently have a lower O 2•− and ROS production after activation [ 179 ]. In neutrophils, H 2 O 2 generation in phagosomes activates the enzyme myeloperoxidase, catalysing the production of HOCl, which is oxidant and antimicrobial, and contributes greatly to the lysis of microbes [ 178 ]. After infection, excessive neutrophil activity causes tissue damage, so their deactivation or cell death is physiologically important and tightly regulated. This process is called pathogen-induced cell death and is also dependent on NOx activity [ 180 ]. Mononuclear phagocytes (primarily monocytes and macrophages) do not express MPO and thus contain more H2O2in their phagosomes. 8. Macrophages, Neutrophils and Superoxide Anion 8.1. Macrophages Macrophages, discovered by Ilya Metchnikoff in the late 19th century, were long considered an important part of the effector cells of the immune system. Metchnikoff won the Nobel Prize in 1908 for his description of phagocytosis and went so far as to propose that the key to immunity was to “stimulate phagocytes” [ 181 ]. Irrespective of their role in the immune system, macrophages clear about 2 × 10 11 erythrocytes daily and are also involved in the removal of cellular debris generated during tissue remodelling, thereby eliminating cells that have undergone apoptosis. These clearance processes are a vital metabolic contribution, without which the host would not survive [182]. Macrophages (from Greek: large eaters, makros + phagein), are the first immune cells to encounter invading pathogens, and their goal is to engulf microbes, dead cells, foreign substances, cancer cells and cellular debris by phagocytosis [ 183 ]. These phagocytes are found essentially in all tissues, and historically, they have been given various names, as histiocytes, Kupffer cells, alveolar macrophages, microglia and others, all of which are part of the mononuclear phagocytic system [ 184 ]. They play a key role in nonspecific defence (also called as innate immunity) and help specific defence mechanisms (adaptive immunity) by recruiting other immune cells (lymphocytes) [ 185 ]. If a person has dysfunctional macrophages, this causes diseases, such as chronic granulomatous disease, which leads to frequent infections, as this first line of defence against infection is not available [ 186 ]. Int. J. Mol. Sci. 2023,24, 1841 33 of 44 Human macrophages are about 21 µ m in diameter and are produced by the differentiation of monocytes in tissues [187]. Macrophage activation is a response to a wide range of stimuli, including the nature of microbial agents, damaged cells, activated lymphocytes and inflammatory cells [ 188 ]. Classically activated macrophages require a priming signal in the form of interferon IFNγ [ 189 ]. Priming of macrophages with IFNγ reprograms cellular responses to other cytokines, such as type I IFNs and IL-10. Alternatively activated macrophages do not require any priming, because interleukins IL-4 and/or IL-13 can act as enough stimuli [190,191] . Alternatively activated macrophages change their morphology and chemical secretion pattern as a result [192]. The bioplasticity of macrophages is one of their main characteristics, resulting in extreme heterogeneity under physiological but also pathological conditions. Macrophages can be classified into two distinct subsets: M1, or classically activated, and M2, or alternatively activated [ 193 ]. This plasticity induces a phenomenon called polarisation, which regulates the functionality of macrophages and the role they will play in the tissues that host them [194]. M1 macrophages are pro-inflammatory and polarized by lipopolysaccharides (LPS) and/or cytokines, such as IFNγ and the granulocyte-macrophage colony-stimulating factor GM-CSF, and produce pro-inflammatory cytokines, such as interleukin-1 β IL-1 β , IL-6, IL-12, IL-23 and TNFα [ 194 ]. They secrete high levels of TNFα , IL-12 and IL-23 cytokines. M1 macrophages produce intracellular nitric oxide • NO by inducible nitric oxide synthase iNOS and superoxide anion O 2•− and subsequent reactive species, which are cytotoxic to microbial agents and fight infection [195]. M2 macrophages are polarized and activated upon exposure to certain cytokines such as IL-4, IL-10 or IL-13, subsequently producing polyamines and/or proline, which are involved in cell proliferation and collagen production [ 196 ]. These M2 macrophages are associated with wound healing and tissue repair. The secretion of the inflammationinhibiting cytokine IL-10 is the hallmark of M2 macrophages, arginase-1 and more organic compounds [ 197 ]. This phenotype can be further subdivided into M2a, M2b and M2c, based on their specific functions. All three subtypes have anti-inflammatory properties, but M2a and M2b macrophages are considered immunoregulatory and are known to mediate the Th-2 response. M2c cells are immunosuppressive and are involved in extracellular matrix ECM remodelling. M2a and M2c secrete growth factors that promote the formation of new blood vessels (angiogenesis) and tissue regeneration [198]. Finally, it is increasingly clear that macrophages are involved in the progression of pathological conditions, including cancer, cardiovascular disease, obesity and wound healing [ 196 ]. In cancer, M1 macrophages have anti-tumour functions, while M2 macrophages support angiogenesis, invasion and metastasis of neoplastic cells. Most tumour-associated macrophages (TAM) adopt an M2-type phenotype, and their presence correlates with a poor prognosis [199,200]. Alongside O 2•− production, macrophages employ several direct antimicrobial mechanisms in the phagosome, such as reactive nitrogen species RNS, as well as delivery of cathepsins and other hydrolases [ 201 – 204 ]. Indirect mechanisms involve inflammasome activation and the secretion of cytokines and chemokines, which aim to organize subsequent innate and adaptive immune responses, as well as MHC-dependent presentation of pathogen-derived antigens [205]. When macrophages initiate the bacterial recognition process, O 2•− production starts in different cellular compartments [ 201 ]. One of the most important goals is the lysis of phagocytosed bacteria by the oxidative burst generated by the NADPH oxidase NOX2. The oxidative burst is the rapid release of O 22from various cell types, especially macrophages and neutrophils, and it requires a 10to 20-fold increase in oxygen consumption by NOX activity. The oxidative burst in phagocytes is usually associated with killing bacteria, but in the case of alveolar macrophages, they usually produce lower levels of ROS than neutrophils and may need to be activated to exert their bactericidal properties. Instead, Int. J. Mol. Sci. 2023,24, 1841 34 of 44 their transient oxidative burst regulates the inflammatory response by triggering the synthesis of cytokines for redox signalling, resulting in an influx of activated neutrophils and macrophages. 8.2. Neutrophils Neutrophils are a main cellular component of the innate immunity and play a dual role because they provide a rapid and non-specific response to the infectious progression and mediate between the innate and the adaptive immune systems. Neutrophils are short-lived granulocytes derived from pluripotent hematopoietic stem cells from bone marrow [ 206 ]. Most haematopoiesis is related to granulopoiesis, and almost 60% of bone marrow leukocytes are granulocyte precursors [207]. Neutrophils are the main leukocytes circulating in the blood, and the first innate immune cells to be recruited to a focus of infection. Here, neutrophils have several defence strategies at their disposal, such as the production of superoxide O 2•− , the release of antimicrobial factors and the formation of neutrophil extracellular traps (NET) [208]. There are two main granule populations in mature neutrophils: (i) azurophils are the first to develop during granulopoiesis, they contain the enzyme myeloperoxidase (MPO) and other proteolytic enzymes (cathepsins, proteinase-3, elastase), antimicrobial defensins and bactericidal proteins [ 209 ]; and (ii) a specific type of granules (peroxidase negative) that mature during differentiation contain membrane proteins, such as lactoferrin and collagenase, and are receptors for chemotactic peptides, cytokines, opsonins and adhesion proteins [ 210 ]. Mature neutrophils are released into the bloodstream, where they circulate for 10–24 h before migrating to tissues, where they remain for the next 1–2 days before undergoing apoptosis and being cleared by macrophages [ 211 ]. Neutrophils released into the systemic circulation constitute most of the circulating leukocyte cell population. Under normal conditions, the number of mature neutrophils is almost constant, but during an infectious process their population can increase up to 10-fold [212]. Some circulating neutrophils move through the walls of postcapillary veins through transient interactions with endothelial cells [ 213 ]. Their role is to look for signs of tissue damage, inflammation or the invading microorganisms themselves, as well as the presence of chemotactic or chemoattractant signals derived from the host and/or pathogen [ 214 ]. In the presence of pathogens, different host cells (such as monocytes and macrophages) secrete inflammatory and neutrophil chemoattractant mediators (such as the leukotriene LTB4 involved in inflammation, the interleukin IL-8, and the chemokine CXCL6, a small cytokine belonging to the CXC chemokine family, also known as granulocyte chemotactic protein 2 GCP-2) which bind to specific receptors on the neutrophil surface [ 215 ]. These signals direct neutrophils from the intravascular space to the site of infection in tissues. Another important fact is neutrophil priming described in the early 1980s. Its classical definition is the ability of a primary agonist, under stimulatory concentrations, to enhance superoxide production in response to a secondary stimulus [ 216 ]. Priming occurs at almost all levels of neutrophil function: adhesion, phagocytosis, cytokine secretion, leukotriene synthesis and degranulation. Priming is induced by cytokines, chemokines, growth factors, lipid-derived signalling molecules and physical cell–cell contact and adhesion [ 217 ]. Following recognition of chemotactic signals and/or priming of neutrophils, neutrophils leave the peripheral circulation by transmigration through the endothelial wall, a process termed extravasation [209]. At the site of infection, neutrophils bind and ingest the invading micro-organisms by phagocytosis. Two main mechanisms account for the microbicidal properties of neutrophils: (i) The production of O 2•− . Neutrophils possess the enzyme NOX which, when activated, produces superoxide anion, with strong antimicrobial properties. Superoxide can be released outside the cell, or inside the cell (in the phagosome); (ii) The coordinated release of proteolytic and antimicrobial granule content. The release of the contents of the primary and secondary granules has important antimicrobial significance. The granules contain MPO, lactoferrin, lysosomes and NGAL [ 218 ]. The Int. J. Mol. Sci. 2023,24, 1841 35 of 44 enzyme myeloperoxidase MPO forms hypochlorous acid HOCl [ 176 ] after reaction of chloride anion with hydrogen peroxide. HOCl oxidises tyrosine residues to form the tyrosyl radical [128]. Because of their common origin, neutrophils and macrophages have common functions (phagocytosis) and similar kinetic performance during infections [ 218 ]. Neutrophils can influence macrophage differentiation into proor anti-inflammatory subtypes [ 218 ]. Interferonγ released by activated neutrophils induces macrophage activation [ 219 ]. Neutrophils release MPO, which is taken up by residential macrophages expressing macrophage mannose receptors (MMR). The interaction between MPO and MMRs leads to the release of ROS (reactive oxygen species) and proinflammatory cytokines (IL-6, IL-8, TNFα , IL-1, GM-CSF) by macrophages. The release of TNFα , IL-1 β , G-CSF and GM-CSF at the site of macrophage infection increases the survival of recruited neutrophils from 6–12 h to 24–48 h [ 220 ]. The cytokine IL-17 is an element of the innate immune system released by CD4+ Th17, NK cells and neutrophils. IL-17 acts on neutrophils by increasing their number, survival and recruitment to the site of infection [ 221 ]. Neutrophils are the transport vehicle for intracellular pathogens carrying antigens to DCs and participate in the activation of the T-cell immune response by DCs [ 222 ]. The reaction of neutrophils and macrophages during infection triggers the production of innate defence regulatory peptide 1 IDR1, with similar activity to defensins or cathelicidins [ 223 ]. IDR1 stimulates the antimicrobial activity of macrophages [224]. Along with activation of innate or adaptive immune cells, neutrophils, by releasing arginase or ROS, can inhibit NK-cell or T-cell activation by depriving extracellular levels of L-arginine, necessary for T-cell activation [225]. 9. Conclusions Superoxide is produced mainly through metabolic reactions in which oxygen molecules are consumed. The control of O 2•− represents for the cell the neural point in the balance between oxidants and antioxidants. In a physiological equilibrium, reactive oxygen species (ROS) are useful substances involved in cell signalling. However, in a pathological state, ROS can be dangerous if the synthesis of these molecules is initiated in an uncontrolled manner. Consequently, they perform two tasks in the metabolism of the cell. The mitochondrial transport chain ETC generates superoxide under physiological conditions, increasing ROS production and oxidative stress. Mitochondria are the main source of intracellular O 2•− production, which can lead to mtDNA damage and increased superoxide production. Other internal sources that can induce superoxide are NADPH oxidase NOX, xanthine oxidase XO, lipoxygenase, cyclooxygenase, and cytochrome P450 CYP/cytochrome P450 reductase POR. Reactive stress occurs when an excess of free radicals overwhelms the body and cannot be neutralized by the antioxidant mechanism (enzymes or peptides, such as glutathione or superoxide dismutase (SOD), etc.). At the same time, ROS-induced toxicity is beneficial for the oxidative destruction of microbial pathogens during the activation of the innate immune system via specialized immune cells, such as neutrophils or macrophages. Author Contributions: Conceptualization, C.M.C.A. and C.A.J.; investigation, C.M.C.A. and C.A.J.; writing—review and editing, C.M.C.A., C.A.J., J.M.P.d.l.L., E.P.-L. and F.J.P.; supervision, C.M.C.A., C.A.J. and J.M.P.d.l.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) del Gobierno de Canarias, Project ProID2020010134, Caja Canarias, Project 2019SP43, the Spanish Ministry of Economy and Competitiveness (Grant PID2019-105838RB-C31) and the State Plan for Scientific, Technical Research and Innovation 2021–2023 from the Spanish Ministry of Science and Innovation (project PLEC2022-009507). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Int. J. Mol. Sci. 2023,24, 1841 36 of 44 Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. 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