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Published online 2023 Jan 17. doi: 10.3390/ijms24031841
PMCID: PMC9916283
PMID: 36768162
Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity
Celia María Curieses Andrés,1 José Manuel Pérez de la Lastra,2,* Celia Andrés Juan,3 Francisco J. Plou,4 and Eduardo Pérez-Lebeña5
Claudio Santi, Academic Editor
Author information Article notes Copyright and License information PMC Disclaimer
Associated DataData Availability Statement
Abstract
Classically, superoxide anion O2•− and reactive oxygen species ROS play a dual role. At the physiological balance level, they are a by-product of O2 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 O2•−, 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 O2•− and how it is harnessed by the innate immune system to produce lysis of microbial agents.
일반적으로 슈퍼옥사이드 음이온 O2와 활성 산소종 ROS는 이중적인 역할을 합니다. 생리학적 균형 수준에서는 세포 신호 전달에 필요한 산소 환원의 부산물이며, 병리학적인 수준에서는 질병과 세포 사멸, 괴사, 페로옵토시스, 파이로옵토시스 및 자가포식 세포 사멸을 유발할 수 있으므로 유해한 것으로 간주됩니다.
이번 개정판에서는
슈퍼옥사이드 O2--의 주요 특징,
생성 경로,
산화되는 생체 분자,
생체 분자의 변형과 독성에 어떻게 기여하는지를
이해하는 데 중점을 두었습니다.
생물체에서 생성되는
대부분의 슈퍼옥사이드 제거를 담당하는 효소인
슈퍼옥사이드 디스뮤타아제의 역할을 연구합니다.
동시에,
과산화물과 유도된 라디칼에 의해 유도된 독성은
선천성 면역이 활성화되는 동안
호중구 또는
대식세포와 같은 특수 면역 세포에 의해 포획되는
미생물 병원균의 산화적 사멸에 유익합니다.
궁극적으로 이 리뷰에서는
산소(O2)와 관련된 화학적 원리와
선천 면역계가 산소를 어떻게 활용하여 미생물 병원체를 용해시키는지에 대해 자세히 설명합니다.
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 self-control 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 O2 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 non-radicals [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 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 O2•− triggers a cascade of ROS, some of which, such as H2O2, 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].
O2•− 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.
의학계에서는 최근 몇 년 동안 세포 스트레스와 자유 라디칼 연구에 큰 관심을 보이며, 인간의 삶의 질을 개선하고 수많은 질병의 기원을 이해할 수 있는 세포 자기 제어 메커니즘에 대한 지식을 심화시키는 데 초점을 맞추고 있습니다 [1].
산화 스트레스는
죽상동맥경화증, 만성 폐쇄성 폐질환, 알츠하이머병, 암 등
많은 질병의 구성 요소입니다[2].
동시에
ROS는
세포의 생존, 성장, 증식 및 분화, 면역 반응 등
다양한 생물학적 기능에 필수적입니다.
그러나 이러한 종의 역할을 이해하는 데 있어
주요 장애물 중 하나는
주로 매우 짧은 수명과 세포 내 여러 항산화제의 존재로 인해
생체 내에서 ROS/RNS를 감지하는 적절한 방법이 부족하다는 것입니다 [3].
실제로
라디칼은
미토콘드리아 전자 수송 사슬과
사이토크롬 P450에서 말단 전자 수용체로 O2를 사용하는 결과로
대부분의 유기체에서 지속적으로 생성됩니다 [4].
반응성 종이라는 용어는
자유 라디칼과 비라디칼의 두 가지 유형의 분자를 지칭합니다[5].
이 분자 세트는
세포 대사의 결과로 형성되며
정상적인 생리적 과정과 병리학적 과정 모두에서 발생하는
활성 산소 종 ROS와
활성 질소 종 RNS로
생물학적 시스템에서 대표됩니다.
그 외에도
염소 RClS 및
브롬 RBrS와 같은 다른 원소의 반응성 종도 존재하지만,
ROS와 RNS는 산화 환원 생물학에 관여하는 두 가지 주요 그룹입니다 [6].
슈퍼옥사이드 음이온은
산소 분자가 전자를 획득할 때 형성되는
1차 산소 라디칼입니다.
O2--의 초기 형성은
일련의 ROS를 촉발하며,
그 중 일부는 세포 신호의 핵심 분자로 작용하고
H2O2와 같은 다른 분자는 손상을 입힙니다.
궁극적으로
이러한 분자의 생물학적 영향은
ROS의 양, 세포 방어 및 세포 적응 능력에 의해 결정됩니다[7].
O2--는
생체 내에서 산화 스트레스를 일으키는
가장 중요한 활성 산소 종 ROS 중 하나이며
미토콘드리아 호흡 사슬의 부산물로 생성됩니다[8].
과산화물은 전하를 띠기 때문에
막 투과성이 낮고
음이온 채널을 통과하지만
이는 비효율적이며
과산화물이 생성되는 생리적 구획에서 대부분 반응합니다.
활성 산소 종(ROS)은
산소의 환원으로부터
외인성 또는 내인성적으로 생성되는 반응성이 높은
산소 함유 화학 물질 그룹으로,
라디칼과 비라디칼을 모두 포함하며
그 중 하나가 바로 슈퍼옥사이드입니다.
체내에 존재하는 ROS는
대부분 내인성이지만
자외선,
전리 방사선,
공해, 술 및 담배 섭취,
약물 및 독성 물질과 같은 외부 자극에 반응하여 생성될 수도 있습니다[9], 그림 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].
반응성 종과 자유 라디칼 및 기타 반응성 산소, 질소 및 염소 종의 명명법.
ROS를 제어하기 위해 신체는
효소 및 비효소 항산화제를 포함한
여러 가지 항산화 메커니즘을 사용합니다[10].
비효소성 저분자 항산화 화합물에는
세포 글루타티온, 비타민 C 및 E, 베타카로틴, 폴리페놀 및 요산이 포함됩니다.
항산화 효소에는
슈퍼옥사이드 디스뮤타제, 카탈라아제, 글루타치온 환원효소, 글루타치온 퍼옥시다아제 등이 있습니다.
SOD는
슈퍼옥사이드가 H2O2로 돌연변이되는 것을 촉매합니다.
포유류 세포에는
Mn-SOD,
세포질 Cu,
Zn-SOD 및
세포 외 Cu, Zn-SOD의 세 가지 형태의 SOD가 포함되어 있습니다.
MnSOD는
미토콘드리아에 가장 많이 존재하는 반면,
Cn, Zn-SOD는 세포질에 우세합니다 [11].
카탈라아제는
H2O2를 H2O로 환원하는 것을 촉매하는 중요한 항산화 효소입니다.
글루타티온 퍼옥시다아제는
H2O2의 분해를 위한 또 다른 중요한 효소입니다.
과일과 채소를 통해 정기적으로 섭취하는 폴리페놀은
페놀산, 플라보노이드, 스틸벤 및 리그난의 네 가지 주요 부류를 포함하여
폴리페놀 구조(방향족 고리에 여러 개의 수산기가 있는)를 가진
다수의 수산기 페놀 단위가 특징인 천연 유기 화합물의 큰 계열입니다 [12].
지금까지의 증거와 연구는
암, 심혈관 질환 및 신경 퇴행성 질환 예방에 있어
폴리페놀의 역할을 뒷받침합니다[13].
폴리페놀의 유익한 효과의 상당 부분은
세포 신호 경로의 조절을 기반으로 합니다[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 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.
슈퍼옥사이드 라디칼 음이온 O2--.
O2--는 17개의 전자와 음전하를 띠는 두 개의 산소 원자로 구성된 분자 산소 O2의 환원된 형태입니다( 그림 2). 슈퍼옥사이드는 전자의 이동에 의한 산소의 환원에 의해 호흡기에서 생성되는 최초의 종으로, 다양한 세포 시스템에서 생성되는 최초의 종 중 하나입니다. O2--는 모든 살아있는 호기성 유기체에서 형성되며 신호 전달 물질, 독성 물질 또는 자연적으로 분해되는 무해한 중간체로 작용할 수 있습니다. 생체 내에서는 두 가지 유형의 효소인 슈퍼옥사이드 환원효소 SOR과 슈퍼옥사이드 디스뮤타제 SOD에 의해 그 수치가 제한됩니다.
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.
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.
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].
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.
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.
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 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 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 non-enzymatically.
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.
Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen.
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].
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.
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.
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.
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.
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.
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 molybdo-flavoenzymes and is released by a calcium-activated protease during hypoxia. XO is 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].
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.
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.
Non-Biochemical sources of superoxide [25].
Int J Mol Sci. 2023 Feb; 24(3): 1841.
Published online 2023 Jan 17. doi: 10.3390/ijms24031841
PMCID: PMC9916283
PMID: 36768162
Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity
Celia María Curieses Andrés,1 José Manuel Pérez de la Lastra,2,* Celia Andrés Juan,3 Francisco J. Plou,4 and Eduardo Pérez-Lebeña5
Claudio Santi, Academic Editor
Author information Article notes Copyright and License information PMC Disclaimer
Associated DataData Availability Statement
Abstract
Classically, superoxide anion O2•− and reactive oxygen species ROS play a dual role. At the physiological balance level, they are a by-product of O2 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 O2•−, 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 O2•− 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 self-control 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 O2 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 non-radicals [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 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 O2•− triggers a cascade of ROS, some of which, such as H2O2, 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].
O2•− 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.
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 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.
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.
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.
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].
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.
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.
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 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 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 non-enzymatically.
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.
Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen.
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].
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.
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.
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.
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.
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.
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 molybdo-flavoenzymes and is released by a calcium-activated protease during hypoxia. XO is 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].
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.
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.
Non-Biochemical sources of superoxide [25].
Int J Mol Sci. 2023 Feb; 24(3): 1841.
Published online 2023 Jan 17. doi: 10.3390/ijms24031841
PMCID: PMC9916283
PMID: 36768162
Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity
Celia María Curieses Andrés,1 José Manuel Pérez de la Lastra,2,* Celia Andrés Juan,3 Francisco J. Plou,4 and Eduardo Pérez-Lebeña5
Claudio Santi, Academic Editor
Author information Article notes Copyright and License information PMC Disclaimer
Associated DataData Availability Statement
Abstract
Classically, superoxide anion O2•− and reactive oxygen species ROS play a dual role. At the physiological balance level, they are a by-product of O2 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 O2•−, 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 O2•− 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 self-control 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 O2 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 non-radicals [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 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 O2•− triggers a cascade of ROS, some of which, such as H2O2, 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].
O2•− 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.
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 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.
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.
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.
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].
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.
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.
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 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 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 non-enzymatically.
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.
Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen.
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].
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.
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.
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.
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.
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.
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 molybdo-flavoenzymes and is released by a calcium-activated protease during hypoxia. XO is 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].
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.
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.
Non-Biochemical sources of superoxide [25].
Int J Mol Sci. 2023 Feb; 24(3): 1841.
Published online 2023 Jan 17. doi: 10.3390/ijms24031841
PMCID: PMC9916283
PMID: 36768162
Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity
Celia María Curieses Andrés,1 José Manuel Pérez de la Lastra,2,* Celia Andrés Juan,3 Francisco J. Plou,4 and Eduardo Pérez-Lebeña5
Claudio Santi, Academic Editor
Author information Article notes Copyright and License information PMC Disclaimer
Associated DataData Availability Statement
Abstract
Classically, superoxide anion O2•− and reactive oxygen species ROS play a dual role. At the physiological balance level, they are a by-product of O2 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 O2•−, 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 O2•− 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 self-control 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 O2 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 non-radicals [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 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 O2•− triggers a cascade of ROS, some of which, such as H2O2, 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].
O2•− 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.
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 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.
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.
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.
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].
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.
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.
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 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 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 non-enzymatically.
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.
Superoxide radicals are produced in complexes I and III of the electron transport chain by transferring electrons to molecular oxygen.
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].
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.
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.
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.
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.
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.
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 molybdo-flavoenzymes and is released by a calcium-activated protease during hypoxia. XO is 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].
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.
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.
Non-Biochemical sources of superoxide [25].