In the previous article, we looked at three major processes through which reactive oxygen species (ROS) are produced in the body: NADPH oxidase activity during immune responses, oxidative phosphorylation involved in ATP production, and the metabolic pathway leading to uric acid formation and excretion. All three are essential to the normal functioning of our bodies and the maintenance of our health. When bacteria and other harmful microorganisms invade, however, the body needs powerful weapons to destroy them. It is here that the NADPH oxidase complex comes into play, producing various reactive oxygen species for precisely this purpose. This process is known as the respiratory burst, or oxidative burst. Let's take a closer look at how phagocytes produce the radicals they use to destroy the bacteria they have engulfed.
Phagocytosis by white blood cells: an early defence of innate immunity
First, let's briefly examine phagocytosis in macrophages and neutrophils, the white blood cells in which the respiratory burst takes place. Neutrophils account for approximately 50–70% of the white blood cells circulating in our blood, while macrophages are found mainly in tissues. Both are important components of innate immunity, responding rapidly when bacteria, viruses or other foreign invaders enter the body. They move towards the invading pathogens and engulf them whole, enclosing them within a membrane-bound sac called a phagosome. The phagosome then fuses with a lysosome, which contains enzymes capable of breaking down pathogens through hydrolysis, forming a phagolysosome. Inside this compartment, the pathogen is digested and processed. Useful molecules, such as amino acids, are returned to the cytoplasm for reuse, while the remaining water-soluble waste is expelled from the cell. Once its task is complete, the phagolysosome is dismantled.
During this process, NADPH oxidase operating in the phagosomal membrane produces ROS, which exert powerful antimicrobial effects inside the phagolysosome, helping to destroy the pathogens it contains.
NADPH oxidase
NADPH and NADPH oxidase
NADPH (nicotinamide adenine dinucleotide phosphate) is a coenzyme involved in cellular redox reactions. We can think of it as an electron carrier that transports and delivers high-energy electrons. It plays an important supporting role in numerous biochemical reactions, primarily acting as a reducing agent by donating electrons to other molecules. NADPH is closely related to NADH, another coenzyme that carries electrons during energy-producing metabolic pathways such as glycolysis, the TCA cycle and cellular respiration, including the electron transport chain and oxidative phosphorylation. Both are derived from niacin, or vitamin B3, and play essential roles in cellular metabolism and redox balance.
NADPH oxidase is an enzyme complex that uses NADPH as an electron donor, transferring electrons to oxygen and thereby reducing it. This reaction leads to the production of reactive oxygen species, including superoxide radicals and hydrogen peroxide.
The NOX family
The NADPH oxidase family, commonly referred to as NOX or Nox, consists of seven known members: NOX1–NOX5 and the two dual oxidases, DUOX1 and DUOX2 (also referred to as Nox6 and Nox7) [1]. These homologous enzymes are widely distributed across different cells and tissues, each with distinct functions and mechanisms of activation. Their roles include host defence (NOX2), the formation of otoconia in the inner ear (NOX3), iodination during thyroid hormone synthesis (DUOX2), and the regulation of vascular tone (NOX2) [2]. NOX proteins are also involved in regulating fundamental physiological processes, including cell growth, differentiation, cell death and cytoskeletal remodelling.
So what roles do the ROS produced by NOX actually play? Broadly speaking, there are two. The first is their long-established role in host defence, particularly in the antimicrobial activity of phagocytes. The second is their involvement in cell signalling, whose importance has attracted increasing attention in recent years. ROS generated by NOX can interact with proteins in specific and reversible ways, altering their activity, cellular location and half-life. In non-phagocytic cells, ROS are also known to regulate signalling pathways by influencing kinase and phosphatase activity or gene transcription. Unlike superoxide radicals, whose negative charge makes it difficult for them to cross biological membranes, hydrogen peroxide (H₂O₂) is relatively stable and can pass through membranes. By oxidising thiol groups in proteins, H₂O₂ can bring about a wide range of physiological effects and participate in numerous signalling pathways. I will explore the role of H₂O₂ as a signalling molecule in more detail in another article. For now, however, the defining function of NOX remains the production of reactive oxygen species.
The mechanism for producing reactive oxygen species
The flavocytochrome b558 complex and four additional components
Let's examine the components that make up the NADPH oxidase complex in phagocytes. At the centre of this enzyme complex is a glycoprotein called gp91phox. The term phox is derived from phagocytic oxidase, referring to the oxidase found in phagocytic cells. gp91phox is a membrane-bound protein better known as NOX2. It associates with a smaller protein, p22phox, to form the flavocytochrome b558 complex (gp91phox + p22phox), which constitutes the central catalytic machinery of NADPH oxidase.
Apart from the flavocytochrome b558 complex, another group of components exists separately in the cytoplasm rather than being attached to the membrane. These include p47phox, p67phox, p40phox and Rac, a small GTP-binding protein. In resting cells, they remain separate from the membrane-bound complex. When stimulation occurs, however — through bacterial or fungal infection, inflammatory cytokines or other signalling molecules — these components move to the membrane and bind to flavocytochrome b558, allowing the enzyme complex to become active. The physical separation of these two groups can be seen as one of the body's carefully evolved safeguards against the uncontrolled production of reactive oxygen species. Indeed, p40phox and p47phox normally exist in a self-inhibited state and undergo regulatory changes, including phosphorylation, during activation. Such a complex system, ensuring that the oxidase components assemble into an active enzyme only in response to appropriate signals, presumably developed over a long period of evolution.
The production of superoxide radicals
Once all six components have assembled around the flavocytochrome b558 complex in the phagosomal membrane, the system is ready to begin producing reactive oxygen species. Electrons are taken from NADPH in the cytoplasm and transferred through the cofactors FAD and haem within flavocytochrome b558. NADPH releases two electrons and one proton, becoming oxidised to NADP⁺. While the proton remains in the cytoplasm, the two electrons are transported across the phagosomal membrane and ultimately transferred to two oxygen molecules, producing two superoxide radicals inside the phagosome. These radicals become powerful antimicrobial agents, contributing to the destruction of the pathogens captured by the phagocyte.
2O₂ + NADPH → 2O₂•⁻ + NADP⁺ + H⁺
The production of hydrogen peroxide (H₂O₂)
The superoxide radicals produced in this way can react with one another, either spontaneously or through a reaction catalysed by superoxide dismutase (SOD), forming hydrogen peroxide and molecular oxygen.
O₂•⁻ + O₂•⁻ + 2H⁺ → H₂O₂ + O₂
Several antioxidant enzymes can convert hydrogen peroxide into harmless water. Among the most important are glutathione peroxidase and catalase.
Glutathione peroxidase: H₂O₂ + 2GSH → 2H₂O + GSSG
Catalase: 2H₂O₂ → 2H₂O + O₂
More powerful oxidants: hypochlorous acid and hydroxyl radicals
Hydrogen peroxide can be converted into harmless water by antioxidant enzymes, but it can also undergo another reaction. In the presence of myeloperoxidase (MPO), hydrogen peroxide reacts with chloride ions (Cl⁻) to produce hypochlorous acid, a powerful antimicrobial oxidant. Hypochlorous acid is also the active disinfecting substance associated with household chlorine bleach. It is therefore a formidable ally in eliminating invading pathogens. Yet there is another side to this reaction. When MPO is released outside cells, it can sometimes produce hypochlorous acid inappropriately, damaging the body's own tissues and contributing to disease. Such activity can be particularly harmful in chronic inflammatory conditions.
Myeloperoxidase: H₂O₂ + Cl⁻ + H⁺ → HOCl + H₂O
Superoxide radicals, hydrogen peroxide and hypochlorous acid are all powerful oxidants produced during the phagocyte's response to invading microorganisms. Together, they contribute to the destruction of bacteria. But this is not the end of the process. Hydroxyl radicals, among the most reactive and potentially destructive ROS known, can also be generated. When iron or copper ions are present, hydrogen peroxide can react with them to form hydroxyl radicals. These reactions are associated with Fenton chemistry and the Haber–Weiss reaction.
Fenton reaction: Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + •OH
Haber–Weiss reaction: O₂•⁻ + H₂O₂ → OH⁻ + •OH + O₂
The production of ROS is an essential part of our immune defence against harmful pathogens. However, if these reactive molecules are produced in excessive amounts, the balance between ROS and antioxidant defences can be disrupted, leading to oxidative stress. Even when ROS are deliberately produced for a beneficial purpose, therefore, antioxidant enzymes play an important role in controlling their activity and limiting the damage they might otherwise cause.
NOX gene mutations and chronic granulomatous disease (CGD)
We have now seen how ROS contribute to antimicrobial defence, and how NADPH oxidase lies at the beginning of this process. The importance of this enzyme's normal function becomes particularly clear when we consider a serious inherited disorder known as chronic granulomatous disease (CGD). In this condition, mutations occur in genes encoding components of the NADPH oxidase complex, such as gp91phox, preventing the enzyme from producing ROS normally. As a result, phagocytes cannot eliminate pathogens effectively, leading to recurrent and sometimes severe bacterial or fungal infections. Poorly regulated inflammatory responses can also cause immune cells to accumulate and form structures known as granulomas. In severe cases, the disease can be life-threatening. CGD provides a striking example of just how indispensable ROS are to our immune defence system.
[References]
[1] NADPH oxidases: an overview from structure to innate immunity-associated pathologies
[2] Nox proteins in signal transduction
Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model.
Activation and assembly of the NADPH oxidase: a structural perspective
NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives
NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology
Chronic Granulomatous Disease
[Image sources]
Image 2. NADPH Oxidase, Figure 1. CC BY 3.0.
