Oxygen-dependent bactericidal mechanisms

Assembly and activation of NADPH oxidase

In a resting cell, enzyme elements are dissociated to prevent autodamage. Specific granules contain flavocytochrome b558 in their membranes (gp91phox, p22phox subunits, and the Rap1a protein). The cytosol contains p47phox, p67phox, p40phox proteins, and the Rac-2 GTPase.

The assembly process is triggered by scavenger receptors upon contact with a target. Granules fuse with the phagosome, and membrane phosphoinositides (PI-3,4P₂ and PI-3P) serve as "anchors" for cytosolic components.

Stages of assembly on the phagolysosome membrane:

1. Protein kinase C (PKC) phosphorylates p47phox, activating its PX and SH3 domains.
2. The PX domain binds to membrane PI-3,4P₂, and the SH3 domain contacts p22phox.
3. The translocated p47phox "pulls" the p67phox protein into the membrane.
4. The p40phox adaptor protein is additionally anchored to membrane PI-3P.
5. The Rac-2 GTPase loses its inhibitor (GDI), inserts into the membrane, and binds to p67phox.

Generation of reactive oxygen species (ROS)

The assembled oxidase changes conformation: gp91phox begins to interact with the NADPH cofactor (a glycolysis product). Electron transfer is initiated: from NADPH to FAD, and then via two hemes to molecular oxygen.

As a result, oxygen is reduced to a superoxide anion (O₂⁻). This short-lived radical enters the phagolysosome. Next, the enzyme superoxide dismutase combines two such molecules, forming hydrogen peroxide (H₂O₂).

Together with iron ions, these substances produce the hydroxyl radical (OH), a potent oxidant that breaks peptide bonds, oxidizes sulfhydryl groups, and induces lipid peroxidation of the bacterial wall, leading to microbial death.

Myeloperoxidase system

Myeloperoxidase (MPO) is a marker enzyme of neutrophil azurophilic granules (up to 5% of total cellular protein). It forms a potent bactericidal system together with hydrogen peroxide and halide ions.

Pathogen destruction mechanisms:

• With iodide (I⁻): oxidized to molecular iodine, which binds protein sulfhydryl groups.
• With chloride (Cl⁻): converted to hypochlorous acid (HOCl) with the participation of H₂O₂. It reacts with amino acids to form toxic chloramines.
• Radicals and singlet oxygen: HOCl is oxidized by superoxide, releasing singlet oxygen and the hydroxyl radical. Singlet oxygen destroys polyunsaturated fatty acids of membranes and participates in the formation of bactericidal ozone.

Reactive nitrogen species

In addition to oxygen radicals, phagocytes synthesize toxic nitrogen derivatives. They are formed by the oxidation of the amino acid arginine to citrulline.

In phagocytes, the inducible isoform of the enzyme — macrophage NO synthase (iNOS) — is responsible for this process. Its primary stimulator is interferon-gamma (IFNγ). As a result of enzyme activity, nitric oxide (NO) is released, which significantly enhances the microbicidal potential of the cell.

Class and exam questions

What else to study on this topic

• Structure and functions of PX and SH3 domains in binding cytosolic proteins.
• Mechanisms of Rac-2 GTPase phosphorylation and the role of the GDI inhibitor.
• Chemical reactions of spontaneous radical dismutation in the phagolysosome.
• Pathogenesis of chronic granulomatous disease.
• Arginine metabolic pathways: the balance between arginase and iNOS activity.

Other topics in the "Innate Immunity" section

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