The phagocyte NOX2 NADPH oxidase in microbial killing and cell signaling

Abstract

The phagocyte NADPH oxidase possesses a transmembrane electron transferase comprised of gp91phox (aka NOX2) and p22phox and two multicomponent cytosolic complexes, which in stimulated phagocytes translocate to assemble a functional enzyme complex at plasma or phagosomal membranes. The NOX2-centered NADPH oxidase shuttles electrons from cytoplasmic NADPH to molecular oxygen in phagosomes or the extracellular space to produce oxidants that support optimal antimicrobial activity by phagocytes. Additionally, NOX2-generated oxidants have been implicated in both autocrine and paracrine signaling in a variety of biological contexts. However, when interpreting experimental results, investigators must recognize the complexity inherent in the biochemistry of oxidant-mediated attack of microbial targets and the technical limitations of the probes currently used to detect intracellular oxidants.

Figure 1.. Flavocytochrome b₅₅₈

Flavocytochrome b₅₅₈, composed of gp91phox (aka NOX2) and p22phox, acts as the redox center of the phagocyte NADPH oxidase. The current model for human gp91phox includes six transmembrane helices and both amino (NH2) and carboxy termini on the cytoplasmic face of the membrane. Residing between two intramembrane helices, two inequivalent heme groups (purple polygons) transport electrons across the membrane from cytosolic NADPH. There are three N-linked carbohydrate chains extracellularly and two cytoplasmic loops (B and D). The carboxy terminal region includes both FAD and NADPH binding sites. The current model for human p22phox has both amino and carboxy termini on the cytoplasmic side of the membrane and lacks glycosylation. The carboxy region of p22phox contains a proline-rich region (PRR) that associates with p47phox during oxidase assembly. Figure from [8] and used with permission.

Figure 2.. Oxidase assembly

Multiple intermolecular interactions among the individual components and between the PX domains of p47phox and p40phox with PI(3,4)P2 in plasma membrane and PI(3)P in phagosomal membranes, respectively, mediate assembly of the NADPH oxidase in stimulated neutrophils. Shown are some of the interactions, including those between proline rich regions (PRR) and src homology domains (SH3), activation domain of p67phox (AD), and tetratricopeptide repeats (TPR) on p67phox with their binding partners (reviewed in detail in [8]) that mediate assembly. For ease of illustration, interactions of the cytoplasmic complexes present in resting neutrophils are omitted. Figure from [8] and used with permission.

Figure 3.. MPO-dependent events in human neutrophil phagosomes

During phagocytosis, human neutrophils assemble and activate the NADPH oxidase and recruit granules to fuse with nascent phagosomes. Electrons transferred into the phagosome by the NADPH oxidase reduce molecular oxygen to superoxide anion (O₂^•−) and H₂O₂. The voltage-gated proton channel Hv1 delivers protons into the phagosome to compensate the charge generated by electron transfer from cytoplasm and thus sustain oxidase activity. The cystic fibrosis transmembrane conductance regulator (CFTR), with some contribution from chloride channel 3 (ClC3) and the potassium-chloride cotransporter (KCC3), provides chloride (Cl⁻) from the neutrophil cytoplasm. MPO, delivered by fusion of azurophilic granules, and H₂O₂, generated by the oxidase, react to yield Compound I (Cpd I) that in turn catalyzes the oxidation of Cl⁻ to produce hypochlorous acid (HOCl) or bleach. HOCl reacts with targets both from host and microbe to generate a variety of reactive products, including monochloramines (NH₂Cl) and protein chloramines (PNHCl), which can then decompose to form aldehydes. Collectively, the granule proteins, acting in both their native and oxidant-modified forms, along with HOCl and its derivatives act synergistically to attack microbial targets. Figure from [40] and used with permission.