The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems

Abstract

The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.

Fig. 1

Electron micrograph of neutrophil. A section through a neutrophil (about 10 μm in diameter) containing 10 S. aureus within phagocytic vacuoles taken after about 30 s. after mixing the cells and bacteria. Granules can be seen in the cytoplasm and degranulating into the vacuoles in which granule contents can also be seen.

Fig. 2

Model of gp91^phox model. The six transmembrane helices and NH₂-and COOH- terminal tails are arranged as indicated. The two non-identical haems are located within the membrane as shown. The haem with E_m7 =−265 mV is toward the outer face of the membrane coordinated between His115 and His222. The inner haem, E_m7 =−225 mV is coordinated by His101 and His209. Arg54, which is hydrogen bonded to the propionate side chain of the outer haem, is indicated. The cytosolic NADPH and FAD-binding regions are shown toward the C-terminus of the protein. In the membrane, gp91^phox is tightly associated with the p22^phox subunit (not shown).

Fig. 3

Structural motifs in the NOX/DUOX family. Structural motifs in the family members are present as indicated. See text for details.

Fig. 4

Electron transfer pathways within flavocytochrome b₅₅₈. The seven electron transfer steps are numbered. Electron transfer takes place from NADPH in the cytosol, across the membrane to the phagocytic vacuole. See text for details.

Fig. 5

The energetics of electron flow within flavocytochrome b₅₅₈. Note that the overall electron transfer from NADPH (E_m =−317 mV) to oxygen ($E_{\mathrm{m}}{\mathrm{O}}_2^{-}∕{\mathrm{O}}_2=-160\mathrm{mV}$) is energetically favourable, transfer from the inner haem to the outer haem is not. In addition, the two electron transfer steps from FAD to haem during the reaction cycle (steps 2 and 5 in Fig. 3) are not equivalent.

Fig. 6

Regions of p40^phox, p47^phox and p67^phox involved in protein/protein interactions. Serine residues of p47^phox that are phosphorylated during oxidase activation are indicated with arrows. See text for details of the structural motifs.

Fig. 7

Schematic representation of the components of the active NADPH oxidase. Cytosolic factors, p47^phox and p67^phox are phosphorylated and translocate from the cytosol to the membrane where they interact with the flavocytochrome b and with p21^rac which is normally maintained in the cytosol in its GDP bound state in association with GDI. The interaction of these factors might induce electron transport by inducing a conformational change, providing the substrate NADPH access to its binding site.

Fig. 8

Equilibrium model for NADPH oxidase activation. This model is a simplified version of the kinetic model developed in reference [47]. In this model, the anionic amphiphile causes a change in conformation in p47^phox that allows it to associate with flavocytochrome b₅₅₈. This association allows the high affinity binding of p67^phox and rac resulting in an intermediate activation state where electron transfer can occur from NADPH to FAD. The formation of this intermediate state is relatively rapid. A subsequent slow step, possibly involving a conformational change in flavocytochrome b₅₅₈, results in the fully active oxidase. The occupancy of each activation state may be influenced by additional factors, such as Mg²⁺ [49].

Fig. 9

Proposed toxic products of NADPH oxidase in neutrophils. The oxidase generates superoxide which dismutates to form hydrogen peroxide. The peroxide and superoxide can react to produce hydroxyl radicals in the presence of metal ions. Hydrogen peroxide can also serve as substrate for myeloperoxidase mediated oxidation of halides. Reactive oxygen species and hypohalous acids could produce damaging interactions with cellular constituents.

Fig. 10

Schematic representation of ion fluxes and influence on pH of phagocytic vacuole. The interior of the cytoplasmic granules is maintained at a pH of about 5.0 by V-ATPases [123]. The degranulation of these contents into the vacuole causes it to become acidic. The superoxide and its dismutation product, peroxide, become protonated, consuming protons in the vacuole and driving the pH up. The passage of electrons across the vacuolar membrane to produce ${\mathrm{O}}_2^{-}$ generates a charge across the membrane that must be compensated by the passage of other ions for electron transport to continue. The nature of the ions compensating this charge has a profound effect on the pH in the vacuole. The movement of protons prevents the consumption of those within the vacuole and the pH is not elevated whereas other ions such as Cl⁻ from, or K⁺ to, the vacuole cause it to rise.