Assembly Of Phagocyte NADPH Oxidase Research Articles

Assembly of phagocyte NADPH oxidase: A concerted binding process?

BackgroundThe phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67phox, p47phox, p40phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase. MethodsA wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47phox–p67phox complex. ResultsThe data presented here are consistent with the absence of a catalytic role of the p47phox–p67phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core. Conclusion and general significanceThe activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.

Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47phox Phox Homology (PX) Domain Revealed by NMR

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.