Activation of the leukocyte NADPH oxidase subunit p47(phox) by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47(phox)

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2(-) from oxygen using NADPH as electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47(phox) and p67(phox) migrate to the plasma membrane, where they associate with cytochrome b(558), a membrane-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47(phox), an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47(phox) leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47(phox). A similar conformational change occurs when p47(phox) is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b(558) during the activation process.