Conformational changes of the leukocyte NADPH oxidase subunit p47phox during activation studied through its intrinsic fluorescence

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2(-) from oxygen using NADPH as the 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-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. In whole cells and under certain circumstances in the cell-free system the phosphorylation of p47(phox) mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47(phox) may be an important part of the activation mechanism. We show here that the total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47(phox) substantially decreased when p47(phox) was treated with anionic amphiphiles. A similar decrease in fluorescence was also observed when p47(phox) was phosphorylated with protein kinase C. Furthermore, a red shift of emission maximum and an increase of quenching by ionic quenchers and acrylamide were observed in the presence of activators. These results indicate the occurrence of a conformational change in the protein structure of p47(phox). We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b(558) during the activation process. (C) 1998 Elsevier Science B.V. All rights reserved.