CONFORMATIONAL-CHANGES OF THE LEUKOCYTE NADPH OXIDASE SUBUNIT P47(PHOX) 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 c atalytic activity when the cells are exposed to appropriate stimuli. D uring activation, the cytosolic oxidase components p47(phox) and p67(p hox) migrate to the plasma membrane, where they associate with cytochr ome b(558), a membrane-integrated flavohemoprotein, to assemble the ac tive oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachi donic acid, as an activating agent. In whole cells and under certain c ircumstances in the cell-free system the phosphorylation of p47(phox) mediates the activation process. It has been proposed that conformatio nal changes in the protein structure of cytosolic factor p47(phox) may be an important part of the activation mechanism. We show here that t he total protein steady-state intrinsic fluorescence (an emission maxi mum of 338 nm) exhibited by the tryptophan residues of p47(phox) subst antially 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 a crylamide were observed in the presence of activators. These results i ndicate the occurrence of a conformational change in the protein struc ture of p47(phox). We propose that this alteration in conformation res ults in the appearance of a binding site through which p47(phox) inter acts with cytochrome b(558) during the activation process. (C) 1998 El sevier Science B.V. All rights reserved.