Fluorescent labeling of the leukocyte NADPH oxidase subunit p47(phox): evidence for amphiphile-induced conformational...

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 b558, 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 (SDS) or arachidonic acid, as an activating agent. 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. The purpose of the present study was to develop an approach to directly monitor conformational changes in p47(phox) when treated with amphiphiles. Cysteines in recombinant p47(phox) were covalently labeled with a sulfhydryl-reactive, environmentally sensitive, fluorescent probe N, N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethyleneamine (IANBD). A series of mutant p47(phox) proteins in which the individual cysteine (C98, C111, C196, and C378) was replaced with alanine revealed that all four cysteines of p47(phox) are reactive to IANBD. We found that anionic amphiphiles elicited a dose-dependent increase in fluorescence at an emission maximum of 537 nm from IANBD-labeled p47(phox). Furthermore, a blue shift of emission maximum and a decrease in quenching by the ionic quencher, potassium iodide, were observed in the presence of amphiphiles. These results indicate that the amphiphile-mediated increase in fluorescence from IANBD-labeled p47(phox) is due to the conformational change as seen in the leukocyte NADPH oxidase activation. We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b558 during the activation process. In addition, recombinant p67(phox) or a peptide containing proline-rich sequence of p22(phox) (residues 149-162) induces the attenuation of the amphiphile-mediated enhancement of fluorescence from IANBD-labeled p47(phox). This supports the notion that both p67(phox) and p22(phox) influence the conformation of p47(phox).