Fluorescent Labeling of the Leukocyte NADPH Oxidase Subunit p47phox: Evidence for Amphiphile-Induced Conformational Changes

Abstract

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O−2from 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 p47phoxand p67phoxmigrate 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 p47phoxmay 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 p47phoxwhen treated with amphiphiles. Cysteines in recombinant p47phoxwere covalently labeled with a sulfhydryl-reactive, environmentally sensitive, fluorescent probeN,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine (IANBD). A series of mutant p47phoxproteins in which the individual cysteine (C98, C111, C196, and C378) was replaced with alanine revealed that all four cysteines of p47phoxare 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 p47phox. 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 p47phoxis 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 p47phoxinteracts with cytochrome b558during the activation process. In addition, recombinant p67phoxor a peptide containing proline-rich sequence of p22phox(residues 149–162) induces the attenuation of the amphiphile-mediated enhancement of fluorescence from IANBD-labeled p47phox. This supports the notion that both p67phoxand p22phoxinfluence the conformation of p47phox.