The purpose of this project is to define the structure and biochemistry of the phagocyte NADPH oxidase, the electron transport chain responsible for production of microbicidal oxidants. This oxidase is comprised of membrane bound and cytosolic proteins which interact in the activated phagocyte to catalyze reduction of molecular oxygen to superoxide anion. Defects in any of several genes encoding oxidase factors cause chronic granulomatous disease (CGD) characterized by a failure to produce superoxide and recurrent life threatening infections. Knowledge of the structure of the oxidase and the biochemistry of oxidase activation are critical to longterm efforts aimed at gene therapy for CGD, development of novel agents to augment or blunt the inflammatory response, and to a molecular appreciation of signal transduction in diverse cell types. We have reconstituted oxidase activity in a cell-free system comprised of purified cytochrome b (an oligomer of gp9lphox and p22phox subunits) and recombinant p47phox, p67phox, and GTP-bound forms of rac1 or rac2 (low molecular weight guanine nucleotide binding proteins) enabling detailed biochemical studies which were not previously possible. We have demonstrated that these protein components are necessary and sufficient for activation of the oxidase and have shown that cytochrome b contains flavin and heme redox centers. Cytochrome domains responsible for binding flavin and NADPH have been identified based on sequence homology to well characterized flavoproteins. Thus, cytochrome b is the sole electron transporting component of the oxidase and the cytosolic factors appear to function as regulatory elements that induce conformational changes in the cytochrome which enable it to transfer electrons. In the intact cell p47phox becomes heavily phosphorylated within a 10 kDa COOH- terminal region leading to interactions with a COOH-terminal domain of gp9lphox (residues arg-gly-val-his-phe-ile-phe). Structural properties of side chains of arginine, valine, phenylalanine, and isoleucine within this sequence motif contribute significantly to protein interactions leading to assembly of the oxidase.