This project explores the structure and biochemistry of the phagocyte NADPH oxidase, the redox chain responsible for production of microbicidal oxidants. A basic understanding of the biochemistry of this oxidase is critical to longterm efforts aimed at gene therapy for chronic granulomatous disease (CGD), and for development of novel agents to modulate the inflammatory response. We have produced a recombinant flavocytochrome b558 (an oligomer of gp91phox and p22phox subunits) that for the first time enables reconstitution of NADPH oxidase in vitro solely from recombinant proteins. This observation is important for several reasons: a) it identifies unequivocally the essential protein components of the oxidase (flavocytochrome b558, p47phox, p67phox, and rac1 or rac2 GTPases); b) it demonstrates the feasibility of gene therapy for flavocytochrome b558- deficient forms of CGD using recombinant constructs derived from the same gp91phox and p22phox cDNAs; and c) it provides a model system for evaluating structure-function relationships and the significance of specific amino acid substitutions in patients with CGD due to a defective flavocytochrome b558. Using oligopeptides modeled from gp91phox as candidate inhibitors of oxidase assembly, we identified regions of the integral membrane gp91phox (residues 35-48, 287-299, and 304-318) which appear to be cytoplasmic contact sites for p47phox. Thus, these gp91phox domains are analogous to the cytoplasmic COOH-terminal domain of gp91phox (amino acid residues 559-565) previously identified as a membrane docking site for p47phox. Through a similar approach we identified a domain of p47phox (amino acids YRRNSVRF, residues 324-331) that is essential for early interactions of p47phox and flavocytochrome b558. Further analysis suggests that the NH2 and COOH-terminal aromatic residues within this p47phox sequence play an essential role in activation of the oxidase. Complex formation with p47phox was shown to protect the flavocytochrome b558 heme prosthetic groups against destruction by amphipathic detergents. In the absence of p47phox these agents lead to heme destruction, release, and covalent adduct formation between heme degradation products and apoflavocytochrome b558. Hence, p47phox appears capable of cross-linking multiple domains of flavocytochrome b558 and these interactions may contribute to electron transfer by inducing conformational changes in the redox domains of flavocytochrome b558.
