Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to f orm the active enzyme. Patients with chronic granulomatous disease (CGD) ar e defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox o r p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal am ounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In p horbol-myristate-stimulated neutrophils and in a cell-free translocation as say with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408- ->Glu, and Glu568-->Lys substitutions was strongly disturbed. Only a Thr341 -->Lys substitution, residing in a region of gp91-phox involved in flavin a denine dinucleotide (FAD) binding, sup ported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp 91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-depend ent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD, Because we found no electro n transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical fo r electron transfer, Apparently, an Intimate relation exists between domain s of gp91-phox involved in electron transfer and in p47/p67-phox binding.