MUTATION AT HISTIDINE-338 OF GP91(PHOX) DEPLETES FAD AND AFFECTS EXPRESSION OF CYTOCHROME B(558) OF THE HUMAN NADPH OXIDASE

Defective NADPH oxidase components prevent superoxide (O-2(radical ani on)) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subun it of cytochrome b(558) and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that conta ins binding sites for NADPH, FAD, as well as heme. We here report a ra re X-linked CGD patient whose neutrophils entirely failed to produce O -2(radical anion), but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membr anes was as low as that of X91(0) patients, suggesting complete deplet ion of FAD in his gp91(phox). This was in agreement with the finding t hat a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome mo del. The loss of FAD could not be corrected even after addition of rea gent FAD or a FAD-rich dehydrogenase fraction isolated from normal neu trophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critica l residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.