THE LEUKOCYTE NADPH OXIDASE SUBUNIT P47(PHOX) THE ROLE OF THE CYSTEINE RESIDUES

The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes t he reduction of oxygen to O-2(-) at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the func tional role of the four cysteines in p47(PHOX), one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47(PHOX)-defici ent Epstein-Barr virus-transformed B lymphoblasts, and O-2(-) producti on by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O-2(-) production by C196A was significantly larger than seen with wild type , The other two mutants (i.e., C111A and C378A) differed from wild typ e not only in maximum O-2(-) production, but also in the time required for activation, which was considerably delayed with both of these mut ants. The similarity in the time courses of oxidase activation with th e C111A and C378A mutants, and the finding that C378A occurs in the se quence CSE, raises the possibility that these cysteines may be involve d in redox regulation of oxidase activity. (C) 1998 Academic Press.