The Leukocyte NADPH Oxidase Subunit p47PHOX: The Role of the Cysteine Residues

The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O−2at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47PHOX, one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47PHOX-deficient Epstein–Barr virus-transformed B lymphoblasts, and O−2production 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−2production by C196A was significantly larger than seen with wild type. The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O−2production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity.