The cytosolic subunit p67(phox) contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase

The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67(phox), but not p47(phox) or Rac2. Purified recombinant p67(phox) was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [P-32]NADPH dialdehyde in a specific fashion. The activity of recombinant p67(phox) in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67(phox) contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase.