An activation domain in p67phox (

Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b 558 phox that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67 phox showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199 -210. p67 phox mutated at single amino acid residues within this region showed diminished activity, and p67 phox V204A was completely inactive. The effects of mutations on activity were independent of p47 phox , and mutations did not affect the binding of p67 phox to Rac. In the presence of wild-type p67 phox , the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67 phox , but not by p47 phox or Rac. The V204A mutant competed with native p67 phox for translocation to neutrophil plasma membrane, indicating that p67 phox V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b 558 by an activation domain in p67 phox .During the respiratory burst, neutrophils generate superoxide and secondarily generate reactive oxygen species (H 2 O 2 , O 2 . , and HOCl) that together participate in killing invading microorganisms (1-4). Superoxide generation is catalyzed by a multicomponent enzyme, the respiratory burst oxidase (NADPH oxidase). The enzyme consists of a plasma membrane-associated flavocytochrome b 558 composed of two subunits, gp91 phox (where phox is phagocytic oxidase) and p22 phox . The former contains all of the electron-carrying groups (one FAD and two hemes) needed to transfer electrons from NADPH to molecular oxygen as well as a candidate NADPH-binding site (5-8). In addition, three cytosolic factors, p47 phox , p67 phox , and Rac, are needed for optimal activity. In resting cells, p47 phox and p67 phox

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b 558 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Herein, the interaction between Rac and p67 phox is explored using functional and physical methods.