Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox).
Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b558 and the cytosolic proteins p47(phox), p67(phox), and Rac. p47(phox) functions as an adaptor protein; it increases the affinity of p67(phox) and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582), implying that p67(phox) and/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67(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 b558 by an activation domain in p67(phox).
Han
C H
CH
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322, USA.
Freeman
J L
JL
Lee
T
T
Motalebi
S A
SA
Lambeth
J D
JD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D017931:DNA Primers; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; C048756:cytochrome b 555; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins