Activation domain in P67phox regulates the steady state reduction of FAD in gp91phox.
An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
Han
C H
CH
Department of Biochemistry, Swiss Federal Institute of Technology in Zurich,Universitatstrasse 16, 8092 Zurich, Switzerland.
Lee
M H
MH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D017931:DNA Primers; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
D017931:DNA Primers; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D005182:Flavin-Adenine Dinucleotide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009245:NADH Dehydrogenase