Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly.
The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.
Li
Xing Jun
XJ
Groupe de Recherche et d'Etude du Processus Inflammatoire, Université Joseph Fourier, Laboratoire d'Enzymologie, Centre Hospitalier Universitaire, Grenoble CHU 38043, Cedex 9, France.
Fieschi
Franck
F
Paclet
Marie-Hélène
MH
Grunwald
Didier
D
Campion
Yannick
Y
Gaudin
Philippe
P
Morel
Françoise
F
Stasia
Marie-José
MJ
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D041321:Chromosomes, Human, X; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006105:Granulomatous Disease, Chronic / Q000235:genetics; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000502:physiology; D010088:Oxidoreductases / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013778:Tetrazolium Salts / Q000378:metabolism
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; C079789:neutrophil cytosol factor 67K; C013574:iodonitrotetrazolium; C036888:cytochrome b558; D010088:Oxidoreductases; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D007930:Leucine