Published online before print October 23, 2006

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(Journal of Leukocyte Biology. 2007;81:238-249.)
© 2007 by Society for Leukocyte Biology

Leu505 of Nox2 is crucial for optimal p67^phox-dependent activation of the flavocytochrome b₅₅₈ during phagocytic NADPH oxidase assembly

Xing Jun Li^*, Franck Fieschi, Marie-Hélène Paclet^*, Didier Grunwald, Yannick Campion^*, Philippe Gaudin^*, Françoise Morel^* and Marie-José Stasia^*,1

^* Groupe de Recherche et d’Etude du Processus Inflammatoire, Université Joseph Fourier, Laboratoire d’Enzymologie, Centre Hospitalier Universitaire, Grenoble, France;
Institut de Biologie Structurale, CEA/CNRS/Université Joseph Fourier, Laboratoire des Proteines Membranaires, Grenoble, France; and
Département Réponse et Dynamique Cellulaire/Commissariat à l’Energie Atomique, Grenoble, France

¹Correspondence: GREPI, EA UJF Laboratoire d’Enzymologie, Grenoble CHU 38043, Cedex 9, France. E-mail mjstasia{at}chu-grenoble.fr

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 p67^phox, p47^phox, and Rac1proteins, we found that the K_m 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 p67^phox 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 -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 p67^phox interaction with cytochrome b₅₅₈, thus affecting NADPH access to its binding site.

Key Words: gp91^phox • PLB-985 cells • X91⁺-CGD • -helical loop • cytosolic C-terminal tail