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Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b₅₅₈ activation states

Marie-Hélène Paclet^*,1, Sylvie Berthier^*, Lauriane Kuhn, Jérôme Garin and Françoise Morel^*

^* GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble, Grenoble, France; and

Laboratory Chimie des protéines, ERIT-M-CEA Grenoble, Grenoble, France

¹Correspondence: GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble BP 217, 38043, Grenoble Cedex 9, France. E-mail: mhpaclet{at}chu-grenoble.fr

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b₅₅₈, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b₅₅₈ purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O₂^– ·s^–1·mol heme b^–1), indicating that cytochrome b₅₅₈ has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (50 mol O₂^– ·s^–1·mol heme b^–1). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b₅₅₈ conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b₅₅₈ activation states.—Paclet, M-H., Berthier, S., Kuhn, L., Garin, J., Morel, F. Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b₅₅₈ activation states.

Key Words: NADPH oxidase complex • MRP • p67-phox

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