NADPH oxidase 5 (NOX5) is a homologue of the gp91 phox subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EFhands, Ca 2؉ -binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca 2؉ elevations. In this study, we have analyzed the mechanism of Ca 2؉ activation of NOX5. In a cell-free system, Ca 2؉ elevations triggered superoxide production by NOX5 (K m ‫؍‬ 1.06 M) in an NADPH-and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca 2؉ . Flow dialysis experiments showed that NOX5 N terminus contained four Ca 2؉ -binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EFhands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca 2؉ , whereas the more C-terminal pair bound Ca 2؉ with high affinity. Ca 2؉ binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca 2؉ -dependent way. Our results indicate that the Ca 2؉ -induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca 2؉ -activated enzymes.

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The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase.