Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5).

Abstract NADPH oxidase 5 (NOX5) is a homologue of the gp91phox 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 EF-hands, Ca2+-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca2+ elevations. In this study, we have analyzed the mechanism of Ca2+ activation of NOX5. In a cell-free system, Ca2+ elevations triggered superoxide production by NOX5 (Km = 1.06 μm) 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 Ca2+. Flow dialysis experiments showed that NOX5 N terminus contained four Ca2+-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca2+, whereas the more C-terminal pair bound Ca2+ with high affinity. Ca2+ 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 Ca2+-dependent way. Our results indicate that the Ca2+-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 Ca2+-activated enzymes.