Reactive oxygen species (ROS) have been known for a long time to play important 
roles in host defense against microbial infections. In addition, it has become 
apparent that they also perform regulatory roles in signal transduction and cell 
proliferation. The source of these chemicals are members of the NOX family of 
NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase 
homologue that is most abundantly expressed in colon epithelial cells, requires 
the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX 
activating protein 1), as well as the flavocytochrome component p22(phox) for 
maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is 
tightly repressed in the resting state, NOX1 produces superoxide constitutively 
at low levels. These levels can be further increased in a stimulus-dependent 
manner, yet the molecular details regulating this activity are not fully 
understood. Here we present the first quantitative characterization of the 
interactions made between the cytosolic regulators NOXO1 and NOXA1 and 
membrane-bound p22(phox). Using isothermal titration calorimetry we show that 
the isolated tandem SH3 domains of NOXO1 bind to p22(phox) with high affinity, 
most likely adopting a superSH3 domain conformation. In contrast, complex 
formation is severely inhibited in the presence of the C-terminal tail of NOXO1, 
suggesting that this region competes for binding to p22(phox) and thereby 
contributes to the regulation of superoxide production. Furthermore, we provide 
data indicating that the molecular details of the interaction between NOXO1 and 
NOXA1 is significantly different from that between the homologous proteins of 
the phagocytic oxidase, suggesting that there are important functional 
differences between the two systems. Taken together, this study provides clear 
evidence that the assembly of the NOX1 oxidase complex can be regulated through 
reversible protein-protein interactions.