| Reactive oxygen species (ROS), including nitric oxide (NO), superoxide (O2-), and hydrogen peroxide (H 2O2) are essential for combating pathogens and maintaining healthy cells. The enzyme family of NADPH oxidases (NOXs), which includes NOX5 and dual oxidase (Duox), generates superoxide and hydrogen peroxide, respectively.;NADPH Oxidase 5 (NOX5) and Dual oxidase (Duox) are members of the NADPH oxidase (NOX) family of Ca2+ binding enzymes.;NOX enzymes are responsible for the production of reactive oxygen species (ROS), which play important roles in cell signaling and homeostasis. Ca 2+ binds to both NOX5 and Duox via their EF hands, which induces a conformational change of the enzyme facilitating the exposure of hydrophobic amino acids. These hydrophobic regions then recognize and bind the dehydrogenase domain (DH) of the same enzyme. Both enzymes are regulated by Ca2+ flux through its self-contained EF-hand domain (EFD). How Ca 2+ binding induces their conformational change, including hydrophobic residue exposure, remains largely unknown.;To elucidate the mechanism, EFDs of NOX5 and Duox were expressed and purified using recombinant protein technology. The overall Ca2+ binding affinity is similar for both enzymes (Ka = 2.09 x 10 5). The thermodynamic properties measured by isothermal titration calorimetry (ITC) of Duox-EFD revealed an exothermic reaction followed by an endothermic reaction for the 1st and 2nd Ca2+ ion binding respectively, displaying a negative linkage effect. On the other hand, Ca2+ binding to the N-terminal of NOX5's EFD (NOX5-N-EFD), containing the same number of EFs, was overall exothermic where the two Ca2+ bindings were somewhat independent. The rate of Ca2+ binding dissociation for Duox-EFD (1.44 s-1) is much slower than that of the N-terminal NOX5-EFD (> 300 s-1). Ca2+ binding also stabilized both enzymes with melting temperature differences (DeltaTm) = 16.8 °C, 30.4 °C and 38.2 °C for NOX5-N-EFD, NOX5-C-EFD and, Duox-EFD, respectively, using differential scanning calorimetry (DSC). The Ca2+ induced hydrophobicity was assessed using 8-anilino-1-naphthalene sulfonic acid (ANS). The implications of binding differences to the enzymes' activation mechanisms are discussed. |
