Reactive oxygen species (ROS) are highly reactive oxygen molecules which are found to have an important role in different disease states, so investigation of their role in diseases and in their prevention is of great importance. ROS such as superoxide and hydrogen peroxide produced from specific enzymes like NADPH oxidases (Nox) are regarded in maintaining bodily homeostasis and regulating cellular functions including immunity, signal transduction, hormone synthesis, cell differentiation and ion transport. Although Nox enzymes share a common mechanism of electron transfer, their activation mechanism for superoxide production varies from enzyme to enzyme. Among Nox family enzymes, ROS produced by NADPH oxidase 5 (Nox5) and Dual oxidase (Duox) are regulated by Ca²⁺ flux which allows their self-contained EF-hand domain (EFD) to bind to the dehydrogenase domain (DH). A recent study on Nox5 showed that the regulatory EF-hand domain (REFBD) peptide sequence within the DH domain interacts with EFD in a Ca²⁺dependent manner through hydrophobic interactions. However, the interplay of EFD-DH domain-domain interactions is still unclear.

Here, different EFD and DH constructs were used to study EFD-DH domain-domain interactions. The DH constructs containing the REFBD sequence (DH 605-719) and without REFBD sequence (DH 665-719) were prepared and their expression and solubility were enhanced by fusing with a maltose binding protein (MBP). The binding of DH to EFD was studied by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Fluorescence studies showed that the EFD and its C-terminal half domain mimic (EFD_C) interact with DH 605-719 in Ca²⁺-dependent manner with a binding constant of (8.6 ± 1.21) × 10⁵ M^-1 and (2.65 ± 0.32) × 10⁵, respectively. Characterization using steady state fluorescence, anisotropy and dynamic quenching measurements showed that EFD’s C-terminal half domain mimic (EFD_C) binds to the DH 605-719 through hydrophobic interactions. Thermodynamic parameters measured by ITC for Nox5’s EFD/DH 605-719 revealed a strong exothermic reaction (ΔH-14.3 ± 0.8 kcal/mol) indicating non-covalent interactions in complex formation. The data obtained these experiments the proposed Nox5’s docking model.

Here, we also attempted to express the full-length of Duox’s EFD domain. By fusing the protein with MBP, the resulted Duox’s EFD became soluble. ANS fluorescence of the partially purified Duox’s EFD showed that upon Ca²⁺, a conformational change is induced causing exposure of hydrophobic residues. Though further purification is required, this is the first time that Duox’s EFD has been expressed.