Abstract
The study of reactive oxygen species (ROS) generated by non‐phagocyte NADPH oxidase 5 (NOX5) and dual oxidase (DUOX) is of growing importance for vascular physiology and disease development. Their ROS activities appear to be regulated by their self‐contained EF‐hand domains (EFDs). Previously, we demonstrated that Ca2+ induces conformational change on NOX5‐EFD and suggested that its N‐ and C‐terminal halves behave differently, including conformational change, kinetics of binding, and chemical stability. Here we extended the study by using separated half domain proteins and characterized their Ca2+ and Mg2+ binding using fluorescence and isothermal titration calorimetry. The key residues for Ca2+ binding in each EF‐hand motif of the half domain proteins were mutated to determine the impacts on Ca2+ and Mg2+ binding. Since domain‐domain interaction is essential for ROS production, interaction of NOX5 EFD to flavoprotein was only observed in the presence of Ca2+. Furthermore, EFD enhances the cytochrome C reduction of flavoprotein. For DUOX‐EFD, the preliminary data suggests that a dramatic conformational change is not essential based on fluorescence data. Currently, we are performing experiments to identify the peptide sequence that interacts with its EFD and to investigate possible dimer formation that might play roles in DUOX ROS production. This work is supported in part by Cottrell College Science Award from Research Corporation.
Published Version
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