Characterization of the Metal Binding in NADPH Oxidase 5 by Fluorescence, Isothermal Titration Calorimetry, and Circular Dichroism

Abstract

Superoxide generated by non‐phagocytic NADPH oxidases (NOXs) plays roles in disease development and cancer. The activity of NOX5 appears to be regulated by its self‐contained calcium binding domain (CaBD). To study its calcium binding properties, we isolated the N‐ and C‐terminal halves of CaBD (N‐ / C‐CaBD), and separately studied their metal binding. Isothermal titration calorimetry reveals a positive cooperation for both halves, with differences in enthalpy values ranging from −30 to −40 kJ/mol. Using mutants whose Glu residue (−z) in each EF‐hand was replaced with Gln, we determined the semi‐microscopic binding constants of Ca2+ for the 1st and 2nd EF‐hands; the result indicates a weak Ca2+ binding to the 1st EF‐hand is enhanced by the binding of Ca2+ to its 2nd EF‐hand. No Mg2+ binding was seen in N‐CaBD. The binding of Ca2+ and Mg2+ to C‐CaBD are exothermic, and Ca2+ binding is only 2–3 fold tighter than Mg2+. Our findings suggest that in the resting cells, the 3rd and 4th EF‐hands are Mg2+‐bound; increasing Ca2+ levels affects the EF‐hands of N‐terminal half. Our results imply that Ca2+ binding to the 1st and 2nd EF‐hands is more critical in controlling NOX5 activity. To investigate structural dependency between half domains that affects metal binding, we created CaBD mutants, such as CaBD(E31Q/E64Q) and CaBD(E99Q/E143Q), and determined their metal binding affinities. This work is supported in part by NSF‐CCLI grant.