Characterization of the 1(st )and 2(nd )EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

BACKGROUND: Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1(st )and 2(nd )EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants. RESULTS: The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca(2+ )binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10(5 )and 5.0 × 10(2 )M(-1 )with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1(st )non-canonical EF-hand is largely enhanced by the binding of Ca(2+ )to the 2(nd )canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca(2+ )binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg(2+)-binding in NCaBD. CONCLUSIONS: We demonstrated that the 1(st )non-canonical EF-hand of NOX5 has very weak Ca(2+ )binding affinity compared with the 2(nd )canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca(2+ )binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca(2+ )specific. GRAPHICAL ABSTRACT: