Characterization of the 1 stand 2 ndEF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

Abstract

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 stand 2 ndEF-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 5and 5.0 × 10 2M -1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1 stnon-canonical EF-hand is largely enhanced by the binding of Ca 2+to the 2 ndcanonical 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 stnon-canonical EF-hand of NOX5 has very weak Ca 2+binding affinity compared with the 2 ndcanonical 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: . © 2011 Wei et al.