title: Characterization of the 1st and 2nd EF-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 1st and 2nd 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 Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ 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 Mg2+-binding in NCaBD.
  
  
  CONCLUSIONS
  We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. GRAPHICAL
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026, USA. cwei@siue.edu.
authors.forename: Chin-Chuan
authors.initials: CC
authors.lastname: Wei
authors.forename: Nicole
authors.initials: N
authors.lastname: Reynolds
authors.forename: Christina
authors.initials: C
authors.lastname: Palka
authors.forename: Kristen
authors.initials: K
authors.lastname: Wetherell
authors.forename: Tiffany
authors.initials: T
authors.lastname: Boyle
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Yang
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr