Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain of NOX5 contains four EF-hands, each binds one calcium ion. To better understand how calcium affects the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (N-CaBD) and its calcium-site knock-out mutants. ITC measurement for N-CaBD reveals a featureless positive Ca2+ cooperative binding. E31Q and E64Q bind only one Ca2+ ion with the association constants equal to 6.5 x 105 and 1.0 x103 M-1 and ?H values of -14 and -8 kJ/mol, respectively. The Stern-Volmer quenching and CD spectra indicate that the mutations do not alter the protein structure, however, upon Ca2+ binding the a-helix content increases. Thus, we conclude that N-CaBD contains two Ca2+-specific EF-hands, in which the Ca2+ binding affinity to the 1st  non-canonical EF-hand is largely increased by the Ca2+ binding to its 2nd canonical EF-hand.

Characterization of the 1<sup>st</sup> and 2<sup>nd</sup> EF-hands of NADPH oxidase 5 reveals its calcium specific binding properties

243rd National Meeting (2012)

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Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 reveals its calcium specific binding properties

Characterization of the 1^st and 2^nd EF-hands of NADPH oxidase 5 reveals its calcium specific binding properties