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Characterization of the Metal Binding in NADPH Oxidase 5 by Fluorescence, Isothermal Titration Calorimetry, and Circular Dichroism
Author(s) -
Palka Christina Diana,
Reynolds Nicole,
Tatro Allison,
Wei Chin-Chuan
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.756.9
Subject(s) - isothermal titration calorimetry , chemistry , circular dichroism , titration , binding site , metal , crystallography , calmodulin , isothermal microcalorimetry , binding constant , calorimetry , enthalpy , fluorescence , ef hand , stereochemistry , calcium , biochemistry , inorganic chemistry , organic chemistry , thermodynamics , physics , quantum mechanics
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 Ca 2+ for the 1 st and 2 nd EF‐hands; the result indicates a weak Ca 2+ binding to the 1 st EF‐hand is enhanced by the binding of Ca 2+ to its 2 nd EF‐hand. No Mg 2+ binding was seen in N‐CaBD. The binding of Ca 2+ and Mg 2+ to C‐CaBD are exothermic, and Ca 2+ binding is only 2–3 fold tighter than Mg 2+ . Our findings suggest that in the resting cells, the 3 rd and 4 th EF‐hands are Mg 2+ ‐bound; increasing Ca 2+ levels affects the EF‐hands of N‐terminal half. Our results imply that Ca 2+ binding to the 1 st and 2 nd 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.