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Mechanism of Calcium‐Induced Electron Transfer in NADPH Oxidase 5
Author(s) -
Wei ChinChuan,
Motl Nicole,
Wetherell Kristen,
Levek Kelli,
Reynolds Nicole,
Williams Tiffany
Publication year - 2010
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.24.1_supplement.463.17
Subject(s) - chemistry , circular dichroism , flavoprotein , isothermal titration calorimetry , conformational change , calcium , electron transfer , biophysics , stereochemistry , biochemistry , photochemistry , enzyme , organic chemistry , biology
Superoxide generated by non‐phagocyte NADPH oxidase 5 (NOX5) is of growing importance for vascular physiology and pathology. NOX5 consists of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. Its activity appears to be regulated by a self‐contained Ca 2+ binding domain (CaBD). Here, we generated recombinant CaBD and E99Q/E143Q mutant, and characterized them using fluorescence spectroscopy. Ca 2+ binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at cys107. The circular dichroism spectra indicated no significant change in the secondary structures of CaBD upon metal binding. We concluded that the C‐terminal half of CaBD has a higher calcium binding affinity, a higher chemical stability, and a slower calcium dissociation. The Mg 2+ ‐bound CaBD was also investigated, and the results indicate that its structure is similar to the apo form, but the rate of Mg 2+ dissociation is close to that of calcium dissociation. Our data suggest that N‐ and C‐terminal halves of CaBD are not completely structurally independent. It has been suggested that the interaction between the CaBD and flavoprotein domains is essential for superoxide production. However, the interaction sites are still unclear. Here, we have generated several flavoprotein fragments and are investigating the domain‐domain interactions by fluorescence spectrometry. We also are determining the metal binding affinities of CaBD's individual halves using isotherm titration calorimetry. This work is supported in part by Cottrell College Science Award (CCSA) from Research Corporation.