title: Conformational States and kinetics of the calcium binding domain of NADPH oxidase 5.
abstract: Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca(2+) binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca(2+) binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize 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 changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca(2+) dissociation from the N-terminal half, followed by a slow Ca(2+) dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of CaBD has a higher Ca(2+) binding affinity, a higher chemical stability, and a slow Ca(2+) dissociation. The Mg(2+)-bound CaBD was also investigated and the results indicate that its structure is similar to the apo form. The rate of Mg(2+) dissociation was close to that of Ca(2+) dissociation. Our data suggest that the N- and C-terminal halves of CaBD are not completely structurally independent.
authors.affiliation: Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026, USA.
authors.forename: Chin-Chuan
authors.initials: CC
authors.lastname: Wei
authors.forename: Nicole
authors.initials: N
authors.lastname: Motl
authors.forename: Kelli
authors.initials: K
authors.lastname: Levek
authors.forename: Liu Qi
authors.initials: LQ
authors.lastname: Chen
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Yang
authors.forename: Tremylla
authors.initials: T
authors.lastname: Johnson
authors.forename: Lindsey
authors.initials: L
authors.lastname: Hamilton
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr