Characterization of the Calcium Binding Domain of NADPH Oxidase 5 (NOX5)

Abstract

Superoxide generated by non-phagocyte NADPH oxidases (NOXs), such as NOX5, is of growing importance for vascular physiology and pathology. NOX5 enzyme consists of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. It appears to be regulated by self-contained Ca2+ binding domains (CaBD), which contains four EF-hands motifs. Previously we demonstrated that this calcium binding gates the heme reduction in NOX5, possibly thorough the CaBD-flavorprotein interaction. To better understand its structure and function, here we characterized the metal binding properties of the recombinant CaBD by fluorescent spectroscopy. Our data revealed that CaBD binds to Ca2+, Mg2+, terbium (Tb3+) in the range of μM to mM. The data are further supported by the studies using the site-directed labeled CaBD. The rate of calcium association was too fast to be determined by a stopped-flow device, but the dissociation rate constant was determined to be 5 s−1 at 20 °C. The ANS titration and Stern-Volmer plots suggested that there was a significant conformational change upon the metal bindings. Interestingly, the spectra of circular dichorism indicated otherwise no change on the context of its secondary structure. However, this conformational change can be observed using the Surface Plasmon Resonance with the CaBD immobilized in the sensor chip. Because our data and other studies suggest there are two different types of calcium bindings in CaBD, currently we are performing the similar studies using N- and C-terminal halves of CaBD (aa 1-78 and 79-184), and mutants. We also are investigating the metal bindings in the CaBD of Dual Oxidase (DUOX), in which its hydrogen peroxide activity is controlled by calcium binding.