Reactive oxygen species (ROS) produced by NADPH oxidases (Noxs) play an important role in gene expression, protein biosynthesis, apoptosis, kinase activation, and oxygen sensing. The ROS-producing activity of Nox5 is regulated by Ca2+ flux through its self-contained EF-hands domain (EFD). The Ca2+ binding to EFD is essential for the interaction with Nox5’s dehydrogenase (DH) domain, which gates the electron transfer from NADPH to FAD to heme. However, the kinetics of electron transfer are not well understood and its study is hindered by dehydrogenase protein expression within inclusion bodies. Here we have prepared several dehydrogenase domain constructs, which contain the putative calmodulin (CaM), EFD, and/or Caveolin binding sites. The dehydrogenase (aa. 404-719) was expressed and refolded using an in-column refolding approach. Its electron transfer activity (NADPH to FAD) was monitored by cytochrome C assay, as a surrogate for the electron acceptor. The rate of electron transfer was enhanced two fold in the presence of Ca2+-bound EFD. This domain-domain interaction was confirmed by using isothermal titration calorimetry and fluorescence. Currently we are studying the interplay among Nox5-EFD, Nox5- DH, CaM, and Cav-1 to better understand its electron transfer mechanism.

Expression and characterization of dehydrogenase domain of NADPH oxidase 5

255th National Meeting (2018)

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