Calcium‐Dependent Interactions and Electron Transfer in NADPH Oxidase 5

Abstract

Superoxide generated by NADPH oxidase 5 (NOX 5) is of growing importance for physiology and pathobiology. The NOX5 activity appears to be regulated by its self‐contained calcium binding domain (CaBD), through an interaction with its flavoprotein domain. Here, using fluorescence we showed that such domain‐domain interaction is Ca2+‐dependent. The resulting interaction enhances the electron transfer from NADPH to FAD in cytochrome C assay and gates heme reduction using full‐length NOX5. Our data support the published results showing that CaBD acts as an activator by binding the inhibitory loops in the flavoprotein, and thus lifting the blockage of electron transfer for superoxide production. CaBD contains two half domains, in which the N‐terminal half contains the Ca2+‐specific EF‐hands and The C‐terminal half contains the Ca2+/Mg2+ mixed EF‐hands. The half domains alone showed no interaction with the flavoprotein. However, the calcium knock‐out mutant, CaBD(E99Q/E143Q) in which the E residues at the –z position in 3rd and 4th EF‐hands were replaced with Q, retains its interaction with the flavoprotein, suggesting that its N‐terminal half is critical for regulating electron transfer. Currently we are investigating other constructs, such as CaBD(E31Q/E64Q) and transmembrane‐deleted NOX5, for the studies of domain‐domain interaction and electron transfer. Furthermore, we include calmodulin (CaM) in our existing system to investigate a possible ternary complex formation (CaBD/flavoprotein/CaM) using FRET and for determining its effects on electron transfer as it has been shown that CaM interacts with the flavoprotein. This work is supported in part by National Science Foundation CCLI grant.