Binding Affinity Of Flavins To The Dehydrogenase Domain Of Spnox

NADPH oxidase (NOX) is a multidomain protein that transfers an electron from NADPH bound by the dehydrogenase (DH) domain through the transmembrane (TM) domain to the final electron acceptor, O2, to form superoxide. The dehydrogenase domain also binds FAD cofactor. A recently discovered prokaryotic NOX homolog, SpNOX, has emerged as an ideal model for the study of NOXes because of its robust expression and activity. An SpNOX DH domain-only construct reduces cytochrome c in the absence of its TM counterpart, indicating the construct's electron transport activity. We used this activity to measure Km of flavins to the SpNOX DH-only construct. This data served as a starting point for isothermal titration calorimetry (ITC) experiments to establish the Kd for DH flavin binding. Initial ITC experiments revealed DH precipitation and relatively high mixing heats in the purification buffer, which had been chosen in a previous buffer screen as optimum for crystallization. To optimize the buffer conditions for ITC, we performed dialysis against a range of buffers and observed protein aggregation visually and spectroscopically. Here, we determine the binding affinity of flavins to SpNOX DH domain using a buffer optimized for ITC conditions.