Abstract text (incl. figure legends and references)

Reactive oxygen species (ROS) are crucial for many cellular processes, from cell to cell signaling to redox regulation. In human cells, the NAPDH oxidase (NOX) membrane proteins are a major source of ROS and play a critical role in the innate immune system and redox regulation processes. Yet, there are large gaps in knowledge on crucial aspects of the function of the NOX proteins, especially with regards to electron transfer pathway and reaction mechanism. Structural and in-depth biochemical studies of human NOX proteins have proven difficult due to complications in heterologous expression and purification of active, stable complexes. Therefore, we have used a bacterial NOX homologue from Streptococcus pneumonia (spNOX) to gain insight into the structure and mechanism of NOX proteins. SpNOX is a small membrane of ~47 kDa and can be heterologously expressed in its active state, as confirmed by superoxide dismutase-sensitive cytochrome c reduction. The protein is stable at high concentrations and can be prepared for single particle analysis cryo-EM. Preliminary studies have allowed for the resolving of the low-resolution structure that is consistent with the structure predicted using AlphaFold2.