Does SpNox Directly Produce Hydrogen Peroxide?

NADPH oxidases (NOXs) are a family of transmembrane enzymes that generate reactive oxygen species (ROS), which in humans acts both as a cytotoxic agent in autoimmunity and as a signaling molecule in many physiological processes. Human NOXes can produce different ROS species; for example, NOX2 isoform generates superoxide whereas NOX4 isoform produces mainly hydrogen peroxide. The ROS species produced affects downstream signaling, and misregulation produces disease states. A recently discovered prokaryotic NOX, SpNOX, provides an ideal model system to investigate controls on ROS product. SpNOX was previously shown to produce superoxide. Because superoxide spontaneously dismutes at a fast rate, assays that detect hydrogen peroxide cannot be used to assess direct hydrogen peroxide formation. Instead, as in the case of xanthine oxidase, we estimate total electron flux and also superoxide production; we assume that any difference between these values reflects direct hydrogen peroxide production by the enzyme. We estimate electron flux using three assays: i/ monitoring NADPH consumption using absorbance (340 nm); ii/ monitoring NADPH consumption using fluorescence (ex 345/em 454); iii/ monitoring cytochrome c reduction by the FAD of a dehydrogenase‐domain only construct of SpNOX. Superoxide generation from full‐length SpNOX is estimated by the superoxide‐dismutase‐inhibitable portion of cytochrome c reduction, monitored by absorbance at 550 nm. Here we present our findings regarding relative production of superoxide and hydrogen peroxide by SpNOX, as a first step toward manipulating ROS product in this model system.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.