Crystal structures and atomic model of NADPH oxidase

Significance

Reactive oxygen species (ROS) are far from being only an inevitable byproduct of respiration. They are instead actively generated by NADPH oxidases (NOXs), a family of highly regulated enzymes that underpin complex functions in the control of cell proliferation and antibacterial defense. By investigating the individual catalytic domains, we elucidate the core of the NOX 3D structure. An array of cofactors is spatially organized to transfer reducing electrons from the intracellular milieu to the ROS-generating site, exposed to the outer side of the cell membrane. This redox chain is finely tuned by structural elements that cooperate to control NADPH binding, thereby preventing noxious spills of ROS. Our findings indicate avenues for the pharmacological manipulation of NOX activity.

Abstract

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.