Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function.

烟酰胺腺嘌呤二核苷酸磷酸 （NADPH） 氧化酶 （NOX） 通过介导活性氧的产生在真核细胞的生理学中起主要作用。在细菌中发现了具有 NOX 催化核心的进化远距离蛋白，包括肺炎链球菌 NOX （SpNOX），由于其在去污剂胶束中的高活性和稳定性，它被提议作为研究 NOX 的模型。我们在这里展示了在周转条件下无底物和烟酰胺腺嘌呤二核苷酸 （NADH） 结合的 SpNOX 以及 NADPH 结合的野生型和 F397A SpNOX 的冷冻电子显微镜结构。这些高分辨率结构提供了对电子转移途径的见解，并揭示了受 F397 位移调节的氢化物转移机制。我们进行了结构引导的诱变和生化分析，解释了对 NADPH 缺乏底物特异性，并提出了组成型活性背后的机制。我们的研究提出了 SpNOX 酶活性的结构基础，并阐明了其潜在的体内功能。