NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which — in particular, how calcium triggers electron transfer from NADPH to FAD — is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

NADPH 氧化酶 5 (NOX5) 催化超氧自由基的产生并调节从精子活力到心律的生理过程。 NOX5 过度表达会导致癌症、糖尿病和心血管疾病。 NOX5 是由细胞内钙信号传导激活的，但其潜在的分子机制（特别是钙如何触发从 NADPH 到 FAD 的电子转移）仍不清楚。在这里，我们使用单粒子低温电子显微镜 (cryo-EM) 捕获全长人类 NOX5 在钙结合时的运动。通过结合生物化学、诱变分析和分子动力学 (MD) 模拟，我们解码了 NOX5 激活和电子转移的分子基础。我们发现钙与 EF-hand 结构域的结合增加了 NADPH 动力学，允许 NADPH 和 FAD 之间的电子转移以及超氧化物的产生。我们的结构发现还揭示了对 NOX5 稳定性和酶活性很重要的锌结合基序，揭示了活性氧 (ROS) 产生的调节机制。