Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

呼吸复合体I（NADH：泛醌氧化还原酶）捕获氧化NADH并还原泛醌的自由能，从而驱动质子穿过线粒体内膜并增强氧化磷酸化能力。最近的冷冻电磁分析已经产生了哺乳动物复合体的近乎完整的模型，但其远距离能量耦合机制的分子原理尚有争议。在这里，我们描述了来自小鼠心脏线粒体的复合物I的3.0-A分辨率冷冻EM结构，其底物样抑制剂Piericidin A结合在泛醌结合活性位点上。我们将结构分析与功能和计算研究相结合，以证明竞争性抑制剂结合姿势，并提供证据表明两个抑制剂分子在长的底物结合通道中端对端结合。