Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. ¹³C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD⁺ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

缺氧会停止氧化磷酸化（OXPHOS），导致还原化合物在线粒体基质中积累，从而阻碍脱氢酶。通过同时实时测量分离线粒体中的氧浓度、NADH自发荧光、线粒体膜电位和泛醌还原程度，我们证明了复合物I在急性缺氧条件下利用内源醌氧化NADH。在存在或不存在电子传递系统位点特异性抑制剂的情况下，对缺氧期间提取的代谢物进行¹³ C 代谢追踪或非靶向分析表明，复合物 I 再生的 NAD ⁺被 2-酮戊二酸脱氢酶复合物还原，产生支持线粒体的琥珀酰辅酶 A底物水平磷酸化（mtSLP），释放琥珀酸。复合物 II 在缺氧事件期间双向运行，为复合物 I 提供醌并将富马酸盐还原为琥珀酸盐。我们的结果强调了在缺乏 OXPHOS 的情况下，提供醌对复合物 I 氧化 NADH 维持谷氨酸分解代谢和 mtSLP 的重要性。