Mitochondrial adenosine triphosphate (ATP) synthase uses the proton gradient across the inner mitochondrial membrane to synthesize ATP. Structural and single molecule studies conducted mostly at neutral or basic pH have provided details of the reaction mechanism of ATP synthesis. However, pH of the mitochondrial matrix is slightly acidic during hypoxia and pH-dependent conformational changes in the ATP synthase have been reported. Here we use single-particle cryo-EM to analyze the conformational ensemble of the yeast (Saccharomyces cerevisiae) ATP synthase at pH 6. Of the four conformations resolved in this study, three are reaction intermediates. In addition to canonical catalytic dwell and binding dwell structures, we identify two unique conformations with nearly identical positions of the central rotor but different catalytic site conformations. These structures provide new insights into the catalytic mechanism of the ATP synthase and highlight elastic coupling between the catalytic and proton translocating domains.

线粒体三磷酸腺苷 (ATP) 合酶利用穿过线粒体内膜的质子梯度来合成 ATP。主要在中性或碱性 pH 值下进行的结构和单分子研究提供了 ATP 合成反应机制的详细信息。然而，缺氧期间线粒体基质的 pH 值呈弱酸性，并且已报道 ATP 合酶发生 pH 依赖性构象变化。在这里，我们使用单颗粒冷冻电镜分析了pH 6 时酵母（酿酒酵母）ATP 合酶的构象集合。在本研究中解析的四种构象中，三种是反应中间体。除了典型的催化停留和结合停留结构之外，我们还确定了两种独特的构象，其中心转子的位置几乎相同，但催化位点构象不同。这些结构为 ATP 合酶的催化机制提供了新的见解，并强调了催化域和质子易位域之间的弹性耦合。