Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 113-8656 Tokyo, Japan.
Department of Mathematics, Stockholm University, 106 91 Stockholm, Sweden.
Proc Natl Acad Sci U S A. 2020 Jan 21;117(3):1447-1456. doi: 10.1073/pnas.1909407117. Epub 2020 Jan 2.
The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F (bMF) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other Fs. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F, which also shows intervening dwell that probably corresponds to the short dwell of bMF, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F, bMF showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.
在单分子实验中研究了旋转催化的反应方案和牛线粒体 F（bMF）的扭矩产生机制。在 ATP 饱和浓度下，对附着在 bMFγ 亚基上的单个 40nm 金珠进行高速成像，显示在旋转过程中有 2 种类型的中间停顿，通过短停留和长停留来区分。使用 ATPγS 作为缓慢水解的 ATP 衍生物以及使用具有减缓 ATP 水解的功能突变体βE188D，可清楚地区分这 2 种停顿事件。与不可水解类似物（AMP-PNP）的缓冲交换实验表明，长停顿对应于催化停顿，即水解的等待状态，而短停顿代表哪种催化状态仍然难以确定。催化停顿的角位置确定为距 ATP 结合角度+80°，与其他 F 大多一致。短停顿的位置在距催化停顿 50 到 60°处发现，即距 ATP 结合角度+10 到 20°处。这与显示出中间停顿的人线粒体 F 明显不同，后者可能对应于 bMF 的短停顿，位于距结合停顿+65°处。此外，我们使用磁镊进行了“停止和释放”实验，以揭示γ旋转如何调节结合亲和力和水解平衡。与嗜热 F 类似，bMF 显示出 ATP 亲和力的强烈指数增长，而水解平衡没有明显变化。这表明 ATP 结合过程产生的扭矩大于水解过程。
