Highly coupled ATP synthesis by F1-ATPase single molecules
Highly coupled ATP synthesis by F1-ATPase single molecules
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DOI：
10.1038/nature03277
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发表时间：
2005-02-17
期刊：
影响因子：
64.8
通讯作者：
Noji, H
中科院分区：
文献类型：
--
作者：
Rondelez, Y;Tresset, G;Noji, H
F-1-ATP酶是已知的最小的旋转马达，当它水解ATP时，它以逆时针方向旋转（1-5）。单分子实验（6-9）指出每转三个催化事件，与络合物（10）的分子结构一致。F-1的生理功能是合成ATP。在普遍存在的F0 F1复合物中，这种能量上的上坡反应由F-0驱动，F-0是F-1的伴侣马达，它迫使F-1向后（顺时针）旋转，导致ATP合成（11-13）。在这里，我们设计了一个结合单分子操作和微加工技术的实验来测量这种机械化学转化的产率。单个F-1分子被封闭在飞升大小的密封室中，并使用磁性镊子顺时针旋转。当磁场关闭时，F-1分子以与合成ATP的量成比例的速度进行反时针旋转。在10 Hz时，α（3）β（3）γ亚复合物（F-1（-β））的机械化学偶联效率较低，但在与ε亚基（F-1（+ β））重构后达到77%。我们在这里提供了直接证据，证明F-1的设计目的是将其催化反应与机械旋转紧密耦合。我们的研究结果表明，ε-亚基在ATP合成过程中具有重要的功能。
F-1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP(1-5). Single-molecule experiments(6-9) point towards three catalytic events per turn, in agreement with the molecular structure of the complex(10). The physiological function of F-1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F-0, the partner motor of F-1, which forces the backward ( clockwise) rotation of F-1, leading to ATP synthesis(11-13). Here, we have devised an experiment combining single- molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F-1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F-1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha(3)beta(3)gamma subcomplex (F-1(-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F-1(+epsilon)). We provide here direct evidence that F-1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.