Kasimova Marina R, Grigiene Jurgita, Krab Klaas, Hagedorn Peter H, Flyvbjerg Henrik, Andersen Peter E, Møller Ian M
Optics and Plasma Research Department, Risø National Laboratory, DK-4000 Roskilde, Denmark.
Plant Cell. 2006 Mar;18(3):688-98. doi: 10.1105/tpc.105.039354. Epub 2006 Feb 3.
The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5- to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.
还原型辅酶NADH在线粒体呼吸代谢中起着核心作用。然而，关于线粒体中游离NADH含量的报道稀少且相互矛盾。我们首先使用等温滴定量热法结合荧光光谱法测定了与蛋白质结合的NADH的发射光谱。然后，通过对荧光发射光谱进行光谱分解分析，测量了处于不同代谢状态的活跃呼吸线粒体（来自马铃薯块茎[Solanum tuberosum cv Bintje]）中的NADH含量。线粒体中的大多数NADH与蛋白质结合，其含量在状态3（存在底物+ADP）时较低，在状态2（仅存在底物）和状态4（存在底物+ATP）时较高。相比之下，游离NADH的含量较低但相对恒定，甚至在状态3时略有增加。通过建模，我们表明，与状态4相比，状态3中NADH与线粒体蛋白的平均结合力弱2.5至3倍，可以解释这些结果。这表明存在游离NADH稳态的特定机制，并且线粒体基质中游离NADH的浓度本身在线粒体代谢中不发挥调节作用。这些发现对细胞代谢的解释具有深远影响。
