Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized.
Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized.
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DOI：
10.1074/jbc.m116.771899
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发表时间：
2017-03-24
期刊：
影响因子：
--
通讯作者：
Hirst J
中科院分区：
文献类型：
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作者：
Jones AJ;Blaza JN;Varghese F;Hirst J
呼吸复合体I偶联NADH和泛醌之间的电子转移，通过能量转导膜进行质子转移，以支持驱动ATP合成的质子动力。络合物I的质子泵浦化学计量学(即每两个转移的电子所泵浦的质子数量)支持所有的机械假设。然而，它仍然存在争议，还没有确定任何被用作哺乳动物酶模型系统的细菌酶。在这里，我们描述了一种简单的方法，在稳定的ADP磷酸化条件下，测定倒置膜囊泡中络合物I的质子泵化学计量比。我们的方法利用呼吸链不同部分参与催化的NADH或琥珀酸氧化驱动的ATP合成速率作为质子转移速率的替代，并参考已知的配合物III和IV的化学计量比来确定配合物I的化学计量比。使用从哺乳动物线粒体(来自牛群)和从细菌Paracoccus denitriicans制备的囊泡，我们证明在这两种情况下，每转移两个电子就有四个质子被泵送。通过确认哺乳动物复合体I的四质子化学计量比，并首次证明了细菌复合体的四质子化学计量学，我们建立了脱氮假单胞菌复合体I作为哺乳动物酶的模型系统的实用性。脱氮假单胞菌是第一个被描述的系统，在该系统中，任何复杂的I核心亚单位的突变都可以与定量的质子泵测量相结合，用于机理研究。
Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.