NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae
NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae
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DOI：
10.1074/jbc.m112.435750
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发表时间：
2013-10-18
影响因子：
4.8
通讯作者：
Moeller, Heiko M.
中科院分区：
文献类型：
--
作者：
Nedielkov, Ruslan;Steffen, Wojtek;Moeller, Heiko M.
来自病原体霍乱弧菌的钠离子转运NADH：醌氧化还原酶（Na+-NQR）利用在用泛醌氧化NADH期间释放的自由能来泵送钠离子穿过细胞质膜。Na+-NQR由四个膜结合亚基NqrBCDE和外周NqrF和NqrA亚基组成。NqrA结合泛醌-8以及具有较短异戊二烯基链的醌（泛醌-1和泛醌-2）。在这里，我们表明，醌衍生物2，5-二溴-3-甲基-6-异丙基-对苯醌（DBMIB），一个已知的抑制剂的bc（1）和B（6）f复合物中发现的线粒体和叶绿体，也抑制醌还原Na+-NQR在混合抑制模式。色氨酸荧光淬灭和饱和转移差NMR实验中的Na+-NQR抑制剂（DBMIB或2-n-庚基-4-羟基喹啉N-氧化物）的存在下，表明两个醌类似物配体结合的NqrA亚基与非常相似的相互作用常数，观察到的全酶复合物。我们的结论是醌还原的催化位点位于NqrA。这两个配体结合到彼此直接邻近的扩展结合口袋，如分别通过泛醌-1和DBMIB或2-正庚基-4-羟基喹啉N-氧化物之间的配体间Overhauser效应所证明的。我们建议，一个类似的空间紧密排列的原生醌基板也在体内操作，提高在Na+-NQR的最后的电子转移步骤的催化效率。
The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc(1) and b(6)f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR.