Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling
Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling
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DOI：
10.1074/jbc.m117.781393
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发表时间：
2017-05-12
影响因子：
4.8
通讯作者：
Miyoshi, Hideto
中科院分区：
文献类型：
--
作者：
Ito, Takeshi;Murai, Masatoshi;Miyoshi, Hideto
Na+泵NADH醌氧化还原酶（Na+-NQR）是呼吸链的第一个酶，也是许多海洋细菌和病原菌（包括霍乱弧菌）的主要离子转运蛋白。霍乱弧菌Na+-NQR已被广泛研究，但其与泛醌和抑制剂的结合位点仍存在争议。在这里，使用光反应性泛醌PUQ-3以及两个aurachin型抑制剂[I-125] PAD-1和[I-125] PAD-2和分离的酶的光亲和标记实验，我们证明泛醌环结合到NqrA亚基的区域Leu-32 Met- 39和Phe-131-Lys-138，包括预测的泛醌结合腔的后壁。aurachin的喹诺酮环和烷基侧链分别与Arg-43-Lys-54和Trp-23-Gly-89区域的NqrB亚基结合。这些结果表明泛醌和aurachin型抑制剂的结合位点非常接近，但彼此不重叠。出乎意料的是，尽管PAD-1和PAD-2的抑制作用几乎被NqrB中的某些突变完全消除（即，e. G140 A和E144 C），[I-125] PAD-1和[I-125] PAD-2与突变酶的结合反应性与野生型酶相比没有变化。我们还发现，在某些实验条件下，[I-125] PAD-1和[I-125] PAD-2的光亲和标记增强，而不是在其他抑制剂存在下被竞争性抑制。为了解释这些明显自相矛盾的结果，我们提出了模型的催化反应的Na+-NQR和它的相互作用与抑制剂的基础上的生物化学和生物物理结果报告在这里和以前的工作。
The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [I-125] PAD-1 and [I-125] PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32Met- 39 and Phe-131-Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43-Lys-54 and Trp-23-Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin- type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i. e. G140A and E144C), the binding reactivities of [I-125] PAD-1 and [I-125] PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [I-125] PAD-1 and [I-125] PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced undersomeexperimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.