Strickland Madeleine, Juárez Oscar, Neehaul Yashvin, Cook Darcie A, Barquera Blanca, Hellwig Petra
From the Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CNRS Université de Strasbourg, Strasbourg, France, 67000 and.
Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
J Biol Chem. 2014 Aug 22;289(34):23723-33. doi: 10.1074/jbc.M114.574640. Epub 2014 Jul 8.
Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.
钠泵NADH：泛醌氧化还原酶（Na(+)-NQR）负责维持细菌内膜两侧的钠梯度。这种呼吸酶将钠泵与NADH和泛醌之间的电子传递偶联起来，真核生物中不存在该酶，因此它可能是抗生素的作用靶点。本文表明，泛醌还原位点在构象上与NqrB亚基偶联，NqrB亚基也是电子传递链中的最终辅助因子核黄素的宿主。先前的研究表明，保守的NqrB甘氨酸残基140和141发生突变会影响泛醌还原和钠泵的正常功能。令人惊讶的是，这些突变体并未影响泛醌或其类似物HQNO（2-正庚基-4-羟基喹啉N-氧化物）与Na(+)-NQR的解离常数，这表明这些残基不直接参与泛醌结合位点，但可能控制其可及性。事实上，氧化还原诱导的差示光谱表明，这些突变阻止了泛醌结合所涉及的构象变化，但并未改变与结合的泛醌相对应的信号。此外，本文提供的数据表明NqrA亚基能够结合泛醌，但亲和力较低，与催化无关。还表明Na(+)-NQR含有一个单一的催化泛醌结合位点和另一个可以结合泛醌但无活性的位点。
