Defining the proton entry point in the bacterial respiratory nitric-oxide reductase
Defining the proton entry point in the bacterial respiratory nitric-oxide reductase
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DOI：
10.1074/jbc.m704615200
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发表时间：
2008-02-15
影响因子：
4.8
通讯作者：
Ardelroth, Pia
中科院分区：
文献类型：
--
作者：
Flock, Ulrika;Thorndycroft, Faye H.;Ardelroth, Pia
细菌呼吸道一氧化氮还原酶（NOR）是O-2还原、质子泵、血红素铜氧化酶超家族的成员。尽管一氧化氮还原是一个高度放能反应，但NOR不是质子泵，而不是像血红素-铜氧化酶那样从膜的细胞质（膜电位负）侧吸收质子，NOR从膜的周质（膜电位正）侧获得其底物质子。这种非生电质子转移的分子细节尚未解决，因此在这项研究中，我们探索了保守的表面谷氨酸（Glu-122）在催化亚基（NorB）中的质子途径中的作用。确定了用Ala、Gln或Asp取代Glu-122对用O-2（NOR的替代和实验上易处理的底物）还原的NOR变体的单次营业额的影响。电子转移耦合到质子摄取绑定O-2是严重和特异性抑制在E122 A和E122 Q的变体，建立在这个位置的质子化侧链的重要性。在E122 D突变体中，质子摄取被保留，但它与观察到的向活性位点提供质子的基团的pK（a）显著增加有关。这表明Glu-122在定义这种质子供体方面很重要。第二个附近的谷氨酸（Glu-125）也需要电子转移耦合到质子摄取，进一步强调了该区域的NorB在质子转移的重要性。由于Glu-122被预测位于NOR的周质表面附近，因此结果提供了强有力的实验证据，该残基有助于定义非生电“E-途径”的孔径，该途径用于将质子从周质递送到NOR中的掩埋活性位点。
The bacterial respiratory nitric-oxide reductase (NOR) is a member of the superfamily of O-2-reducing, proton-pumping, heme-copper oxidases. Even although nitric oxide reduction is a highly exergonic reaction, NOR is not a proton pump and rather than taking up protons from the cytoplasmic (membrane potential-negative) side of the membrane, like the heme-copper oxidases, NOR derives its substrate protons from the periplasmic (membrane potential-positive) side of the membrane. The molecular details of this non-electrogenic proton transfer are not yet resolved, so in this study we have explored a role in a proposed proton pathway for a conserved surface glutamate (Glu-122) in the catalytic subunit (NorB). The effect of substituting Glu-122 with Ala, Gln, or Asp on a single turnover of the reduced NOR variants with O-2, an alternative and experimentally tractable substrate for NOR, was determined. Electron transfer coupled to proton uptake to the bound O-2 is severely and specifically inhibited in both the E122A and E122Q variants, establishing the importance of a protonatable side chain at this position. In the E122D mutant, proton uptake is retained but it is associated with a significant increase in the observed pK(a) of the group donating protons to the active site. This suggests that Glu-122 is important in defining this proton donor. A second nearby glutamate (Glu-125) is also required for the electron transfer coupled to proton uptake, further emphasizing the importance of this region of NorB in proton transfer. Because Glu-122 is predicted to lie near the periplasmic surface of NOR, the results provide strong experimental evidence that this residue contributes to defining the aperture of a non-electrogenic "E-pathway" that serves to deliver protons from the periplasm to the buried active site in NOR.