The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli
The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli
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DOI：
10.1074/jbc.m302361200
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发表时间：
2003-07-11
影响因子：
4.8
通讯作者：
Yu, CA
中科院分区：
文献类型：
--
作者：
Gong, X;Xie, T;Yu, CA
利用叠氮基泛醌衍生物3-叠氮基-2-甲基-5-甲氧基[ H-3]-6-癸基-1，4-苯醌（[H-3] azido-Q）研究了泛醌与蛋白质的相互作用，并鉴定了大肠杆菌NADH：泛醌氧化还原酶（复合物I）中的泛醌结合位点。在黑暗中与20倍摩尔过量的[H-3]叠氮基-Q温育后，纯化的复合物I没有显示出活性损失。在0 ℃下用长波长紫外光照射孵育的样品10分钟导致NADH：泛醌氧化还原酶活性降低40%。用[H-3]叠氮基-Q标记的复合物的SDS-PAGE，随后分析亚基之间的放射性分布，揭示亚基NuoM被大量标记，表明该蛋白质含有Q结合位点。当[H-3]叠氮基-Q-标记的NuoM通过制备性SDS-PAGE从标记的还原酶中纯化时，通过蛋白酶K消化标记的亚基的高效液相色谱，获得保留时间为41.4min的3-叠氮基-2-甲基-5-甲氧基-6-癸基-1，4-苯醌连接的肽。该肽具有NH 2-VMLIAILALV-的部分NH 2-末端氨基酸序列，其对应于NuoM的氨基酸残基184 - 193。使用Toppred亲水性分析的NuoM的二级结构预测表明，Q结合肽与位于跨膜螺旋5中间朝向膜的细胞质侧的拟议Q结合基序重叠。使用PHDhtm亲水性图，标记的肽位于跨膜螺旋4中朝向膜的周质侧。
An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[ H-3]-6-decyl-1,4-benzoquinone ([H-3] azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH: ubiquinone oxidoreductase ( complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [H-3] azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degreesC caused a 40% decrease of NADH: ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [H-3] azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [H-3] azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4- benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184 - 193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.