Asymmetric binding of the high-affinity QH•- ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy
Asymmetric binding of the high-affinity QH•- ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy
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DOI：
10.1021/bi034010z
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发表时间：
2003-05-20
期刊：
影响因子：
2.9
通讯作者：
MacMillan, F
中科院分区：
文献类型：
--
作者：
Grimaldi, S;Ostermann, T;MacMillan, F
将在1-或4-羰基碳上用C-13（I =1/2）选择性标记的泛醌-2（UQ-2）掺入到来自大肠杆菌的泛醇氧化酶bo（3）中，其中天然醌（UQ-8）已被预先去除。在高亲和力醌结合位点（QH（.-））中产生稳定的阴离子自由基，研究使用多频（9，34，和94 GHz）电子顺磁共振（EPR）光谱。相应的光谱揭示了显着的差异，在C-13超精细耦合表明一个强烈的不对称的自旋密度分布在醌头基。通过与以前蛋白质和有机溶剂中标记泛半醌的结果进行比较，得出结论：QH（.-）很可能通过单侧氢键或强不对称氢键网络与蛋白质结合。这一观察结果进行了讨论，关于Q（H）在酶中的功能，并与其他蛋白质结合的半醌自由基的信息进行了对比。
Ubiquinone-2 (UQ-2) selectively labeled with C-13 (I =1/2) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed. The resulting stabilized anion radical in the high-affinity quinone-binding site (QH(.-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy. The corresponding spectra reveal dramatic differences in C-13 hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup. By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that QH(.-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network. This observation is discussed with regard to the function Of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals.