Study on ubiquinol reaction site of cytochrome bo in Escherichia coli.

用系统合成的泛喹酚类似物探索了大肠杆菌细胞色素bo和bd的底物结合部位。Q_2H_2衍生物对细胞色素bo的表观K_m值远低于相应的6-正癸基衍生物。与相同碳数的饱和正烷基相比，类异戊二烯结构的疏水性较小，因此，天然的类异戊二烯侧链除了增加分子的疏水性外，似乎在与苯二酚的结合中发挥了特殊的作用。无论侧链结构如何，2-甲氧基-3-乙氧基类似物的V&lt；max&gt；值均大于2-乙氧基-3-甲氧基类似物。这一结果不仅表明2-位上的甲氧基比3-位上的甲氧基更能被结合部位识别，而且侧链结构不影响对苯二酚环部分的结合。对5-位不同取代基的类似物的给电子活性进行了系统的分析，发现5-甲基对活性有重要影响。在与细胞色素BD的平行研究中，得到了类似的观察结果，除了几乎所有的喹酚类似物，但不是Q_1，在较高浓度下引起显著的底物抑制。这些结果表明，结构上不相关的两个末端氧化酶具有相同的结构性质，即对苯二酚的氧化点。此外，为了研究细胞色素b酶的苯醌结合部位，建立了制备可光解的2-叠氮基-Q_2和3-叠氮-Q_2的简便合成工艺。

Substrate binding sites of the Escherichia coli cytochromes bo and bd were probed with systematically synthesized ubiquinol analogues. The apparent K_m values of Q_2H_2 derivatives to the cytochrome bo were much lower than that of the corresponding 6-n-decyl derivatives. The isoprenoid structure is less hydrophobic than the saturated n-alkyl group with the same carbon number, therefore, the native isoprenoid side chain appears to play a specific role in quinol binding besides simply increasing hydrophobicity of the molecule. The V_<max> values of 2-methoxy-3-ethoxy analogues were greater than that of 2-ethoxy-3-methoxy analogues irrespective of the side chain structure. This result indicates not only that a methoxy group in the 2-position is recognized more strictly than the 3-position by the binding site, but also that the side chain structure does not affect binding of the quinol ring moiety. Systematic analysis of the electron-donating activities of the analogues with different substituents in the 5-position revealed that the 5-methyl group is important for the activity. In the parallel studies with the cytochrome bd, similar observations were obtained except that almost all quinol analogues, but not Q_1, elicited a remarkable substrate inhibition at higher concentrations. These results indicate that the structurally unrelated two terminal oxidases share common structural properties for the quinol oxidation site. In addition, to investigate the quinone binding site of cytochrome bo enzyme, convenient synthetic procedures which enable the preparation of photolable 2-azido-Q_2 and 3-azido-Q_2 have been established.