Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane.
Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane.
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DOI：
10.1042/bj20081345
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发表时间：
2009-01-15
期刊：
影响因子：
--
通讯作者：
Jeuken LJ
中科院分区：
文献类型：
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作者：
Weiss SA;Bushby RJ;Evans SD;Henderson PJ;Jeuken LJ
我们开发了一种简单的类天然表面束缚膜系统来研究细胞色素 bo3 (cbo3)（大肠杆菌中的一种末端氧化酶）的活性。系留膜由大肠杆菌内膜提取物与含有不同量的 cbo3 底物 ubiquinol-10 (UQ-10) 的额外大肠杆菌脂质混合组成。系留膜是通过将囊泡自组装到用胆固醇衍生物功能化的金电极上而形成的。使用循环伏安法监测细胞色素 bo3 活性，并通过 UQ-10 介导电子转移至 cbo3。该系统对氧的表观 KM 为 1.1±0.4 μM，与全细胞实验和纯化 cbo3 的文献值非常一致。增加膜中亲脂性 UQ-10 的浓度会导致 cbo3 活性增加。 cbo3 与长链泛醌的活性似乎与之前使用短链底物类似物（例如 UQ-1）的报道不同，因为使用 UQ-10 没有观察到典型的 Michaelis Menten 动力学。因此，这种类似天然的膜模型为跨膜酶与疏水性底物的相互作用提供了新的见解，这与使用亲水性 UQ 类似物的研究形成鲜明对比。
We have developed a simple native-like surface-tethered membrane system to investigate the activity of cytochrome bo3 (cbo3), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo3 substrate ubiquinol-10 (UQ-10). Tethered membranes are formed by self assembly from vesicles onto gold electrodes functionalised with cholesterol derivatives. Cytochrome bo3 activity was monitored using cyclic voltammetry with electron transfer to cbo3 mediated by UQ-10. The apparent KM for oxygen with this system is 1.1±0.4 μM, in good agreement with literature values for whole cell experiments and for purified cbo3. Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo3 activity. The activity of cbo3 with long chain ubiquinones appears to be different to previous reports using short chain substrate analogues such as UQ-1 in that typical Michaelis Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.