Euro Liliya, Belevich Galina, Verkhovsky Michael I, Wikström Mårten, Verkhovskaya Marina
Helsinki Bioenergetics Group, Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Finland.
Biochim Biophys Acta. 2008 Sep;1777(9):1166-72. doi: 10.1016/j.bbabio.2008.06.001. Epub 2008 Jun 9.
Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.
对复合体I膜结构域中亚基NuoM和NuoN的氨基酸序列分析揭示了一种明显的共同模式，包括两个预计位于膜内的赖氨酸，它们对醌还原酶活性很重要。将此模式中氨基酸残基E144、K234、K265和W243的定点突变引入大肠杆菌复合体I的染色体基因nuoM中。在膜和纯化的复合体I中研究了突变复合体I的活性。醌还原酶活性在K234A、K234R和E144A中几乎丧失，在W243A和K265A中降低，但在E144D中不变。所有这些突变体的复合体I每摩尔FMN含有1摩尔紧密结合的泛醌，与野生型酶一样。突变酶E144D、W243A和K265A对罗利他汀具有野生型敏感性，并且复合体I具有完全的质子泵浦效率。值得注意的是，膜结构域中的亚基NuoL和NuoH在跨膜螺旋中似乎也含有保守的赖氨酸残基，这可能为质子转运机制提供线索。基于膜亚基中赖氨酸的静电相互作用，提出了复合体I质子转运的一个初步原理。
