Tocilescu Maja A, Fendel Uta, Zwicker Klaus, Dröse Stefan, Kerscher Stefan, Brandt Ulrich
Molecular Bioenergetics Group, Medical School, Cluster of Excellence Frankfurt Macromolecular Complexes, Center for Membrane Proteomics, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):625-32. doi: 10.1016/j.bbabio.2010.01.029. Epub 2010 Feb 1.
Iron-sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only approximately 7A in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q1 and Q2 that were coupled to proton pumping. Apparent Km values for Q1 and Q2 were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron-sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.
复合物I（质子泵NADH：醌氧化还原酶）的铁硫簇N2是辅酶Q的直接电子供体。在49 kDa亚基中，距离仅约7埃处，在先前表征的醌结合口袋底部发现了一个高度保守的酪氨酸。为了深入了解该残基的功能，我们在解脂耶氏酵母的复合物I中将其替换为六种不同的氨基酸。所有六个突变体的线粒体膜都含有完全组装的复合物I，其对正癸基泛醌表现出非常低的dNADH：泛醌氧化还原酶活性。对于最保守的替换Y144F，未检测到复合物I的电子顺磁共振光谱有变化。值得注意的是，观察到与质子泵偶联的辅酶Q1和Q2具有高dNADH：泛醌氧化还原酶活性。Q1和Q2的表观Km值显著增加，并且我们发现对复合物I抑制剂癸基喹唑啉胺（DQA）和鱼藤酮具有明显抗性。我们得出结论，Y144直接结合辅酶Q的头部基团，最有可能是通过芳香羟基与辅酶Q羰基之间的氢键。这使得底物与其电子供体铁硫簇N2处于理想距离，以便在复合物I的催化循环中进行有效的电子转移。
