Flemming Dirk, Hellwig Petra, Lepper Simone, Kloer Daniel P, Friedrich Thorsten
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, D-79104 Freiburg, Germany.
J Biol Chem. 2006 Aug 25;281(34):24781-9. doi: 10.1074/jbc.M602499200. Epub 2006 Jun 28.
The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.
来自大肠杆菌的NADH：泛醌氧化还原酶（复合体I）由13个亚基组成，分别称为NuoA至NuoN，并含有一个FMN和9个铁硫簇作为氧化还原基团。从NADH到泛醌的电子传递与质子通过膜的转运通过一种未知机制偶联。氧化还原诱导的傅里叶变换红外差光谱表明，位于NuoB上的铁硫簇N2的氧化伴随着酸性氨基酸的质子化。在此，我们描述了NuoB上保守酸性氨基酸突变的影响。所有突变体中都组装了复合体，但在突变体E67Q、D77N和D94N中电子传递活性完全丧失。从这些突变体中分离出的复合体含有N2，尽管数量减少。在突变体E67Q的复合体中未检测到与N2氧化偶联的酸性氨基酸的质子化。然而，保守突变E67D和D77E并未干扰酶活性，并且在E67D突变体中可检测到由于酸性氨基酸质子化产生的信号。我们讨论了Glu(67)可能参与与N2氧化还原反应偶联的质子途径。
