Nehls U, Friedrich T, Schmiede A, Ohnishi T, Weiss H
Heinrich-Heine-Universität Düsseldorf, Institut für Biochemie, Federal Republic of Germany.
J Mol Biol. 1992 Oct 20;227(4):1032-42. doi: 10.1016/0022-2836(92)90519-p.
ubiquinone oxidoreductase, the respiratory chain complex I of mitochondria, is an assembly of some 25 nuclear-encoded and 7 mitochondrially encoded subunits. The complex has an overall L-shaped structure formed by a peripheral arm and an elongated membrane arm. The peripheral arm containing one FMN and at least three iron-sulphur clusters constitutes the NADH dehydrogenase segment of the electron pathway. The membrane arm with at least one iron-sulphur cluster constitutes the ubiquinone reducing segment. We are studying the assembly of the complex in Neurospora crassa. By disrupting the gene of a nuclear-encoded subunit of the membrane arm a mutant was generated that cannot form complex I. The mutant rather pre-assembles the peripheral arm with all redox groups and the ability to catalyse NADH oxidation by artificial electron acceptors. The final assembly of the membrane arm is blocked in the mutant leading to accumulation of complementary assembly intermediates. One intermediate is associated with a protein that is not present in the fully assembled complex I. The results demonstrate that the two arms of complex I are assembled independently on separate pathways, and gave a first insight into the assembly pathway of the membrane arm. It is also shown for the first time that the obligate aerobic fungus N. crassa can grow and respire without an intact complex I. Gene replacement in this fungus is therefore a tool for investigation of this complex.
泛醌氧化还原酶，即线粒体呼吸链复合体I，由约25个核编码亚基和7个线粒体编码亚基组装而成。该复合体具有由外周臂和细长的膜臂形成的整体L形结构。包含一个FMN和至少三个铁硫簇的外周臂构成了电子传递途径的NADH脱氢酶部分。带有至少一个铁硫簇的膜臂构成了泛醌还原部分。我们正在研究粗糙脉孢菌中该复合体的组装。通过破坏膜臂的一个核编码亚基的基因，产生了一个不能形成复合体I的突变体。该突变体反而预先组装了带有所有氧化还原基团的外周臂，并具有通过人工电子受体催化NADH氧化的能力。膜臂的最终组装在突变体中受阻，导致互补组装中间体的积累。一种中间体与一种在完全组装的复合体I中不存在的蛋白质相关。结果表明，复合体I的两个臂在不同的途径上独立组装，并首次深入了解了膜臂的组装途径。还首次表明，专性需氧真菌粗糙脉孢菌在没有完整的复合体I的情况下也能生长和呼吸。因此，该真菌中的基因替换是研究这个复合体的一种工具。
