Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity
Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity
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DOI：
10.1074/jbc.m305819200
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发表时间：
2003-10-24
影响因子：
4.8
通讯作者：
Brandt, U
中科院分区：
文献类型：
--
作者：
Garofano, A;Zwicker, K;Brandt, U
线粒体质子易位 NADH：泛醌氧化还原酶（复合体 I）将两个电子从 NADH 到泛醌的转移与四个质子穿过线粒体内膜的易位耦合起来。 PSST 亚基是铁硫簇 N2 最有可能的载体，已被认为在泛醌还原和质子泵浦中发挥着至关重要的作用。为了探索该亚基的功能，我们生成了解脂耶氏酵母同源物（NUKM 蛋白）中所有八个高度保守的酸性残基的定点突变体。突变体D99N和D115N分别仅具有野生型催化活性的5%和8%。在这两种情况下，复合物 I 均稳定组装，但纯化酶的电子顺磁共振谱显示 N2 信号减少（约 50%）。就复合物 I 催化活性而言，当天冬氨酸单独变为谷氨酸或甘氨酸时，获得了几乎相同的结果。其他保守酸性残基的突变对催化活性的影响较小，并且不会阻止铁硫簇 N2 的组装。这排除了 PSST 亚基中作为该氧化还原中心的第四配体的所有保守酸性残基。根据与水溶性[NiFe]氢化酶同源小亚基的结构相似性对结果进行了讨论。
Mitochondrial proton-translocating NADH: ubiquinone oxidoreductase ( complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.