Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex
Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex
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DOI：
10.1016/j.bbabio.2006.09.003
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发表时间：
2006-12-01
影响因子：
4.3
通讯作者：
Brandt, Ulrich
中科院分区：
文献类型：
--
作者：
Abdrakhmanova, Albina;Zwicker, Klaus;Brandt, Ulrich
除14个中心亚基外，来自好氧酵母解脂耶氏酵母的呼吸链复合物I还含有至少24个辅助亚基，其中大多数亚基的特征很差。在这里，我们研究了辅助39-kDa亚基的作用，它属于异源短链脱氢酶/还原酶（SDR）酶家族，含有非共价结合的NADPH。删除编码39 kDa亚基的基因的染色体拷贝大大损害了解脂耶氏酵母中复合物I的组装。我们在核苷酸结合基序中引入了几个定点突变，严重降低了NADPH结合。当NADPH结合Rossman折叠的第二条p-链末端的精氨酸被亮氨酸或天冬氨酸取代时，这种效果最明显。影响核苷酸结合的突变对线粒体膜的特异性催化活性只有轻微或中度影响，但明显使复合物I不稳定。一个突变体表现出温度敏感的表型和显着量的三种不同的亚复合物，即使在更允许的温度下观察。我们得出的结论是，39 kDa的亚基的解脂耶氏酵母在复合物I的组装和稳定性中起着至关重要的作用，结合的NADPH作为一个整体，而不是作为一个催化功能，以稳定的亚基和复合物I。(c)2006 Elsevier B.V.保留所有权利。
In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second p-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function. (c) 2006 Elsevier B.V. All rights reserved.