Albury M S, Affourtit C, Moore A L
Biochemistry Department, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, United Kingdom.
J Biol Chem. 1998 Nov 13;273(46):30301-5. doi: 10.1074/jbc.273.46.30301.
We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.
我们之前已经证明，在粟酒裂殖酵母中表达斑龙芋交替氧化酶可使这些细胞具有抗氰呼吸活性（阿尔伯里，M. S.，达德利，P.，瓦茨，F. Z.，和摩尔，A. L.（1996年）《生物化学杂志》271卷，17062 - 17066页）。利用这个功能表达系统，我们研究了植物交替氧化酶的活性位点，据推测该活性位点包含一个非血红素双核铁中心。一个保守的谷氨酸（Glu - 270），之前被假定为活性位点内的桥连配体，突变为天冬酰胺后催化活性丧失，因为含有E270N突变蛋白的线粒体不表现出抗霉素A抗性呼吸。使用针对交替氧化酶的特异性抗体进行的蛋白质免疫印迹分析表明，E270N突变蛋白以与野生型蛋白相似的方式被靶向运输到粟酒裂殖酵母线粒体并进行加工。含有E270N突变蛋白的线粒体中观察到缺乏抗霉素A不敏感呼吸，可能是由于突变的交替氧化酶不正确地插入线粒体内膜。然而，对分级分离的线粒体进行的蛋白质免疫印迹分析表明，野生型和E270N交替氧化酶都特异性地位于线粒体内膜，这表明错误折叠或缺乏插入不太可能。这些结果提供了首个实验证据来支持交替氧化酶活性位点包含一个偶联双核铁中心的结构模型。
