NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.
NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.
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DOI：
10.1073/pnas.96.7.4149
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发表时间：
1999-03
影响因子：
11.1
通讯作者：
F. Schuler;T. Yano;S. Bernardo;T. Yagi;V. Yankovskaya;T. Singer;J. Casida
中科院分区：
文献类型：
--
作者：
F. Schuler;T. Yano;S. Bernardo;T. Yagi;V. Yankovskaya;T. Singer;J. Casida
质子易位nadh -醌氧化还原酶（EC 1.6.99.3）是呼吸链中最大和最不了解的酶复合体。哺乳动物线粒体酶（也称为复合体I）包含40多个亚基，而反硝化副球菌和嗜热热菌HB-8中结构更简单的细菌对应物（NDH-1）包含14个亚基。从铁硫簇N2到醌的末端电子转移步骤的位置和机制是一个尚未解决的主要问题。在这个关键区域起作用的有效抑制剂是用于解剖nadh -醌氧化还原酶的候选光亲和探针。配合物I和NDH-1对多种结构各异的毒物的抑制非常敏感，包括鱼藤酮、皮利西丁a、布拉他星和嘧螨酯。我们设计（三氟甲基）二氮基[3H]吡啶苯（[3H]TDP）作为我们的光亲和配体，因为它结合了出色的抑制剂效力、合适的光反应基团和高比活性的氚。线粒体电子传递粒子的光亲和标记具有特异性和可饱和性。分离、蛋白测序和免疫沉淀鉴定了高亲和力特异性标记的23-kDa亚基为复合体i的PSST。反硝化假单胞菌和嗜热假单胞菌标记膜的免疫沉淀建立了等效细菌NQO6的光亲和标记。竞争性结合和酶抑制研究表明，PSST特异性高亲和力结合位点的光亲和标记对上述每种高效抑制剂都异常敏感。这些发现表明，线粒体的同源PSST和细菌的NQO6具有保守的抑制剂结合位点，并且该亚基通过铁硫簇N2与醌的功能偶联在电子传递中起关键作用。
The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone.