Schuler F, Yano T, Di Bernardo S, Yagi T, Yankovskaya V, Singer T P, Casida J E
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4149-53. doi: 10.1073/pnas.96.7.4149.
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.
质子转运型NADH-醌氧化还原酶（EC 1.6.99.3）是呼吸链中最大且了解最少的酶复合物。哺乳动物线粒体酶（也称为复合物I）包含40多个亚基，而其结构更简单的细菌对应物（NDH-1），如反硝化副球菌和嗜热栖热菌HB-8中的NDH-1由14个亚基组成。一个主要的未解决问题是从铁硫簇N2到醌的末端电子转移步骤的位置和机制。作用于这一关键区域的强效抑制剂是剖析NADH-醌氧化还原酶的候选光亲和探针。复合物I和NDH-1对多种结构不同的毒物抑制非常敏感，这些毒物包括鱼藤酮、杀粉蝶菌素A、布拉他辛和哒螨灵。我们设计了（三氟甲基）二氮杂萘基[3H]哒螨灵（[3H]TDP）作为我们的光亲和配体，因为它兼具出色的抑制效力、合适的光反应基团以及高比活的氚。线粒体电子传递颗粒的光亲和标记具有特异性且可饱和。分离、蛋白质测序和免疫沉淀确定高亲和力特异性标记的23 kDa亚基为复合物I的PSST。反硝化副球菌和嗜热栖热菌标记膜的免疫沉淀确定了等效细菌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强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，并且该亚基通过将铁硫簇N2与醌功能偶联在电子传递中起关键作用。
