Molecular organization of mitochondrial quinol-fumarate reductase and its role in oxygen adaptation.

成虫肌肉复合体II具有较高的富马酸醌还原酶（QFR）活性，在适应微氧环境的过程中，复合体II在厌氧电子传递中起关键作用。相比之下，幼虫（L3）复合物II的QFR活性远低于成虫酶，并在有氧呼吸中起琥珀酸-泛醌还原酶（SQR）的作用。我们报道了A.suum线粒体中复合体II的阶段特异性亚型，并表明幼虫复合体II的黄蛋白亚基（Fp）和细胞色素b（cybS）的小亚基与成虫不同。在本研究中，已经从厌氧成年A.suum和自由生活的线虫秀丽隐杆线虫中克隆并测序了复合体II的铁硫亚基（Ip）的完整cdna，它与Fp形成复合体II的催化部分。这两种线虫的Ip亚基的氨基酸序列是相似的，特别是在三个富含半胱氨酸的区域周围，这些区域被认为与酶的铁硫簇有关。由于Northern杂交显示成虫的Ip在L3中也有表达，因此也鉴定了黄花蓟马幼虫的Ip。幼虫复合体II的Ip被抗体识别，并通过肽图谱与成体Ip无法区分。deae -纤维素纤维素柱层析纯化的幼虫复合物II中Ip的n -热端42个氨基酸序列与成熟的成虫Ip相同。此外，在PVDF膜上微量分析发现，幼虫的氨基酸组成与成虫的氨基酸组成基本相同。这些结果，加上使用退化引物的RT-PCR同源性探测未能找到幼虫特异性Ip，表明这两种不同阶段特异性形式的A.suum复合物II具有共同的Ip亚基，尽管成虫酶作为QFR，而幼虫酶作为SQR。此外，我们还从黑曲霉中发现了新的化合物nafuretin。Nafuredin在nM级抑制nadh -富马酸还原酶（复合体I+II）的活性，nadh -富马酸还原酶是脐部线粒体中一种独特的厌氧电子传递系统。它在复合体I中竞争醌结合位点，对蠕虫酶表现出高选择性毒性。此外，在绵羊体内试验中，萘脲素对扭曲血蜱具有驱虫活性。因此，我们的研究表明，线粒体复合体I是一个有希望的化疗靶点，nafureredin是一个新的潜在的先导化合物，作为一种从微生物中分离出来的新型驱虫药。少

Complex II of adult Ascaris suum muscle exhibits high quinol-fumarate reductase(QFR)activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval(L3)complex II shows a much lower QFR activity than the adult enzyme, and functions as succinate-ubiquinone reductase(SQR)in aerobic respiration. We have reported the stage-specific isoforms of complex II in A.suum mitochondria, and showed that at least the flavoprotein subunit(Fp)and the small subunit of cytochrome b(cybS)of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit(Ip)of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A.suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cystein-rich regions that are thought to com … More prise the iron-sulfur clusters of the enzyme. The Ip from A.suum Larvae also was characterized because Northern hybridization showed that the adult Ip also is expressed in L3. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-therminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact that homology probing by RT-PCR using degenerated primers failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A.suum complex II share a common Ip subunit, even though the adult enzyme functions as a QFR, while larval enzyme acts as an SQR.In addition, we found novel compound, nafuredin, from Aspergillus niger. Nafuredin inhibits NADH-fumarate reductase(complexes I+II)activity, a unique anaerobic electron transport system in hilminth mitochondria, at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials using sheep. Thus, our study indicates that mitochondrial complex I is a promising target for chemotherapy and that nafuredin is a new potential lead compound as a novel anthelmintic isolated from microorganisms. Less