Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation
Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation
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DOI：
10.1046/j.1365-2958.2002.02875.x
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发表时间：
2002-04-01
影响因子：
3.6
通讯作者：
Cole, JA
中科院分区：
文献类型：
--
作者：
Brondijk, THC;Fiegen, D;Cole, JA
大肠杆菌K-12的NAP操纵子编码一个周质硝酸还原酶(NAP)，编码7种蛋白质。周质中的催化络合物napA-napB被认为是通过膜结合的细胞色素NAPC接收来自对苯二酚池的电子。与NapA、B和C一样，第四种多肽Napd对Nap活动也是必不可少的。然而，其余三个多肽NAPF、G和H被预测为编码非血红素、铁硫蛋白，没有一个是NAP活性所必需的，它们的功能目前尚不清楚。利用两种膜结合硝酸还原酶缺陷的菌株以及泛醌或菜醌生物合成缺陷的菌株，研究了生理底物向NAP的相对生长速率和电子转移速率。这些数据表明，NAP与尿喹酚氧化的偶联比与泛喹酚氧化的偶联更有效。相反，对第二组突变体进行的平行实验表明，硝酸还原酶A与泛喹酚的结合比与甲喹酚的结合更有效。用框内缺失的ubiCA、menBC、NAPC、NAPF和napGH基因组合构建了另外三组菌株。NAPF、nAPG和napH在从菜籽酚到napAB复合体的电子转移中没有作用，但在Ubi(+)Man(-)背景下，NAPF、napGH或napFGH的缺失都会导致硝酸盐依赖生长的完全丧失。泛喹酚向NaAB的电子转移完全依赖于NapGH，而不依赖于NAPF。NAPC是泛喹酚和尿喹酚向NaAB转移电子所必需的。这一结果清楚地证明了从泛喹酚到纳巴酚的电子转移是必需的，但不是NAPF，而是NapG和H。在Ubi(+)Man(+)菌株中，由于失去NAPF而导致的生物质产量下降，这意味着NAPF在与泛喹酚的氧化相耦合的能量守恒作用中。我们认为，当电子从泛喹酚转移到NAPC时，NapG和H形成一个节能的喹酚脱氢酶，作为质子泵的组成部分或在Q循环中发挥作用。
The nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and also defective in either ubiquinone or menaquinone biosynthesis. The data reveal that Nap is coupled more effectively to menaquinol oxidation than to ubiquinol oxidation. Conversely, parallel experiments with a second set of mutants revealed that nitrate reductase A couples more effectively with ubiquinol than with menaquinol. Three further sets of strains were constructed with combinations of in frame deletions of ubiCA , menBC , napC , napF and napGH genes. NapF, NapG and NapH were shown to play no role in electron transfer from menaquinol to the NapAB complex but, in the Ubi (+) Men (-) background, deletion of napF , napGH or napFGH all resulted in total loss of nitrate-dependent growth. Electron transfer from ubiquinol to NapAB was totally dependent upon NapGH, but not on NapF. NapC was essential for electron transfer from both ubiquinol and menaquinol to NapAB. The results clearly established that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi (+) Men (+) strain implicates NapF in an energy- conserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energy- conserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC.