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BIOCHEMICAL GENETIC ANALYSIS OF THE LINKAGEFACTOR OF PHOTOSYNTHETIS AND NITROGEN FIXATION IN PURPLE BACTERIA

紫色细菌光合作用与固氮连锁因子的生化遗传学分析

基本信息

批准号：
07640862
负责人：
SAEKI Kazuhiko
金额：
$ 1.34万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640862/
关键词：
RHODOBACTER FERREDOXIN ELECTRON-TRANSPORT NITROGEN-FIXATION NADH MEMBANE-PROTEIN-COMPLEX

项目摘要

Nitrogen fixation is one of the most important biological processes, because it is the main gateway of nitrogen atom in biosphere. Biological nitrogen fixation is catalyzed by nitrogen enzyme which reduce nitrogen gas to ammonium ion with hydrolysis of ATP.Despite the wealth of knowledge on structure, enzymology and synthesis of nitrogenase itself, little is known about how reducing power is supplied to the enzyme except in the genetically well studied bacterium Klebsiella pneumonia. We have studied the electron transport pathway to nitrogenase in the purple photosynthetic bacterium Rhodobacter capsulatus which is taxonomically close to rhizobia. First, we have site-specifically engineered R.capsulatus ferredoxin I that is the primary electron donor to nitrogenase. With series of engineered genes and a purified products, the unique structural feature of this group of ferredoxins were related to their extremely low redox potential. Second, we analyzed the R.capsulatus rnf genes that are essential for nitrogen fixation under illuminated conditions. RnfA protein was shown to span the chromatophore membrane with its odd-numbered hydrophilic regions exposed to periplasm, whereas RnfB and RnfC proteins were revealed to situate at the periphery of the chromatophore membranes. The contents in cellular fractions indicated that the three proteins stabilize each other, supporting a hypothesis that the Rnf products are subunits of a membrane complex. Finally, we detected homologs of rnf genes in Haemophilus influenzae, Vibrio alginolyticus and E.coli. Close comparisons revealed that RnfC has potential binding sites for NADH and FMN which are similar to those found in proton-translocating NADH-quinone oxidoreductases and that RnfA,RnfD and RnfE show similarity to subunits of sodium-translocating NADH-quinone oxidoreductases. We predict that the putative Rnf complex represents a novel family of energy-coupling NADH-oxidoreductases.

固氮是生物圈中最重要的生物过程之一，是氮原子进入生物圈的主要通道。生物固氮是由氮酶催化的，它通过ATP的水解将氮气还原为铵离子。虽然人们对固氮酶的结构、酶学和自身合成有丰富的知识，但除了基因研究很好的肺炎克雷伯菌外，人们对固氮酶是如何提供还原动力的知之甚少。我们研究了在分类学上与根瘤菌相近的紫色光合细菌Rhodbacter Capsatus中固氮酶的电子传递途径。首先，我们有定点工程的包裹体铁氧还蛋白I，它是固氮酶的主要电子供体。这组铁氧化还蛋白具有一系列工程基因和一系列纯化产物，其独特的结构特征与其极低的氧化还原电位有关。其次，我们分析了在光照条件下对固氮起重要作用的胶囊核糖体rnf基因。RnfA蛋白横跨着着色体膜，其奇数亲水区域暴露在周质中，而RnfB和RnfC蛋白则位于着色体膜的外围。细胞组分中的含量表明这三种蛋白质相互稳定，支持RNF产物是膜复合体的亚单位的假设。最后，我们检测了流感嗜血杆菌、溶藻弧菌和大肠杆菌中rnf基因的同源性。进一步的比较表明，RnfC与NADH和FMN具有潜在的结合位点，与质子转运型NADH-醌氧化还原酶相似，RnfA、RnfD和RnfE与钠转运型NADH-QOX还原酶的亚基相似。我们预测，假定的RNF复合体代表了一个新的能量偶联NADH氧化还原酶家族。