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

固氮是氮原子进入生物圈的主要通道，是生物过程中最重要的过程之一。生物固氮是由固氮酶催化的，它通过水解ATP将氮气还原为铵离子，尽管对固氮酶本身的结构、酶学和合成有丰富的知识，但除了在基因上研究得很好的肺炎克雷伯氏菌外，对固氮酶如何提供还原力知之甚少。研究了分类上与根瘤菌相近的紫色光合细菌Rhodobacter capsulatus的电子传递途径。首先，我们有位点特异性工程改造的荚膜红球菌铁氧还蛋白I，它是固氮酶的主要电子供体。通过一系列的基因工程和纯化产物，发现这类铁氧化还原蛋白的独特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固氮所必需的R.capsulatus rnf基因。显示RnfA蛋白跨越色素细胞膜，其奇数的亲水区域暴露于周质，而RnfB和RnfC蛋白被发现在色素细胞膜的外围。细胞组分中的含量表明，这三种蛋白质相互稳定，支持了Rnf产物是膜复合物亚基的假设。最后，我们在流感嗜血杆菌，溶藻弧菌和大肠杆菌中检测到rnf基因的同源物。密切的比较表明，RnfC具有潜在的结合位点的NADH和FMN，这是在质子易位的NADH-醌氧化还原酶中发现的那些相似，RnfA，RnfD和RnfE显示类似的钠易位的NADH-醌氧化还原酶的亚基。我们预测，推定的RNF复合物代表了一个新的家庭的能量耦合NADH-氧化还原酶。

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.