Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study. We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis. We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.

中文翻译：

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同源生产，一步纯化，和Na的证明⁺运输通过从RNF复杂醋酸杆菌伍氏醋酸杆菌，用于向生物燃料C1基板的产乙酸转换的模型

能量载体氢以及温室气体二氧化碳的捕获和储存是人类当前面临的两个主要问题。已经开发出化学催化剂，但是直到最近，一组将氢和二氧化碳转化为乙酸盐，甲酸盐或生物燃料（如乙醇）的厌氧细菌才是产乙酸细菌。这些生物催化剂以最高的速率从H2 + CO2甚至一氧化碳中产生液态有机氢载体甲酸。自养，氢氧化和减少CO2的乙酸原通常具有专门的新陈代谢，以催化CO2的减少，即Wood-Ljungdahl途径（WLP）。WLP不会产生净ATP，但会与膜结合的呼吸链相连，从而使ATP合成与CO2固定耦合。自发现这些细菌以来，呼吸酶的性质一直是一个谜，在本研究中尚未阐明。我们从模型产乙酸木杆菌中得到了铁氧还蛋白：NAD氧化还原酶（Rnf复合物）的His标记变体，从细胞质膜中溶解了该酶，并通过Ni2 + –NTA亲和层析纯化。将该酶掺入人工脂质体中，并催化Na +转运与铁氧还蛋白依赖性NAD还原反应。我们使用纯化的酶得到的结果不仅证实了木桐的Rnf复合物是Na +依赖性的，而且还首次证明了这种膜包埋的分子引擎在木桐的膜上产生了Na +梯度，从而可以用于ATP合成。我们提出了Rnf复合物的同源生产和纯化的协议。该酶催化电子转移驱动的Na +出口，因此，我们的研究提供了人们期待已久的生化证据，证明Rnf复合物是一种呼吸酶。