The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii
The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii
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DOI：
10.1128/jb.00357-18
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发表时间：
2018-08
影响因子：
3.2
通讯作者：
Lars Westphal;A. Wiechmann;Jonathan P. Baker;N. Minton;V. Müller
中科院分区：
文献类型：
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作者：
Lars Westphal;A. Wiechmann;Jonathan P. Baker;N. Minton;V. Müller
铁氧还蛋白和NAD+是厌氧细菌的关键电子载体，但在能量上，它们并不等同，因为铁氧还蛋白的氧化还原电位低于NADH/NAD+对。通过对woodii乙杆菌的突变体研究，我们发现Rnf的主要功能是将铁氧还蛋白和NAD+的细胞池强力连接起来。当铁氧还蛋白大于NADH时，从铁氧还蛋白到NAD+的逸出电子流产生化学渗透势。这对自养生长过程中的能量节约是必不可少的。当NADH大于铁氧还蛋白时，Rnf起相反的作用。这个反应对于在低能底物上生长提供还原的铁氧还蛋白是必不可少的，这是生物合成和二氧化碳还原必不可少的。我们的研究为广泛存在于细菌中的膜结合离子转运Rnf复合体的细胞功能提供了新的视角。Rnf复合物是一种呼吸酶，可催化还原的铁氧还蛋白氧化为NAD+的还原，该反应的负自由能变化用于产生跨膜离子梯度。在一类厌氧产乙细菌中，Rnf复合体被认为是能量节约和自养生长所必需的。我们在这里描述了一种对这类模型细菌——伍迪醋酸杆菌进行无标记诱变的方法，这种方法使我们能够删除rnf基因并测试它们在体内的作用。rnf突变体不能在H2 + CO2上生长，也不能从H2 + CO2中产生乙酸或ATP，并且铁氧还蛋白：NAD+氧化还原酶活性和Na+易位也完全丧失，这支持了rnf复合物是这种代谢中唯一的呼吸酶的假设。出乎意料的是，突变体也不能在低能基质上生长，如乙醇或乳酸。这些底物的氧化不耦合于铁氧还蛋白的还原，而只耦合于NAD+，我们推测生长表型是由还原的铁氧还蛋白的损失引起的，而还原铁氧还蛋白是生物合成和二氧化碳还原所必需的。伍迪的电子分叉氢化酶减少了铁氧还蛋白，事实上，向培养物中添加H2可以恢复乙醇和乳酸的生长。这与NADH的氧还原是由Rnf复合物催化的反向电子传递驱动的假设是一致的，这使得Rnf复合物对于低能底物的生长也是必不可少的。铁氧还蛋白和NAD+是厌氧细菌的关键电子载体，但在能量上，它们并不等同，因为铁氧还蛋白的氧化还原电位低于NADH/NAD+对。通过对woodii乙杆菌的突变体研究，我们发现Rnf的主要功能是将铁氧还蛋白和NAD+的细胞池强力连接起来。当铁氧还蛋白大于NADH时，从铁氧还蛋白到NAD+的逸出电子流产生化学渗透势。这对自养生长过程中的能量节约是必不可少的。当NADH大于铁氧还蛋白时，Rnf起相反的作用。这个反应对于在低能底物上生长提供还原的铁氧还蛋白是必不可少的，这是生物合成和二氧化碳还原必不可少的。我们的研究为广泛存在于细菌中的膜结合离子转运Rnf复合体的细胞功能提供了新的视角。
Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+. When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria. ABSTRACT The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates. IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+. When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.