Electron-bifurcating enzymes in the energy metabolism of the model acetogen, Acetobacterium woodii

模型产乙酸菌伍氏醋杆菌能量代谢中的电子分叉酶

• 批准号: 221575835
• 负责人: Professor Dr. Volker Müller
• 金额: --
• 依托单位: Arbeitskreis Molekulare Mikrobiologie und Bioenergetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/221575835?language=en
• 关键词: Electron; bifurcating; enzymes; energy; metabolism

Up to the year 2008, only two mechanisms to drive endergonic cellular reactions were known. Cytoplasmic reactions are usually driven by hydrolysis of ATP (or other nucleotides) and membrane-bound processes by the electrochemical ion potential across the membrane. In 2008, a third mechanism was discovered: soluble enzymes that drive endergonic redox reactions by coupling to an exergonic redox reaction. Usually one electron donor, but two different electron acceptors are used. The electrons coming from the donor are bifurcated: one goes energetically „downhill“ and this provides the energy for the other electron going energetically „uphill“. Bifurcation of electrons usually requires a flavin, and therefore this process is also referred to as flavin-based electron bifurcation (FBEB). The discovery of FBEB has changed our understanding of anaerobic metabolism fundamentally. It does not only increase the energetic efficiency of anaerobic metabolism in general but in particular allows autotrophic growth of acetogenic bacteria, an ecophysiological and evolutionary important group of strictly anaerobic bacteria, on H2+CO2. The pathway involved is considered „ancient“ and maybe one of the oldest on earth since it is the only one that combines carbon dioxide fixation with the synthesis of ATP. Therefore, FBEB may be an ancient mechanism to overcome energetic barriers in metabolism. The acetogenic bacterium Acetobacterium woodii conserves energy by carbonate respiration (acetogenesis) or caffeate respiration. The respiratory chain is identical in both cases and consists of a ferredoxin-NAD:oxidoreductase (Rnf) and an ATP synthase that are coupled via a transmembrane electrochemical Na+ gradient. Reduction of ferredoxin with H2 as electron donor is highly endergonic. In the first funding period we have isolated and characterized a tetrameric, electron bifurcating hydrogenase that overcomes the barrier by FBEB and reduces ferredoxin and NAD simultaneously; mutagenesis experiments revealed that this hydrogenase is essential for autotrophy. During caffeate respiration, there is an additional electron-bifurcating enzyme, the caffeyl-CoA reductase/Etf complex that couples caffeyl-CoA reduction with NADH as electron donor to simultaneous ferredoxin reduction. The caffeyl-CoA reductase/Etf complex was purified and characterized, its 3D structure was solved and enabled first steps into a structure-based analysis of electron flow and energy coupling by site-directed mutagenesis. The lactate dehydrogenase/Etf complex is the third electron-bifurcating enzyme discovered; it enables A. woodii to grow on lactate. Since the encoding genes are widespread in bacteria, this seems to be a common way of lactate oxidation in anaerobes. In the next funding period, we will built on these discoveries and unravel electron flow and energetic coupling in these enzymes as well as study their physiological importance by genetic means.

直到2008年，已知只有两种机制驱动吸能细胞反应。细胞质反应通常由ATP（或其他核苷酸）的水解和跨膜电化学离子电位的膜结合过程驱动。2008年，第三种机制被发现：可溶性酶通过耦合到放能氧化还原反应来驱动放能氧化还原反应。通常使用一种电子供体，但使用两种不同的电子受体。来自供体的电子被分叉：一个能量“下坡”，这为另一个能量“上坡”的电子提供能量。电子的分叉通常需要黄素，因此这个过程也被称为黄素基电子分叉（FBEB）。FBEB的发现从根本上改变了我们对无氧代谢的认识。它不仅提高了一般厌氧代谢的能量效率，而且特别允许产乙酸细菌（严格厌氧细菌的生态生理学和进化的重要群体）在H2+CO2上的自养生长。这条途径被认为是“古老的”，可能是地球上最古老的途径之一，因为它是唯一一条将二氧化碳固定与ATP合成相结合的途径。因此，FBEB可能是一种古老的克服能量代谢障碍的机制。产乙酸细菌伍氏醋酸杆菌通过碳酸盐呼吸（产乙酸）或咖啡酸呼吸保存能量。呼吸链在两种情况下是相同的，并且由铁氧还蛋白-NAD：氧化还原酶（Rnf）和ATP合酶组成，所述铁氧还蛋白-NAD：氧化还原酶（Rnf）和ATP合酶通过跨膜电化学Na+梯度偶联。用H2作为电子供体还原铁氧还蛋白是高度吸能的。在第一个资助期内，我们已经分离和表征了一种四聚体的电子分叉氢化酶，它克服了FBEB的障碍，同时减少了铁氧还蛋白和NAD;诱变实验表明，这种氢化酶对自养是必不可少的。在咖啡酸呼吸过程中，还有一种额外的电子分叉酶，咖啡酰辅酶A还原酶/Etf复合物，它将咖啡酰辅酶A还原与作为电子供体的NADH结合起来，同时还原铁氧还蛋白。咖啡酰-CoA还原酶/Etf复合物进行了纯化和表征，其3D结构得到解决，并通过定点诱变实现了基于结构的电子流和能量耦合分析的第一步。乳酸脱氢酶/Etf复合物是发现的第三种电子分叉酶，它使A.伍迪在乳酸盐上生长。由于编码基因在细菌中广泛存在，这似乎是厌氧菌中乳酸氧化的常见方式。在下一个资助期内，我们将在这些发现的基础上，解开这些酶中的电子流和能量耦合，并通过遗传手段研究它们的生理重要性。