Fueling CO2-fixation by detoxifying CO, what are the secrets behind the electron-bifurcating hydrogenase/formate dehydrogenase from homoacetogens?

将CO2化学还原为甲酸盐解决了我们现代社会面临的两个挑战：（1）捕获大气温室气体CO2;（2）通过使用甲酸盐作为能量载体，安全长期储存通过可再生能源（例如风能，太阳能......）产生的能量。这种对化学家来说难度很大的反应是在标准温度和压力下在微生物中不断进行的。我们的提案旨在破译甲酸脱氢酶（Fdh）催化的生物CO2还原背后的技巧，解决关键问题：酶如何通过使用低电位电子固定CO2？为此，我们将使用一种模式生物，它利用CO2固定进行碳同化和能量获取：同型产乙酸细菌。在自然环境中，它们在有机物回收和初级生产中发挥着关键作用;在生物技术中，这些"生物转化器"将废气（例如钢厂的合成气）转化为生物燃料。由于废气含有大量的一氧化碳（CO），一种已知的Fdh和氢化酶抑制剂，细胞CO2固定应该崩溃。然而，Clostridium autoethanogenum进化出一种天才的策略，通过HytA-E/Fdh复合物将有毒的CO转化为CO2还原的燃料。来自NADPH和还原铁氧还蛋白的电子，在CO解毒过程中产生，通过电子分叉/汇合，在HytA-E中合并，这是最近在厌氧菌中描述的生物化学概念。HytA-E中这种技巧的分子基础尚不清楚，但一种新型的含钨蝶呤的Fdh应该使用合并的电子来还原CO2。由于CO解毒可以饱和Fdh周转能力，该系统必须使用一种催化剂：[FeFe]-氢化酶（HytA），能够通过将质子还原为H2来排出额外的电子。在H2/CO2条件下（没有CO），HytA用来自H2的电子供给Fdh。该项目的目的是阐明复杂的不同的关键点：哪一个辅因子操作的电子confurcation/bifurcation事件？如果涉及到一个新的辅因子，它的生物合成和整合是如何进行的？氢化酶和Fdh如何相互作用以同步电子再分配？FDH如何减少二氧化碳？我们的工作流程首先在CO下培养不同的同型产乙酸菌，然后进行天然厌氧纯化和HytA-E/Fdh的结晶。结构研究将提供有关全球架构的机器，其辅因子组成，电子途径和参与CO2加氢反应的关键催化残基的见解。然而，为了获得HytA-E/Fdh的机械性质的完整图像，与SPP中的其他基团的整合和相互作用是必不可少的。通过生理学、生物物理学、光谱学、电化学和基于结构的计算进行的补充分析将证实我们的结构假设，并扩展我们对这种革命性能量转换器的看法。

The chemical reduction of CO2 into formate answers to two challenges faced by our modern society: (1) the capture of the atmospheric greenhouse gas CO2; (2) safe long-term storage of the energy generated through renewable sources (e.g. wind, solar…) by using formate as an energy-carrier. Such difficult reaction for chemists is constantly running in microorganisms under standard temperature and pressure. Our proposal aims to decipher the tricks behind this biological CO2-reduction catalyzed by formate dehydrogenase (Fdh), addressing the key question: how enzymes fix CO2 by using low-potential electrons? For this purpose, we will use a model organism that uses CO2-fixation for both, carbon assimilation and energy acquisition: the homoacetogenic bacteria. In natural environments, they play a critical role in organic matter recycling and primary production; in biotechnology, these "bio-converters" turn waste gases (e.g. syngas from steel mill) into biofuels. As waste gases contain high amounts of carbon monoxide (CO), a known inhibitor of Fdh and hydrogenases, cellular CO2-fixation should collapse. However, Clostridium autoethanogenum evolved a genius strategy by converting the poisonous CO as a fuel for the CO2-reduction through the HytA-E/Fdh complex. Electrons from NADPH and reduced ferredoxin, generated during CO­detoxification, are merged in HytA-E through electron bifurcation/confurcation, a biochemical concept recently described in anaerobes. The molecular basis of such trick in HytA-E is unknown, but a new type of tungstopterin-containing Fdh should use the merged electrons to reduce CO2. Since CO-detoxification could saturate Fdh turnover capacities, the system must use an exhauster: an [FeFe]-hydrogenase (HytA), able to evacuate extra electrons by the reduction of protons to H2. Under H2/CO2 condition (without CO), HytA feeds Fdh with electrons from H2. The project aims to elucidate the different key points of the complex: which cofactor operates the electron confurcation/bifurcation event? If a new cofactor is involved, how its biosynthesis and incorporation is performed? How hydrogenase and Fdh cross-talk to synchronize electron repartition? And how does Fdh reduce CO2? Our workflow starts by the cultivation of different homoacetogens under CO, followed by native anaerobic purification and crystallization of HytA-E/Fdh. Structural investigations will provide insights about the global architecture of the machinery, its cofactors composition, electron pathways and the key catalytic residues involved in the CO2-hydrogenation reaction. However, in order to obtain the full image of the mechanistic properties of HytA-E/Fdh, integration and interaction with the other groups among the SPP is indispensable. Complementary analyses through physiology, biophysics, spectroscopy, electro-chemistry and structure-based calculation will confirm our structural hypotheses and expand our views on this revolutionary energy converter.