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

二氧化碳的化学还原为甲酸盐是为了应对现代社会面临的两个挑战：(1)捕获大气中的温室气体二氧化碳；(2)长期安全地储存通过可再生能源(如风能、太阳能…)产生的能量。通过使用甲酸盐作为能量载体。对于化学家来说，这种困难的反应是在标准温度和压力下不断在微生物中进行的。我们的提案旨在破译甲酸脱氢酶(FDH)催化的这种生物二氧化碳还原背后的诀窍，解决关键问题：酶如何通过使用低势电子来固定二氧化碳？为此，我们将使用一种模式生物，它同时使用二氧化碳固定来进行碳同化和能量获取：同乙酰菌。在自然环境中，它们在有机物循环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(例如来自钢铁厂的合成气)转化为生物燃料。由于废气中含有大量的一氧化碳(CO)，一种已知的FDH和氢酶抑制剂，细胞对二氧化碳的固定应该会崩溃。然而，自产乙酸梭菌通过HYTA-E/FDH复合体将有毒的CO转化为二氧化碳还原的燃料，从而进化出一种天才的策略。来自NADPH的电子和还原的铁氧还蛋白在共解毒过程中通过电子分叉/聚合作用合并到HYTA-E中，这是最近在厌氧菌中描述的一个生化概念。HYTA-E中这种技巧的分子基础尚不清楚，但一种新型的含有钨的FDH应该会利用合并的电子来还原二氧化碳。由于CO解毒可能会饱和FDH的周转能力，系统必须使用一个排气器：AN[FeFe]-氢酶(HYTA)，能够通过将质子还原为H2来疏散额外的电子。在H2/CO2条件下(无CO)，HYTA从H2向FDH提供电子。该项目旨在阐明该复合体的不同关键点：哪个辅助因子操作电子分叉/分叉事件？如果涉及到一种新的辅因子，它的生物合成和整合是如何进行的？氢酶和脱氢酶如何相互作用使电子重新分配同步？外佣是如何减少二氧化碳排放的？我们的工作流程首先是在CO下培养不同的高乙酸菌，然后是HYTA-E/FDH的自然厌氧纯化和结晶。结构研究将提供关于该机制的全球架构、其辅因子组成、电子路径以及参与二氧化碳加氢反应的关键催化残基的见解。然而，为了全面了解HYTA-E/FDH的力学性质，SPP中与其他基团的整合和互动是必不可少的。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互补分析，将证实我们的结构假设，并扩大我们对这一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.