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

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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.