Carbon monoxide (CO), one of the critical C1 compounds in the chemical/energy industry, is an abundant and cheap source of carbon and energy^1-3. However, as an anthropogenic air pollutant in industrial flue gas and an agent of human toxicity, CO is problematic. Biocatalytic CO dehydrogenase (CODH)-based oxidation, which is capable of selective and sustainable removal of CO from waste gas mixtures and its conversion to CO₂ at room temperature, is a promisingly feasible and attractive technology for CO detoxification, carbon utilization, and energy generation from CO waste gas that includes, notably, the metal-catalyst inhibitors (ex. sulfur, chlorine, tar). Due to the permanent inactivation of CODHs under oxygen-stress conditions, their utilization for CO conversion under atmospheric conditions is challenging to say the least^4-6. Therefore, we have pursued the development of effective and stable, tunnel-redesigned CODHs that show high CO oxidation rates even in the atmosphere.

To date, the only known aerobic CODH is represented by Mo–Cu CODH enzyme⁷. On the contrary, Ni–Fe CODHs are very sensitive against O₂ except for two O₂-tolerant forms: Carboxydothermus hydrogenoformans (ChCODH-IV)⁸ and Desulfovibrio vulgaris (DvCODH)⁹. ChCODH-II shows the highest activity towards CO but is more sensitive to O₂ than ChCODH-IV. The homodimeric ChCODH-II and ChCODH-IV contain five metal clusters: the [NiFe₃S₄] cluster at the catalytic site (C-cluster), the [Fe₄S₄] cubane-type cluster (B-cluster), and the [Fe₄S₄] cluster in the dimer interface (D-cluster), which is unlike the [Fe₂S₂] D-cluster in DvCODH, implying that the catalytic mechanisms of ChCODHs are similar due to the identical metal cluster contents.

For the design of O₂-tolerant CODH, the first issue is the discovery of CODH’s key substrate tunnel regions related to oxygen transfer. Because the substrate tunnel within CODH’s structure is the only route for O₂ transfer into the deeply buried catalytic site (C-cluster). In our tunnel analysis, we could distinguish the hydrophilic tunnels in ChCODH-II by the presence of water molecules within each tunnel. Two substrate tunnels formed by abundant hydrophilic and hydrophobic residues were readily predicted in ChCODH-II. Approximately 20~30 water molecules were found only in the hydrophilic tunnel of ChCODH-II¹⁰ but not in the hydrophobic gas tunnel. In the hydrophobic tunnel, six tunnel residues (I429, L539, V552, A571, L596, V610) of ChCODH-II aligned with common xenon-coordinating residues in DvCODH¹¹ and MtCODH/acetyl-CoA synthase (ACS)¹². Among these six residues, interestingly, two residues, L596 and V610, were related to those in the hydrophilic tunnels. Moreover, the alignment of ChCODH-II with the xenon-containing structure of ChCODH-V¹³ supported this interpretation. Thus, it seemed that hydrophilic tunnels non-selectively transfer various molecules (such as water, CO, and O₂), whereas hydrophobic tunnels strictly limit water access.

The most exciting point is that the only significantly different tunnel residue was commonly located in a position of hydrophilic gas tunnels between O₂-sensitive ChCODH-II and less O₂-sensitive ChCODH-IV. All less O₂-sensitive CODHs showed a narrow tunnel radius at the position (as a bottleneck). Consequently, we could hypothesize that oxygen molecules can pass through the hydrophilic tunnel and be selectively blocked by obstruction of the common bottleneck position. Notably, the typical bottleneck of substrate tunnels is found in most Ni–Fe CODHs, including CODH/ACS complexes involved in the Wood–Ljungdahl pathway.

We aimed to design a common bottleneck of Ni‒Fe CODH gas tunnels for high O₂ tolerance, achieving up to 148-fold elevation by in-depth analysis of structural and molecular dynamics. Finally, we demonstrated CO conversion from industrial flue gases discharged from Hyundai Steel (one of the world's largest steel producers) under near-atmospheric conditions, as illustrated in the Figure above. This work reveals the A559 residue in all highly O₂-sensitive CODHs from anaerobes. Our work with various syngas mixtures (CO, CO₂, and H₂) has significant biotechnological implications in demonstrating that this single mutation may enhance CO and CO₂ bioconversion in industrial processes.

Developing a stable O₂-tolerant enzyme as a CO-transformation catalyst would be an important stepping stone to effective carbon utilization. We believe this study will provide valuable insights into that biocatalyst and its application for using carbon in real flue gas without pretreatment.

Original paper DOI: 10.1038/s41929-022-00834-y

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