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Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO₂ and play central roles in microbial carbon metabolism. While well-characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here we present the first structural and functional characterisation of a clade B CODH from Ruminococcus flavefaciens (RfCODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo-EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å RfCODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for RfCODH’s severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well-studied ChCODH-II). EPR spectroscopy reveals unique oxidised C-cluster states not previously characterised in CODHs. Mirror tree analysis hints to co-evolution between clade B CODHs and associated ABC transporter substrate-binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO₂ fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III (Cp^BC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. Cp^BC1CODH-III shows moderate CO oxidation (150 U/mg) and CO₂ reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g ≈ 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g ≈ 1.75, g ≈ 1.72), indicative of a bound CO₂. Investigation of maturation effects showed that co-expression of CooCTJ stabilised Cp^BC1CODH-III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well-studied clade F Rhodospirillum rubrum CODH (RrCODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal-binding regions identified via AlphaFold3 modelling and dot plot analysis. Cp^BC1CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity.

The enzymatic interconversion of CO₂ and CO by nickel-iron carbon monoxide dehydrogenases ([NiFe]-CODHs) offers a biocatalytic route to valorize CO₂ into useful chemical feedstocks. However, all characterized CODHs display a pronounced catalytic bias toward CO oxidation, limiting their utility for electrochemical or biological CO₂ reduction. To overcome the constraints imposed by evolutionary specialization, we combined ancestral sequence reconstruction (ASR) with directed evolution to access more promiscuous CODH scaffolds with enhanced evolvability. A phylogenetic analysis of 6,543 CODH sequences was used to reconstruct the common ancestor of clade F (CODHASR0), which shares 63% sequence identity with the well-characterized extant Carboxydothermus hydrogenoformans CODH-II (ChCODH-II). A consensus variant (CODHCON) was additionally generated to decouple the contributions of ancestral reconstruction from consensus-driven thermostabilization. Both ancestral variants displayed markedly reduced catalytic bias toward CO oxidation (5.25- and 2.6-fold, respectively) compared to ChCODH-II (228-fold), consistent with greater catalytic generalism. Error-prone PCR libraries of four CODH variants — CODHASR0, CODHCON, ChCODH-II, and Rhodospirillum rubrum CODH (RrCODH) — were screened for enhanced CO production under anaerobic conditions. Both ancestral variants showed superior evolvability and mutation tolerance relative to ChCODH-II, with CODHCON outperforming CODHASR0, suggesting that consensus sequence generation represents a practical and accessible alternative to full ASR for identifying evolvable starting scaffolds. These findings establish ancestral and consensus sequence reconstruction as a promising framework for engineering biocatalysts under non-native evolutionary pressures. Work in progress.

Photobiocatalytic CO2 reduction represents an attractive approach for conversion of solar light and abundant resources to value-added chemicals. However, the design of suitable systems requires a detailed understanding of the interaction between the artificial photosensitizer and biocatalyst interface. In this work, we investigate the effect of surfactant charge utilized in the preparation of a phenoxazine-based organic molecule nanorod photosensitizer on the interaction with the carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans within biohybrid assemblies for sacrificially driven photobiocatalytic CO2 reduction into CO. Electrophoretic mobility shift assay in conjunction with cryogenic electron microscopy (Cryo-EM) and detailed physicochemical characterization are conducted to understand the interaction at the biohybrid interface in order to suggest a strategy for future functionalization of nanoparticles that fulfills the needs of the biocatalyst for green fuel production.