Beyond Canonical CO Oxidation: Structural and Evolutionary Insights Into a Non‐Canonical Carbon Monoxide Dehydrogenase

ABSTRACT Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO 2 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 biochemical characterisation of a clade B CODH from Ruminococcus flavefaciens ( Rf CODH). 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 Rf CODH 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 Rf CODH's severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well‐studied Ch CODH‐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.