2026/02/15 by Maximilian Böhm, Vivek Srinivas, Benjamin Wiseman +4 · 1 voice
Biochemistry, Genetics and Molecular Biology · Energy · #Hemoglobin structure and function #Metalloenzymes and iron-sulfur proteins #Microbial metabolism and enzyme function
paper · pdf · doi:10.26434/chemrxiv.15000083/v1
openalex publication_date 2026/02/15 · openalex created_date 2026/02/17 · openalex updated_date 2026/07/14
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 ( 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.