Maturation and Catalysis of a Clade E Carbon Monoxide Dehydrogenase Encoded in a CooCTJ‐Operon From <i>Clostridium pasteurianum</i> BC1

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO 2 fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH‐III ( Cp BC1 CODH‐III), from the phylogenetic clade E, co‐expressed with its maturation machinery CooCTJ. Cp BC1 CODH‐III shows moderate CO oxidation (150 U/mg) and CO 2 reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal (g av ≈ 1.97; g 1 ≈ 2.06, g 2 ≈ 1.95, g 3 ≈ 1.91), characteristic of reduced B‐clusters, and C‐clusters at different redox levels (g 3 ≈ 1.76, g 3 ≈ 1.73), indicative of bound CO 2 and OH. Investigation of maturation effects showed that co‐expression of CooCTJ stabilised Cp BC1 CODH‐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 ( Rr CODH) 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 BC1 CODH‐III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity.