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bioRxiv · 10.64898/2026.03.10.710785

Self-Sufficient Maturation and Catalysis of a Clade E CODH Encoded in a CooCTJ-Operon from Clostridium pasteurianum BC1

Böhm, M.·Land, H.
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Abstract

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 (CpBC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. CpBC1CODH-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 {approx} 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g {approx} 1.75, g {approx} 1.72), indicative of a bound CO2. Investigation of maturation effects showed that co-expression of CooCTJ stabilised CpBC1CODH-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. CpBC1CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity

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Böhm, M., Land, H.. 2026-03-12. Self-Sufficient Maturation and Catalysis of a Clade E CODH Encoded in a CooCTJ-Operon from Clostridium pasteurianum BC1. https://doi.org/10.64898/2026.03.10.710785

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