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Maturation and Catalysis of a Clade E Carbon Monoxide Dehydrogenase Encoded in a CooCTJ-Operon From Clostridium
Maximilian Böhm1, Henrik Land1
1Department of Chemistry - Ångström Laboratory, Uppsala University, Uppsala, Sweden.
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 (gav ≈ 1.97; g1 ≈ 2.06, g2 ≈ 1.95, g3 ≈ 1.91), characteristic of reduced B-clusters, and C-clusters at different redox levels (g3 ≈ 1.76, g3 ≈ 1.73), indicative of bound CO2 and OH. 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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