Abstract
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 at g ≈ 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g ≈ 1.75, g ≈ 1.72), indicative of a bound CO 2 . 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
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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 ≈ 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g ≈ 1.75, g ≈ 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
Competing Interest Statement
The authors have declared no competing interest.
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