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Carbon monoxide dehydrogenases (CODHs) are nickel-dependent metalloenzymes that catalyse the reversible interconversion of CO₂ and CO, making them promising biocatalysts for carbon capture and utilisation (CCU) technologies. Despite the immense phylogenetic diversity of CODHs — distributed across eight clades (A–H) in anaerobic bacteria and archaea — biochemical characterisation has been heavily biased towards clades A, E, and F, leaving the functional landscape of the remaining clades largely unexplored.

This thesis investigates CODH diversity and catalytic potential from three complementary angles: bioinformatic exploration of sequence space, biochemical characterisation of underexplored clades, and biotechnological application of CODH in engineered systems.

In Paper I, a large-scale genomic context analysis of the CODH sequence space revealed distinct operon compositions and co-occurrence trends across clades, suggesting that clades A, E, and F are the most likely to harbour efficient CO₂ reduction catalysts, while clades B, C, and D are less likely to do so. Building on these findings, Paper II presents the first biochemical and structural characterisation of a clade B CODH (Ruminococcus flavefaciens CODH; RfCODH), solved by anaerobic cryo-EM and characterised by EPR spectroscopy. RfCODH was found to be incapable of CO₂ reduction, a phenotype rationalised by atypical features of its proton transfer pathway and gas channel architecture, and by its apparent functional association with an ABC transporter system. Paper III describes the characterisation of a clade E CODH from Clostridium pasteurianum BC1 (Cp^BC1CODH-III) that carries a clade F-type operon composition, including a CooCTJ maturation cluster. Notably, this enzyme is catalytically active towards CO–CO₂ interconversion when expressed without its apparent maturation machinery, representing a rare self-sufficient CODH.

To explore the evolutionary plasticity of CODH, Paper IV employs ancestral sequence reconstruction (ASR) combined with directed evolution. Reconstructed ancestral CODHs were found to be more tolerant of mutational changes while maintaining catalytic function compared to extant enzymes, establishing a foundation for future engineering of improved CO₂ reduction catalysts. Finally, Paper V demonstrates the assembly of a modular photobiohybrid catalyst in which CODH is coupled to light-harvesting small organic molecule nanoparticles (Mdots). Surface charge tuning of the Mdots was shown to be critical for productive bioassembly and photocatalytic CO₂ reduction performance.

Collectively, this thesis expands the functional and structural understanding of CODH diversity, identifies key determinants of CO₂ reduction competence, and demonstrates pathways towards biotechnological exploitation of these ancient enzymes for sustainable carbon management.