Investigating the native functions of [NiFe]-carbon monoxide dehydrogenases through genomic context analysis

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO2, representing key targets for biocatalytic CO2 reduction. Despite dramatic differences in catalytic rates and O2 tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1,376 CODH and 1,545 hybrid cluster protein sequences. Around ∼30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E, F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests environmental interdependency. These findings establish clades A, E, F as primary biocatalyst targets while defining regulatory functions for clades C, D, providing a genomics framework for predicting CODH phenotypes.