Targeted Mutagenesis Reveals Noncanonical Genetic Requirements for Carbon Monoxide Oxidation

Carbon monoxide (CO) oxidation is a widespread microbial energy metabolism catalysed by MoCu CO dehydrogenase (CODH). Of the two phylogenetically distinct forms (form‐I and form‐II), only form‐I CODH is functionally verified. CODH activity requires maturation of the [CuSMoO 2 ] active site, linked in the carboxydotroph Afipia carboxidovorans OM5 to the genes coxDEF . CODH maturation remains untested outside of this system. Paraburkholderia terrae COX, a recently isolated carboxydovore, encodes both forms of CODH with a reduced and reorganised cox gene cluster. Using targeted mutagenesis, we identify rcoM , coxL , coxF , and coxG as essential for CO oxidation, whereas coxD, coxE , and mocA are non‐essential. Deletion of the accessory coxDEF genes produced distinct phenotypes compared to A. carboxidovorans OM5, revealing alternative routes for CODH maturation. Both coxL paralogs were deleted singly and in double knockouts, providing the first molecular evidence that form‐II CODH does not contribute to CO oxidation in vivo in these groups of bacteria. Together, these findings show that CODH maturation and function are not universally conserved and caution against inferring CO‐oxidising capacity solely from gene presence. Pb. terrae COX emerges as a genetically tractable model for elucidating the diversity and evolution of metalloenzyme assembly pathways in bacteria.