Entangled hydrophobic and hydrophilic networks in carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase (CODH) is a crucial enzyme involved in the global carbon cycle and fixation by catalysing the reversible interconversion of CO₂ and CO. Although major structural features of the enzyme are known, key connections between the structure and function remain unclear. Here, we employ large-scale unconstrained all-atom molecular dynamics simulations to investigate the key catalytic features of CODH, including (i) the implications of CODH dimerization, (ii) substrate and water transport cavities, (iii) the impact of global and local hydration, and (iv) protonation-dependent local solvation around metallocofactors. The results show that dimerization involves strong interlocking near the metal clusters, with free energy analysis highlighting the role of inter-monomer hydrogen bonding. Importantly, cavity and channel analysis reveals parallel hydrophilic and hydrophobic pathways, which enable efficient CO₂/CO and water transport to the catalytic site. This likely reflects evolutionary optimization of spatially intertwined substrate access and hydration. Enhanced sampling nearly doubled enzyme hydration relative to the crystallographic model, especially near the C-clusters, while His96, Ile567, and Ser525 rearrangements govern distinct hydration pathways around the active site. Furthermore, the protonation state of His93 controls the catalytically essential water network. Our study provides an atomistic view of CODH as a finely tuned molecular nanomachine, in which entangled hydrophilic and hydrophobic networks coordinate substrate and water delivery to the C-cluster, while key protein-matrix residues act as strategic gatekeepers. Collectively, these findings reveal how dimerization, hydration, and local protein matrix cooperatively regulate critical aspects of CODH function.