In view of the depletion of fossil fuel reserves and climatic effects of greenhouse gas emissions, Ni,Fe-containing carbon monoxide dehydrogenase (Ni-CODH) enzymes have attracted increasing interest in recent years for their capability to selectively catalyze the reversible reduction of CO to CO (CO + 2H + 2e CO + HO). The possibility of converting the greenhouse gas CO into useful materials that can be used as synthetic building blocks or, remarkably, as carbon fuels makes Ni-CODH a very promising target for reverse-engineering studies. In this context, in order to provide insights into the chemical principles underlying the biological catalysis of CO activation and reduction, quantum mechanics calculations have been carried out in the framework of density functional theory (DFT) on different-sized models of the Ni-CODH active site. With the aim of uncovering which stereoelectronic properties of the active site (known as the C-cluster) are crucial for the efficient binding and release of CO, different coordination modes of CO to different forms and redox states of the C-cluster have been investigated. The results obtained from this study highlight the key role of the protein environment in tuning the reactivity and the geometry of the C-cluster. In particular, the protonation state of His93 is found to be crucial for promoting the binding or the dissociation of CO. The oxidation state of the C-cluster is also shown to be critical. CO binds to C according to a dissociative mechanism (i.e., CO binds to the C-cluster after the release of possible ligands from Fe) when His93 is doubly protonated. CO can also bind noncatalytically to C according to an associative mechanism (i.e., CO binding is preceded by the binding of HO to Fe). Conversely, CO dissociates when His93 is singly protonated and the C-cluster is oxidized at least to the C redox state.