How the [NiFe4S4] Cluster of CO Dehydrogenase Activates CO2 and NCO−

Ni,Fe‐containing CO dehydrogenases (CODHs) use a NiFe₄S₄] cluster, termed cluster C, to reversibly reduce CO₂ to CO with high turnover number. Binding to Ni and Fe activates CO₂, but current crystal structures have insufficient resolution to analyze the geometry of bound CO₂ and reveal the extent and nature of its activation. The crystal structures of CODH in complex with CO₂ and the isoelectronic inhibitor NCO^? are reported at true atomic resolution (d_min≤1.1 Å). Like CO₂, NCO^? is a μ₂,η² ligand of the cluster and acts as a mechanism‐based inhibitor. While bound CO₂ has the geometry of a carboxylate group, NCO^? is transformed into a carbamoyl group, thus indicating that both molecules undergo a formal two‐electron reduction after binding and are stabilized by substantial π backbonding. The structures reveal the combination of stable μ₂,η² coordination by Ni and Fe2 with reductive activation as the basis for both the turnover of CO₂ and inhibition by NCO^?.