Substrate Activation at the Ni,Fe Cluster of CO Dehydrogenases: The Influence of the Protein Matrix

Carbon monoxide dehydrogenases catalyze the reversible conversion of CO, with two electrons to CO and water at a unique Ni- and Fe-containing duster (cluster C). Structural studies indicate that several highly conserved amino acids in the second coordination sphere of cluster C support the activation of the substrates, CO/CO2 and water, and may be mandatory for catalytic turnover. However, their contribution to substrate activation has been poorly explored. We replaced the three residues with potential direct interaction with the substrates (1567, H93, and K563) and one residue essential for proton/water transfer (H96) and analyzed the associated changes in the structure and reactivity of the enzyme. In addition to the expected exchange of side chains, we observed rearrangements of water molecules as well as the appearance of additional water molecules at the active site. These changes also affect the coordination of cluster C and the hydroxo ligand at Fe, with additional hydroxo/water ligands at Ni. Subsequently, we were able to convert cluster C from a [NiFe4(OH)(mu(3)-S)(4)] cluster to a [Fe-4(mu(3)-S)(4)] cluster by exchanging K563 and a primary coordinating C295. Therefore, the second coordination sphere is important not only for the affinity of the substrates but also for the stability of cluster C. Thus, beyond substrate activation, the residues in the second coordination sphere of cluster C also determine its coordination and stability.