A conserved isoleucine gates the diffusion of small ligands to the active site of NiFe CO-dehydrogenase

Abstract CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO 2 , at a buried NiFe 4 S 4 active site. The substrates, CO and CO 2 , need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. We used site-directed mutagenesis in an extensive manner to study the role of the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Certain substitutions significantly change the biochemical properties of the enzyme (K M for CO, catalytic efficiency, product inhibition constant, catalytic bias…), and increase its resistance to the inhibitor O 2 , showing that isoleucine 563 plays a key role in determining access to the active site. The mutations have the same effects on the rates of binding of CO and O 2 showing that the two molecules follow the same pathway and are not discriminated by the protein matrix. The I563F mutation decreases the bimolecular rate constant of inhibition by O 2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O 2 resistance reported so far.