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 CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, 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 (KM for CO, catalytic efficiency, product inhibition constant, catalytic bias...), and increase its resistance to the inhibitor O2, 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 O2 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 O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far.