Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum

A Site for the binding of exogenously added carbon monoxide has been identi fied at the active site of the Fe-only hydrogenase (CpI) fi om Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been explo ited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate a n irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon mono xide indicates the addition of a single molecule of carbon monoxide. The ab ility to generate crystals of the carbon monoxide bound state of the hydrog enase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identific ation of the site of carbon monoxide binding at the active site of CpI. Car bon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a ter minally bound water molecule in the as crystallized native state of CpI tha t has been previously suggested to be a potential site of reversible hydrog en oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), pr oviding a rational potential mechanism for inhibition of reversible hydroge n oxidation at the active site of CpI.