A site for the binding of exogenously added carbon monoxide has been identified
at the active site of the Fe-only hydrogenase (CpI) from Clostridium
pasteurianum. The binding and inhibition of carbon monoxide have been exploited
in biochemical and spectroscopic studies to gain mechanistic insights. In the
present study, we have taken advantage of the ability to generate an
irreversibly carbon monoxide bound state of CpI. The crystallization and
structural characterization of CpI inhibited in the presence of carbon monoxide
indicates the addition of a single molecule of carbon monoxide. The ability to
generate crystals of the carbon monoxide bound state of the hydrogenase that are
isomorphous to those of the native enzyme has allowed for a direct comparison of
the crystallographic data and an unambiguous identification of the site of
carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an
Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule
in the as crystallized native state of CpI that has been previously suggested to
be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide
at this site results in an active site that is coordinately saturated with
strong ligands (S, CO, and CN), providing a rational potential mechanism for
inhibition of reversible hydrogen oxidation at the active site of CpI.
