Binding of Exogenously Added Carbon Monoxide at the Active Site of the Iron-Only Hydrogenase (CpI) from Clostridium pasteurianum^,

Abstract

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.

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

“…In either of these two mechanisms, it is likely that O 2 attacks the H-cluster in the oxidation level H ox that is assigned as [4Fe-4S] 2ϩ -Fe p (I)Fe d (II). This proposal is based on analogy with observations made with CO, which is known to bind preferentially to H ox relative to H red (giving a characteristic EPRdetectable form known as H ox -CO) (14,17) The observation that CO reacts so much faster than O 2 leads us to question the effectiveness of a 'gas filter' within the enzyme and to suggest instead that more effective gas discrimination occurs in the highly specific region of the active site. In the close proximity of the H-cluster, electronic and steric influences of the protein environment are likely to determine the efficiency of ligand binding.…”

“…First, exogenous CO, known to bind at the distal Fe site (17) and be a competitive inhibitor of H 2 oxidation (21), protects the enzyme against inactivation by O 2 . Second, the rate of inactivation by O 2 is lower at high H 2 levels, in line with the competitive relationship between CO and H 2 .…”

“…Diminished Fe-Fe interactions from the cubane cluster in the H ox air spectrum allow for discrimination of peak III even in FT spectra calculated from a k-range starting at lower values. Contributions to the EXAFS spectra from multiple-scattering (MS) effects of the near-linear Fe-C ϭ O/N arrangements are small (25) (14,17,24,25). In particular, the Fe-Fe distances of 2.52 Å for the 2Fe H moiety (III, inset in Fig.…”

“…The distal Fe is also coordinated by one CO and one CN Ϫ ligand, and an additional binding site is vacant (9) or occupied by an exchangeable O ligand, most likely a water molecule (11). In the structure of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, which is believed to be crystallized in the H red form, the bridging CO is replaced by a terminal CO on Fe d (13 (16)(17)(18). Carbon monoxide is a -acceptor ligand and binds to electron-rich transition metals (19).…”

“…From a molecular orbital perspective, H 2 resembles CO because, by analogy with the Dewar-Chatt-Duncanson model for binding of alkenes to metals (20), back donation of electron density into the antibonding -orbital of molecular H 2 is important for its binding and activation (20). These facts are highly relevant because they form the basis for CO being competitive with H 2 (21) during H 2 oxidation, and therefore, Fe d is likely to be the site for H 2 binding (17). Like CO, O 2 is also a -acceptor ligand and likely to bind to the same site(s); we can anticipate that any such binding could result in the generation of highly reactive oxygen intermediates (22,23).…”