X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli

Abstract

The crystal structure of the membrane-bound O(2)-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H(2)-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O(2)-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oε of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H(2)-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O(2) to H(2)O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O(2) reduction, located near the active site.

Fig. 1.

Crystal structure of the dimeric [NiFe]-hydrogenase 1 from E. coli. The C-terminal anchor helices are not visible in the electron density maps. Electrons resulting from H₂-oxidation are transferred to cytochrome b.

Fig. 2.

(A) As-isolated EcHyd-1 proximal cluster structure. Iron positions are depicted with their corresponding anomalous difference (Δ_anom) electron density peaks (Left) contoured at the 5σ level (purple). The omit map for Glu76 is depicted in light blue contoured at 8σ (the same color codes apply to (B) and (C). A schematic representation of the two conformations for Fe₄ and Glu76 (black/gray and red) is shown on the right side of the figure. (B) H₂-reduced cluster structure. The positions of Glu76 and Fe₄ are as in O₂-sensitive [NiFe]-hydrogenases. The peaks for the iron ions and the omit map are contoured at the 10 and 20σ levels, respectively. (C) The 4-OH-1,4-naphthoquinone/ferricyanide-oxidized structure. Glu76 has the alternative positions already observed in the as-isolated structure, whereas Fe₄ is modeled with two nearby positions bound to S₁, the Sγ and amide N from Cys20 and to either the Sγ of Cys19 or one Oϵ from Glu76. In both cases the iron coordination forms a distorted tetrahedron. The iron peaks and the omit map are contoured at the 6 and 9σ levels, respectively. Hydrogens have not been included in these figures.

Fig. 3.

Schematic representation of the pattern of exchange pathways within the unusual proximal [4Fe-3S] cluster (red lines) in the PC3 state. Labels 1–4 represent Fe atoms (numbered as in Fig. 2B). There is no direct exchange interaction between Fe₃ and Fe₄. By contrast, within a standard [4Fe-4S] pseudocubane cluster, each iron ion magnetically interacts with all the others through superexchange couplings.

Fig. 4.

Molecular orbitals of the favored PC3^H model in the BS13 state: (A) SOMO α and (B) LUMO β; both belong to their respective minority spin orbital set (32). Atoms are represented according to standard color codes.

Fig. 5.

Partial water escape pathway from the active site. Light-gray semitransparent surfaces show hydrophobic cavities that may be accessible to O₂ and H₂. Small and large subunit residues are labeled in blue and black (italics), respectively. Putative waters, resulting from O₂ reduction, are labeled in red for the as-isolated structure, which is shown as a color-coded ball-and-stick model, and in green for the model corresponding to the H₂-reduced structure shown in light gray. Arrows indicate probable displacements of water molecules upon reduction. The waters labeled O1, O3, O4, O5, and OH^- are only present in the structure of the as-isolated enzyme. All the dashed lines are compatible with hydrogen-bonding interactions.