Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase

Abstract

The tungsten-iron-sulfur enzyme acetylene hydratase stands out from its class because it catalyzes a nonredox reaction, the hydration of acetylene to acetaldehyde. Sequence comparisons group the protein into the dimethyl sulfoxide reductase family, and it contains a bis-molybdopterin guanine dinucleotide-ligated tungsten atom and a cubane-type [4Fe:4S] cluster. The crystal structure of acetylene hydratase at 1.26 A now shows that the tungsten center binds a water molecule that is activated by an adjacent aspartate residue, enabling it to attack acetylene bound in a distinct, hydrophobic pocket. This mechanism requires a strong shift of pK(a) of the aspartate, caused by a nearby low-potential [4Fe:4S] cluster. To access this previously unrecognized W-Asp active site, the protein evolved a new substrate channel distant from where it is found in other molybdenum and tungsten enzymes.

Fig. 1.

Overall structure of acetylene hydratase from P. acetylenicus. The stereo representation shows an orientation viewing down the active site channel as seen in Fig. 4A.

Fig. 2.

Domain structure and active site access. (A) Schematic view of the four-domain structure typically found in members of the DMSO reductase family. All members, with the exception of AH, have an active site access pathway along the red cone. AH uses the black cone, entering at the intersection of domains I, II, and III. (B) Stereoview of a superimposition of AH (black) and periplasmic nitrate reductase (red) (5), with exit pathways calculated by CAVER (24). The W atom of AH (blue) and the Mo atom of nitrate reductase (magenta) are shown as spheres.

Fig. 3.

Cofactors and active site of AH. (A) The tungsten atom (blue) is coordinated by the dithiolene groups of both MGD cofactors and the side chain of Cys-141. A water molecule completes the slightly distorted octahedral geometry. This water is also hydrogen-bonded to Asp-13, a residue adjacent to the [4Fe:4S] cluster ligand Cys-12. (B) Above the bound water molecule, a ring of hydrophobic residues forms the bottom of the active site access channel (see Fig. 4).

Fig. 4.

Substrate channel and model for acetylene binding. (A) Surface representation of acetylene hydratase with the substrate access pathway (boxed). The enlarged area (upper box) shows a binding pocket that exactly fits the dimensions of an acetylene molecule (lower box). (B) The binding pocket positions an acetylene molecule directly above the water molecule and Asp-13. (C) Bond distances of 2.04 Å to W and 2.41 Å to the O_δ1 atom of Asp-13 indicate a highly activated water molecule positioned right below a binding pocket for acetylene.