Living on Acetylene. A Primordial Energy Source - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 23

harbors the [4Fe-4S] cluster, ligated by the four cysteine residues Cys9, Cys12,
Cys16, and Cys46. Domain II (residues 65-136 and 393-542) and III (residues
137-327) have an fold with homologies to the NAD-binding fold in dehydro-

genases. Each of these two domains provides hydrogen bonds, needed to bind one
of the MGD cofactors. The interactions are mainly provided by variable loop
regions at the C-termini of the strands of a parallel

ʱʲʱ

ʲ

-sheet. The coordination of
both of the MGD cofactors is completed by domain IV (residues 590-730), which
consists mainly of a seven stranded

-barrel fold [ 21 ].
The overall tertiary structure of acetylene hydratase and the position of the
cofactors, with the two MGDs (P MGD and Q MGD ) in an elongated conformation
and the [4Fe-4S] cluster close to the Q MGD is similar to all other structures of
members of the DMSO reductase published so far [ 21 ]. However, the access from
the surface of the protein towards the putative active site, consisting of the tungsten
ion ligated by the two MGDs with the iron-sulfur cluster in close proximity is
unique for an enzyme of the DMSO reductase family. In all structures of DMSO
reductase family enzymes published so far, the access funnel starts at the pseudo
twofold axis between domain II and III. A shift in the loop region of the residues
327-335 towards the surface of the protein and further rearrangements of the
residues 336-393 seal this entrance point in AH. In other enzymes (e.g., nitrate
and formate reductases), this loop region separates the [4Fe-4S] cluster from
the Mo/W site. In AH, the shift of this loop opens a new access funnel towards
the central tungsten ion at the intersection of domains I, II, and III, allowing the
substrate to reach the central tungsten ion from a totally different direction than in
other enzymes of the DMSO reductase family [ 21 ].

ʲ

4.5 Active Site Setup

While the overall fold of AH is quite similar to other enzymes of the DMSO
reductase family, major rearrangements are found at the active site [ 21 ]. The
tungsten center in its reduced W(IV) state is coordinated by the four sulfur atoms
of the dithiolene moieties of the P MGD and Q MGD cofactors and by one sulfur atom
of a cysteine residue (Cys141), as found in the dissimilatory nitrate reductase.
The sixth ligand position is taken by a tightly coordinated oxygen atom at 2.04

Å
distance from the tungsten ion. Due to a rotation of the P MGD cofactor, the geometry
of the coordination in AH is not square pyramidal or trigonal prismatic, as typically
found in enzymes of this family [ 29 ], but resembles more an octahedral or trigonal
antiprismatic coordination (Figure 2 )[ 21 ].
The access funnel opened by a shift in the loop region of residues 327-335 ends
in a ring of six bulky hydrophobic residues (Ile14, Ile113, Ile142, Trp179, Trp293,
and Trp472) forming a small hydrophobic pocket directly above the oxygen ligand
and an adjacent aspartate residue (Asp13) (Figure 3 ). Asp13, a direct neighbor of
the [4Fe-4S]-coordinating Cys12, forms a tight hydrogen bond of 2.41

to the
oxygen ligand of the W ion. Although it was yet not possible to solve a crystal

Å