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than the original acetylene hydratase in its native tungsten form (AH(W)) but still
converts acetylene to acetaldehyde at a rate of 1.9
mol min
1
mg
1
at 37
C
ʼ
mol min
1
mg
1
of the original AH(W) [
27
]. According to
ICP-MS AH(Mo) contained 0.45-0.51 mol Mo per mol enzyme, 2.7-3.1 mol Fe per
mol enzyme, and no tungsten [
26
-
28
], ruling out that the low activity of
AH(Mo) derives from residual tungsten in the enzyme.
The EPR spectrum of AH(Mo), as isolated under N
2
/H
2
(94 %/6 % v/v) atmo-
sphere, showed a weak signal assigned to a Mo(V) center with
g
x
¼
compared to 14.8
ʼ
1.99,
and
g
z
¼
2.023. The signal size increased upon oxidation with hexacyanoferrate(III),
indicating that AH(Mo) was isolated in a partially oxidized state. Dithionite-reduced
samples of AH(Mo) showed the identical signal of a ferredoxin type [4Fe-4S] cluster
as AH(W) with
g
z
¼
1.978,
g
y
¼
1.920 [
28
]. When comparing
the contribution of secondary structural elements to the total fold in AH(Mo) and
AH(W) by circular dichroism (CD) spectroscopy, only slight differences in the
amount of
2.048,
g
y
¼
1.939, and
g
x
¼
ʱ
-helices (14.3 % in AH(Mo)
versus
11.3 % in AH(W)) and
ʲ
-sheets
(35.4 % in AH(Mo)
versus
39.9 % in AH(W)) were found [
27
].
4.3 Crystallization
A high resolution X-ray structure of acetylene hydratase was solved in 2007 (PDB
2E7Z) [
21
]. The crystallization of AH was performed under a N
2
/H
2
(94 %/6 % v/v)
atmosphere at 20
C using the sitting drop vapor diffusion method. Yellow brownish,
plate-shaped crystals grew within 1-3 weeks from a 10 mg/mL solution of AH in
5 mM HEPES/NaOH pH 7.5 containing 5 mM Na
+
dithionite. 2
L of the protein
solution were mixed with 2.2
ʼ
Lof0.1MNa
+
cacodylate, pH 6.5, containing
0.3 M Mg(acetate)
2
, 21 % PEG 8000, and 0.04 M Na
+
azide. 15 % MPD was added
as cryo protectant before flush freezing of the crystals in liquid N
2
. The crystals
belonged to space group C2 with
a
ʼ
¼
120.8
Å
,
b
¼
72.0
Å
,
c
¼
106.8
Å
,and
124.3
, and contained one monomer per asymmetric unit. The native structure
was solved by single wavelength anomalous dispersion (Fe absorption edge) and
refined to 1.26
ʲ¼
Å
. In total the model consisted of 730 amino acid residues,
880 water molecules, two MGD cofactor molecules, and one [4Fe-4S] cluster.
Additionally, two MPD molecules, one acetate molecule, and one sodium ion have
been identified in the crystal structure of AH [
21
].
4.4 Structural Overview
As expected, from the biochemical and spectroscopic characterization of
the enzyme, the structure is a monomer of 730 amino acids, containing a bis-
molybdopterin-guanine-dinucleotide (bis-MGD) and a cubane [4Fe-4S] cluster.
The two cofactors are buried deep inside a four domain fold, as found typically in
enzymes of the DMSO reductase family (Figure
1
)[
21
]. Domain I (residues 4-60)
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