Environmental Engineering Reference
In-Depth Information
theory [
34
,
44
]. While the hydrophobic channel indeed leads right up to a gate
formed by two bulky amino acid side chains (Phe621 and Met627 in
P. stutzeri
)on
the face of Cu
Z
that is directed towards Cu
A
, the additional space gained through the
loss of the second sulfide, S
2
, in the Cu
Z
*
state would allow the substrate to access
the Cu
1
-Cu
4
edge of the cluster as well.
5.2 Gated Electron Transfer
An unexpected finding in the structure of
P. stutzeri
N
2
OR was the flexibility of
His583, a ligand to Cu
2
of the Cu
A
site (Figure
6
)[
32
]. Based on the available
structural data the 135
flip of the imidazole side chain of this histidine residue
cannot be related to the redox state of the site, and both conformations seem
accessible. However, all structures of gas-pressurized crystals of the
P. stutzeri
enzyme showed the ligand to be coordinated to the metal ion, as did all earlier
observations, where the tetranuclear site was in the Cu
Z
*
state. The flipped-out state
of His583 was thus only observed in form I of N
2
OR, i.e., with an intact [4Cu:2S]
cluster, and in the absence of the substrate N
2
O. As His583 in both its confor-
mations remains hydrogen-bonded to residue Asp576 that is presumed to be the
electron entry site from an external electron transfer protein [
37
], the on-off
coordination to Cu
A
was suggested to form a conformational gate for intra-
molecular electron transfer, with the consequence that electron flow will only be
permitted
after
the substrate is bound at Cu
Z
[
32
,
44
]. Obviously, this intricate
coupling mechanism is tightly linked to the physiological activity of the enzyme,
based on a reduced electron transfer protein as a redox partner. Using small-
molecule reductants such as ascorbate or sodium dithionite, complete reduction
of Cu
A
is swift, but there is no way of controlling at which points the reductants will
interact with the enzyme. The question of gated electron transfer in nitrous oxide
reductase thus touches the distinction between reactivities observed
in vitro
and
those occurring in the actual physiological environment of the bacterial periplasm.
5.3 Activation of Nitrous Oxide
The reduction of N
2
OtoN
2
by nitrous oxide reductase (equation
1
) takes place at
the tetranuclear site, Cu
Z
. The N
2
O molecule observed in the
P. stutzeri
X-ray
structure was not directly coordinated to any of the metal ions of the cluster, but
rather it was fixed by hydrogen bonds to the oxygen atom, originating from His626,
a ligand to Cu
A
, and a structural water molecule (Figure
9
)[
32
]. This structure did
not represent a coordination complex in the classical sense, but rather revealed a
substrate-binding site in close proximity to the metal centers. This binding event is
presumed to be the trigger for His583 to flip back towards the Cu
A
center and enable
electron transfer to the substrate. Nevertheless, the suggestive implication that
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