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revealed that this was associated to the Cu
Z
state, while the single-sulfide species
[4Cu:
-S] represented Cu
Z
*
[
32
,
44
]. The tetranuclear site seems to be rather
susceptible to partial degradation during the isolation procedure of the enzyme,
and while the four copper ions are stably retained, it is in the presence of the
additional sulfide ion bridging atoms Cu
1
and Cu
4
where the differences manifest.
Degradation of the site in the Cu
Z
state leads to formation of Cu
Z
*
, and the
relative abundance of both states tends to vary from one protein batch to another.
In addition, the reverse process, a conversion of Cu
Z
*
to Cu
Z
, had not been achieved
in vitro
, and it is assumed that this is only possible with the help of additional
maturation factors during the biogenesis of the enzyme [
11
]. As a consequence, the
electron density maps obtained from X-ray diffraction data are prone to showing an
average of different forms of the cluster, and this refers in particular to mixtures of
the Cu
Z
and Cu
Z
*
states. The magnitude of a peak in an electron density map
primarily reflects the number of electrons at a given position, and in addition
the value obtained represents an average of all unit cells in the entire crystal.
For a 1:1 mixture of Cu
Z
and Cu
Z
*
within a crystal, this means that the electron
density maximum at the position of atom S
Z2
, where both forms of the cluster differ,
will have half the magnitude of a fully occupied Cu
Z
state. In number of electrons
this corresponds to only 8 of the 16 electrons expected for the element sulfur, but it
also corresponds to a fully occupied oxygen atom, and in the original structures it
was indeed modeled as such. An oxygen atom in the model will satisfy the observed
electron density so that no conspicuous residual peaks will be visible in
F
o
-
F
c
difference electron density maps. Oxygen was modeled in this position in the
structures of
M. hydrocarbonoclasticus
and
P. denitrificans
, but in both cases the
Cu-'O' bond lengths refined to 2.3
ʼ
(PDB ID 1QNI, 1FWX) [
27
,
30
], falling far
more in the expected range of a Cu-S bond [
44
]. The current interpretation of the
existing Cu
Z
structures (Figure
7
) thus suggests that CuZ exists as a [4Cu:2S]
cluster, while Cu
Z
*
only lacks the bridging S
Z2
, but does not contain a water ligand
connecting Cu
1
and Cu
4
. The situation differed slightly in the structure of the
A. cycloclastes
enzyme, where two distinct electron density maxima were
interpreted as water ligands at reasonable bond distances of 2.0-2.1
Å
(PDB ID
2IWF), or where iodide as an inhibitor bound between Cu
1
and Cu
4
in a bridging
manner (PDB ID 2IWK) [
31
]. This enzyme had the spectroscopic properties of a
form II N
2
OR with Cu
Z
in the Cu
Z
*
state, but it did unambiguously show exogenous
ligands binding to the center for the first time.
Å
3.3.2 States of Cu
Z
and Catalytic Properties
Detailed activity assays for N
2
O reductase were first presented for the
P. stutzeri
enzyme [
43
]. As detailed above, the observed activities were highest for the
anoxically isolated form I of the enzyme that we now understand to contain the
intact [4Cu:2S] Cu
Z
cluster (Figure
7
). Subsequently, Snyder and Hollocher
published activities for a 'purple' form of
P. denitrificans
N
2
OR that were two
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