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denitrificans
at 1.6
Å
resolution soon thereafter clarified that the central oxygen in
Cu
Z
was indeed a sulfido species that likely was misinterpreted due to partial
occupancy [
27
,
30
].
Both structures were obtained as a blue form III of the enzyme and interpreted to
show the Cu
Z
*
form of the active center. In 2006, the enzyme from
A. cycloclastes
was crystallized and refined to 1.7
resolution, showing for the first time an
enzyme in form II, which differed from the previous structures in having the Cu
A
site in the oxidized state [
31
]. Note, however, that the copper sites of nitrous oxide
reductase are likely prone to swift photoreduction upon exposure to X-rays, so that
spectroscopic monitoring of crystals will be required before unambiguous assign-
ments of oxidation state can be made. The most recent structural data on N
2
OR
came from
P. stutzeri
, the organism where the enzyme was originally discovered.
Using anoxically isolated protein, purple crystals of form I N
2
OR were obtained
[
51
], and the resulting structure showed several relevant features, including the Cu
Z
state of the active site cluster [
32
].
Å
3.2 Cu
A
: More than an Electron Transfer Center
Beside its role as an electron transfer site in nitrous oxide reductase, further interest
in the Cu
A
site arose from the fact that its spectroscopic features indicated a possible
structural and electronic similarity to one of the copper sites in respiratory cyto-
chrome
c
oxidase [
52
]. Due to a better resolution of the observed spectroscopic
features, nitrous oxide reductase eventually proved to be highly instrumental to
understand the structure and properties of this site, but agreement was only reached
after lengthy debates with the advent of several crystal structures [
53
]. Yet, only the
most recent analyses of the denitrificatory enzyme have revealed intriguing
properties that after all do set the Cu
A
site in N
2
OR apart from the one in
cytochrome
c
oxidase and that help to explain the improved resolution in EPR
spectra of the latter [
32
,
44
].
3.2.1 Spectroscopic Properties of Cu
A
Copper-containing proteins are frequently classified within a scheme of three basic
types according to a proposal by Malkin and Malmstr¨m[
54
]. When the enzyme
nitrous oxide reductase was initially isolated from
P. stutzeri
in different form as
described above [
43
], it was readily apparent that this novel protein would not fit
into the established scheme. For the purple form I of the enzyme, EPR studies at
X-band showed a distinct axial signal with
g
||
¼
2.03, with charac-
teristic hyperfine splittings with seven lines in a 1:2:3:4:3:2:1 intensity ratio that
were not consistent with a mononuclear copper site (
63/65
Cu nuclear spin
I
2.18 and
g
⊥
¼
¼
3/2)
(Figure
4
)[
43
]. The
g
||
region featured seven equidistant lines with
A
||
¼
3.83 mT,
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