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and a similar pattern was observed in the
g
⊥
2.8 mT.
This hyperfine structure disappeared upon reduction of the protein in the absence
of dioxygen to yield to a broad featureless signal with
g
values at 2.18 and 2.06.
Notably, the pink form II of the enzyme that is characterized by an intact Cu
A
site,
but a Cu
Z
site in the Cu
Z
*
state, showed a similar spectrum to form I, but with an
inferior resolution for the hyperfine pattern in both the
g
||
and
g
⊥
regions.
Based on these and further studies, Kroneck and coworkers concluded
that Cu
A
in nitrous oxide reductase was a valence-delocalized [Cu
1.5+
:Cu
1.5+
]
binuclear center, and that the same was true for the corresponding site in cyto-
chrome
c
oxidase [
52
]. This claim was soon contested by the proponents of a
mononuclear Cu
A
site in the respiratory complex [
55
], largely under the impression
of the available spectroscopic data for the respiratory enzyme. While the additional
copper center there was found to show an unusual
g
||
value of 2.18 and lacked the
four-line hyperfine pattern typical for type-I and type-II copper sites unless it was
chemically denatured, the characteristic seven-line pattern (Figure
4
) was never
observed clearly. In retrospect this was ascribed to the additional presence of heme
groups in cytochrome
c
oxidase [
53
,
56
], but more recent data has provided an
alternative explanation (see Section
3.2.3
).
In the absence of a crystal structure, Cu K-edge EXAFS was highly valuable
for obtaining initial information on the architecture of Cu
A
.Astudyonthe
solubilized cupredoxin domain of subunit II in cytochrome
c
oxidase from
Bacillus subtilis
indicated the presence of two coppers, each of which was
coordinated to a single histidine and two cysteines [
57
]. In addition, a direct
metal-metal interaction was observed at a distance of 2.5
region with
A
⊥
¼
.Inafollow-up,the
authors compared the Cu
A
domains of the oxidases of
B. subtilis
and
Thermus
thermophilus
in different redox states, and obtained a Cu-Cu distance of
2.43
Å
Å
for the valence-delocalized oxidized state,
versus
2.51
Å
for the fully-
reduced [Cu
1+
:Cu
1+
] state [
58
].
3.2.2 Three-Dimensional Structure(s) of Cu
A
While the Cu
A
site in N
2
O reductase had proved valuable for understanding
the electronic properties of the center, structural information was first provided
along different lines. Extensive efforts to solve the three-dimensional structure of
respiratory cytochrome
c
oxidase were undertaken by several groups, until the
prokaryotic ortholog from
Paracoccus denitrificans
was solved to 2.8
resolu-
tion in 1995 [
59
], followed shortly thereafter by the eukaryotic enzyme from
bovine heart mitochondria [
60
,
61
]. The same arrangement was also found in a
solubilized form of subunit II of cytochrome
bo
3
oxidase from
Escherichia coli
[
62
]. These structures unequivocally settled the debate concerning the number of
metal ions in Cu
A
, and they underlined that the ligand environment in Cu
A
is
highly conserved.
Å
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