Environmental Engineering Reference
In-Depth Information
by the sulfur of a methionine, at a slightly longer distance. The site thus is of high -
if not perfect - symmetry, and shows the features of electronic delocalization, in
particular the characteristic seven-line hyperfine pattern.
Based on the crystal structures, an excellent biomimetic model complex
was presented soon thereafter [
64
]. While the complex was centrosymmetric and
therefore had perfectly identical geometries for both metals, the observed seven-line
hyperfine pattern in EPR spectroscopy showed a hyperfine splitting of 4.99 mT for
A
||
and 3.63 mT for
A
⊥
, notably larger than for the protein. This was concomitant with a
Cu-Cu distance in the model complex of 2.92
Å
that was significantly longer than the
2.5
determined by EXAFS spectroscopy for Cu
A
[
57
,
65
]. The short distance
between both metals in this center is commonly interpreted as one of the rare cases of
a direct metal-metal interaction in Biology [
57
]. In the structure of
P. denitrificans
N
2
OR determined at 1.6
Å
resolution, the Cu-Cu distance was 2.51
Å
[
30
], but was
significantly longer at 2.63
Å
in the purple form I structure from
P. stutzeri
[
32
]. For
this enzyme,
63
Cu- or
65
Cu-enrichment as well as
15
N-labeling had allowed for a
significantly improved resolution for EPR spectroscopy that then formed the basis for
molecular orbital calculations on a structural core unit consisting of Cu ions with two
ʼ
Å
2
-bridging sulfides and a terminal amine ligand on each metal [
66
]. The obtained
values agreed very well with the experimental data, and subsequent ENDOR studies
helped to refine the picture further (see Section
5.1
)[
67
,
68
].
The Cu
A
center is located in a domain that shows a conserved tertiary structure
commonly termed the cupredoxin fold [
69
,
70
]. Cupredoxins are mononuclear
copper proteins ('type-I copper proteins') that coordinate a single copper ion in a
rigid binding site at the periphery of a 100-140 aa peptide with a characteristic
-
barrel forming a Greek-key motif [
34
]. In cupredoxins, the metal is coordinated by
a cysteine that allows for a LMCT, giving rise to an intense absorption maximum
around 600 nm that conveys the typical color of these 'blue copper proteins'.
Further ligands are two histidines and a fourth ligand that commonly is a methio-
nine, but can also be a different amino acid that modulates the midpoint potential of
the Cu
+
/Cu
2+
redox pair [
71
]. Within the protein chain, one of the histidine ligands
is found in the first third of the sequence, while the other three ligands cluster in a
single loop near the C-terminal that connects the last two
ʲ
strands.
Interestingly, the very same architecture is found both in cytochrome
c
oxidase
and nitrous oxide reductase, where a cupredoxin domain holds the Cu
A
site.
In
P. stutzeri
N
2
O reductase, the ligands to Cu
A
are His583 in the N-terminal part
of the cupredoxin domain, and Cys618, Trp620, Cys622, His626, and Met629 that
all form part of the terminal loop between
ʲ
strands 8 and 9 (Figures
3
and
6
).
This loop is longer than in a typical type-I copper protein, and in fact Sanders-
Loehr, Canters, and coworkers could show that a simple insertion of a Cu
A
-binding
loop into a cupredoxin such as amicyanin led to the assembly of an intact Cu
A
center in a valence-delocalized state [
72
,
73
]. The copper-binding loop of the Cu
A
site in nitrous oxide reductase faces the second metal site, Cu
Z
, and two residues,
Phe621 and Met627 in
P. stutzeri
, form part of the substrate binding pocket, as
detailed below.
ʲ
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