Environmental Engineering Reference
In-Depth Information
A fourth form of N
2
O reductase was described as a 'reconstituted' enzyme,
where Cu was first removed to generate the apo-protein that could subsequently be
replenished with copper. This was achieved by anoxic dialysis against KCN,
followed by the addition of Cu(en)
2
SO
4
[
40
]. This procedure led to an enzyme
with a lower copper content of approximately 4 Cu per homodimer of N
2
OR that
had the spectroscopic features of an intact Cu
A
site, but with Cu
Z
fully absent.
Besides providing evidence that the assembly of Cu
Z
required further maturation
factors, this procedure also gave access to a form of the enzyme in which one of the
metal centers could be studied without interference from the other. The same effect
was reached through a variant strain,
P. stutzeri
MK402, in which the biogenesis of
Cu
Z
was prevented through a Tn5 insertion, leading to an enzyme containing
exclusively Cu
A
, termed form V [
40
].
The Cu content of different preparations of nitrous oxide reductase was
generally between 7 and 8 Cu per dimer, but the exogenous addition of copper
salts to the growth medium yielded a highly active form of the enzyme with an
increased Cu content of 10-12/dimer [
45
,
46
]. This could be rationalized from the
crystal structures that became available at the same time, and showed the Cu
Z
site to
be an entirely novel, tetranuclear copper center, with four metal ions arranged
around a central ligand and coordinated to a total of seven histidine residues at
the hub of the N-terminal
-propeller domain [
27
,
28
,
30
]. Both relevant forms of
Cu
Z
, the Cu
Z
site of N
2
OR form I and the Cu
Z
*
site of form II, contain the same
number of copper ions within the cluster, and even the original assumption that
Cu
Z
*
represents an oxidatively damaged form of Cu
Z
was recently questioned by
the finding that an enzyme with the typical features of N
2
OR form I could be
isolated under oxic conditions from
M. hydrocarbonoclasticus
[
47
].
ʲ
3.1.2 Three-Dimensional Structures
Initial advances towards structural data for the metal sites of nitrous oxide reductase
was made for the Cu
A
site in respiratory cytochrome
c
oxidase or in engineered
cupredoxins as detailed below, but no information was available for the active
site of the denitrificatory enzyme, the Cu
Z
center. In the initial description of the
ortholog from
P. nautica
(
M. hydrocarbonoclasticus
)[
29
,
48
], Cu
Z
was identified
as a novel tetranuclear site coordinated by seven histidines, with a central oxygen
ligand [
28
,
49
,
50
]. This structure was obtained from a preparation corresponding to
form III of N
2
O reductase, with Cu
A
in the reduced state and the distinct blue color
of an absorption band with a maximum at 600 nm that consequently had to originate
from Cu
Z
. This assignment raised immediate criticism from the spectroscopic
community, as an environment consisting exclusively of imidazole nitrogens and
an oxygen lacked the soft, donating ligand required to yield the prominent
ligand
metal charge-transfer transition (LMCT) represented by the blue color
of the preparation. Indeed, a re-determination of the
M. hydrocarbonoclasticus
N
2
OR structure, combined with the structure of the enzyme from
Paracoccus
!
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