Environmental Engineering Reference
In-Depth Information
orders of magnitude higher, but the precise state of the enzyme preparation was
not clearly stated [
79
]. More recent activity data on the form II preparation of
M. hydrocarbonoclasticus
N
2
OR provided further insights [
50
]. Here, the protein
was isolated with the tetranuclear site in the Cu
Z
*
state, and the observed activity of
0.01 U N
2
O was far below the one reported for other orthologs. However, the
enzyme could be activated by complete reduction of Cu
Z
*
to an all-Cu(I) form
using sodium dithionite in conjunction with methyl viologen as a redox mediator
(see Section
3.3
). In this state, the activity of the enzyme was determined to 160 U
N
2
O, and the conclusion drawn was that Cu
Z
*
must be the active form of the Cu
Z
cluster and that reductive activation of the enzyme is a necessary requirement for
catalytic activity [
47
,
78
]. In this mechanism, the activation of the enzyme still is
far slower than the observed catalytic turnover, and it is not straightforward to
reconcile fast oxidation of Cu
Z
*
during N
2
O reduction with the observed, slow
activation of the cluster with dithionite [
44
].
A possible solution for the issue came with the identification of a further state
of the Cu
Z
site that was proposed to be an additional catalytic intermediate.
This species was characterized by an additional absorption maximum at 680 nm
and termed Cu
Z
0
[
80
]. Solomon and coworkers identified Cu
Z
0
as a [3Cu
+
:1Cu
2+
]
form of the cluster that was, however, distinct from the isoelectronic Cu
Z
*
and that
can be rapidly reduced back to the all-Cu(I) state of the site [
81
]. For the mechanism
of nitrous oxide reduction, this suggested that within the catalytic cycle, the fully
reduced state was required to bind and activate the kinetically inert N
2
O molecule,
but that the catalytic one-electron oxidation of the tetranuclear cluster would lead to
Cu
Z
0
rather than the Cu
Z
*
resting state, allowing for a quick reductive reactivation
of the site to keep up with the observed rate of catalysis. At the same time, a single-
turnover experiment, where activated and reduced N
2
OR was incubated with its
substrate in the absence of further reductant, yielded the enzyme in the Cu
Z
*
resting
state, while N
2
O had obtained one electron each from Cu
A
and Cu
Z
to yield the
products N
2
and H
2
O[
81
]. Note that this mechanistic proposal strictly depends on
the availability of the all-Cu(I) state of Cu
Z
, which only seems to be accessible with
the [4Cu:S] Cu
Z
*
site, while in the [4Cu:2S] form, Cu
Z
, even extended incubation
with sodium dithionite and viologens will not reduce all four copper ions [
44
,
47
].
This may be explained with the presence of the additional sulfur donor ligand that
forms highly covalent bonds with Cu
1
and Cu
4
of the cluster and stabilizes the more
oxidized state [
34
,
81
]. The activity reported for the purple form I N
2
OR from
P. stutzeri
would thus either be due to a residual fraction of Cu
Z
*
that was
reductively activated, or it would reflect the catalytic potential of the Cu
Z
itself
that, however, was too low to explain the activities observed
in vivo
[
34
]. In this
mechanism, N
2
O was still suggested to bind to the exposed edge of Cu
Z
*
formed by
Cu
1
and Cu
4
in the absence of a bridging sulfur [
81
-
83
], and the binding mode
observed experimentally [
32
] was proposed to reflect the lower activity of the
purple form I of N
2
OR [
81
].
From a physiological point of view, however, this mechanistic proposal
encounters two major problems. First, the reductive activation of N
2
OR to generate
the all-Cu(I) state of Cu
Z
*
that is a prerequisite for catalysis can only be achieved at
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