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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