Purified and biochemically and/or structurally characterized NosZ enzymes
generally belong to clade I with the notable exception of W. succinogenes cNosZ
[62]. NosZ proteins form homodimers and consist of an N-terminal seven-bladed
b-propeller domain and a C-terminal cupredoxin domain. Each monomer contains a
mixed-valent binuclear Cu A centre and a sulphide-bridged tetranuclear Cu Z centre
that harbours four more copper atoms. The four copper atoms of Cu Z are coordinated by imidazole nitrogen atoms derived from seven conserved histidine residues.
Thus, NosZ is a copper-rich enzyme that binds 12 copper atoms per homodimer.
Each homodimer contains two active sites of N 2 O reduction, and each active site is
composed of Cu A and Cu Z centres that originate from different NosZ monomers.
The shortest intermonomer distance between the Cu A and Cu Z sites is about 10 Å,
whereas the two centres located in the same monomer are 40 Å apart [63–66].
Isolated NosZ enzymes showed K M values for N 2 O in the µM range, which is
way above the atmospheric N 2 O concentration [67, 68]. Therefore, once in the
atmosphere, the chemically stable N 2 O molecule evades biological turnover due to
its low concentration. Usually, the N 2 O reduction activity of NosZ is
oxygen-sensitive and exhibits an alkaline pH optimum. The spectroscopic features
of different NosZ enzyme forms isolated or incubated under different oxygen
regimes have been comprehensively reviewed, and the reader is referred to these
articles for details concerning the structure/function relationships of various oxidation state-dependent molecular forms of the copper centres [67–72]. Notably, the
formation of an inactive (resting) state of the Cu Z centre has been designated Cu Z *.
The Cu Z * form corresponds to a (4Cu1S) [1Cu
2+
–3Cu
1+ ] centre that contains one
sulphur and one oxygen atom from a hydroxide ion or a water molecule [68]. This
form was postulated to result from enzyme inactivation due to the absence of
reductants (see Sect. 5 for putative in vivo electron donors). Furthermore, the Cu Z *
state was obtained in aerobically isolated enzymes and might thus be environmentally relevant. This raised the question as to how the Cu Z * state can be rescued
to restore a fully reduced and catalytically competent form (the [4Cu
1+ ] state). In
vitro, this repair process has been reported to occur by reductive activation, for
example, by donating electrons using a low-potential reductant such as methyl or
benzyl viologen radicals [68, 69]. Thus, it seems mandatory that a dedicated in vivo
electron transfer route to the active site of NosZ exists to enable (re-)activation of
NosZ activity and/or to prevent Cu Z * formation (see Sect. 5).
Evaluation of the literature argues that the copper content in many NosZ
preparations was less than 12 copper atoms per NosZ dimer and that a mixture of
different NosZ forms was present (especially in enzymes obtained from heterologous host cells) despite the fact that the cells were grown under copper-sufficient
laboratory conditions [73 and references therein]. This implies that copper supply
and incorporation are important features in NosZ biogenesis that are, moreover,
embedded in the wider context of cellular copper homoeostasis. In prokaryotes,
copper transport (import/export) and delivery/metallation systems such as cytoplasmic and periplasmic copper chaperones contribute to this tightly controlled
process [74]. Copper toxicity is usually circumvented by fixing Cu(I) in the form of
stable Cu(I)-thiol complexes through copper-binding proteins and/or low molecular
194
J. Simon
generally belong to clade I with the notable exception of W. succinogenes cNosZ
[62]. NosZ proteins form homodimers and consist of an N-terminal seven-bladed
b-propeller domain and a C-terminal cupredoxin domain. Each monomer contains a
mixed-valent binuclear Cu A centre and a sulphide-bridged tetranuclear Cu Z centre
that harbours four more copper atoms. The four copper atoms of Cu Z are coordinated by imidazole nitrogen atoms derived from seven conserved histidine residues.
Thus, NosZ is a copper-rich enzyme that binds 12 copper atoms per homodimer.
Each homodimer contains two active sites of N 2 O reduction, and each active site is
composed of Cu A and Cu Z centres that originate from different NosZ monomers.
The shortest intermonomer distance between the Cu A and Cu Z sites is about 10 Å,
whereas the two centres located in the same monomer are 40 Å apart [63–66].
Isolated NosZ enzymes showed K M values for N 2 O in the µM range, which is
way above the atmospheric N 2 O concentration [67, 68]. Therefore, once in the
atmosphere, the chemically stable N 2 O molecule evades biological turnover due to
its low concentration. Usually, the N 2 O reduction activity of NosZ is
oxygen-sensitive and exhibits an alkaline pH optimum. The spectroscopic features
of different NosZ enzyme forms isolated or incubated under different oxygen
regimes have been comprehensively reviewed, and the reader is referred to these
articles for details concerning the structure/function relationships of various oxidation state-dependent molecular forms of the copper centres [67–72]. Notably, the
formation of an inactive (resting) state of the Cu Z centre has been designated Cu Z *.
The Cu Z * form corresponds to a (4Cu1S) [1Cu
2+
–3Cu
1+ ] centre that contains one
sulphur and one oxygen atom from a hydroxide ion or a water molecule [68]. This
form was postulated to result from enzyme inactivation due to the absence of
reductants (see Sect. 5 for putative in vivo electron donors). Furthermore, the Cu Z *
state was obtained in aerobically isolated enzymes and might thus be environmentally relevant. This raised the question as to how the Cu Z * state can be rescued
to restore a fully reduced and catalytically competent form (the [4Cu
1+ ] state). In
vitro, this repair process has been reported to occur by reductive activation, for
example, by donating electrons using a low-potential reductant such as methyl or
benzyl viologen radicals [68, 69]. Thus, it seems mandatory that a dedicated in vivo
electron transfer route to the active site of NosZ exists to enable (re-)activation of
NosZ activity and/or to prevent Cu Z * formation (see Sect. 5).
Evaluation of the literature argues that the copper content in many NosZ
preparations was less than 12 copper atoms per NosZ dimer and that a mixture of
different NosZ forms was present (especially in enzymes obtained from heterologous host cells) despite the fact that the cells were grown under copper-sufficient
laboratory conditions [73 and references therein]. This implies that copper supply
and incorporation are important features in NosZ biogenesis that are, moreover,
embedded in the wider context of cellular copper homoeostasis. In prokaryotes,
copper transport (import/export) and delivery/metallation systems such as cytoplasmic and periplasmic copper chaperones contribute to this tightly controlled
process [74]. Copper toxicity is usually circumvented by fixing Cu(I) in the form of
stable Cu(I)-thiol complexes through copper-binding proteins and/or low molecular
194
J. Simon