Environmental Engineering Reference
In-Depth Information
4.2 Protein Maturation and Cu
A
Insertion
In
P. stutzeri
, the
nosZ
gene is translated in the cytoplasm, so that its folded - or
partially folded - protein product is exported
via
the Tat pathway. It is currently not
known whether NosZ is exported as a monomer or a dimer, and whether it reaches
the periplasm as a metal-free apo protein or already has pre-assembled copper sites
included. The presence and location of the known maturation factors, however,
strongly indicates that the major part of the assembly process occurs in the
extracellular compartment. Here, the less intricate Cu
A
site is assembled along a
different route, as deletions of any of the genes
nosDFYL
will leave the binuclear
site unaffected, while the resulting protein is fully deplete of NosZ [
11
]. In a metal-
depleted NosZ protein, Cu
A
can be readily reconstituted by the addition of
Cu(II) salts, while Cu
Z
cannot [
43
], and it is assumed that
in vivo
the Cu
A
site
employs a copper chaperone, such as ScoA, that is also required for the assembly of
heme-copper oxidases [
11
]. This is in line with the concept of rack-induced metal
binding by cupredoxin domains and the facile reconstitution of the binuclear CuA
site has also been observed in model proteins with an engineered CuA-binding
loop [
72
,
73
].
4.3 Assembly of Cu
Z
The ABC transporter NosFY and the periplasmic proteins NosD and NosL that
form part of all known gene clusters encoding a functional N
2
O reductase system
are required for assembly of the Cu
Z
active site. This site is substantially more
complex than Cu
A
(Figures
6
and
7
), and consequently its biogenesis is more
intricate, to the point that
in vitro
reconstruction without the help of additional
factors has not been achieved [
7
]. The Cu
Z
center consists of two essential building
blocks, the four copper ions and the two sulfide ions, that must be provided along
different routes.
As mentioned previously, Cu trafficking is a highly regulated process in all
organisms, and specialized chaperones are required to provide the metal, commonly
in the Cu(I) state [
84
]. For N
2
OR, the NosL protein was suggested for this role
[
11
,
78
,
91
]. An NMR structure of NosL is available that shows the apo-state of the
protein, but contains cysteine residues that may well serve as ligands for Cu(I) [
92
].
NosFY is an ABC transporter that shuttles a necessary assembly intermediate from
the cytoplasm to the periplasm, but its actual cargo is unknown to date [
89
]. It is,
however, homologous to the Atm1 type of ABC transporters that are involved in
iron-sulfur cluster biogenesis. Two structures of Atm1 orthologs have become
available most recently and imply that the transported species may be a glutathione
persulfide [
93
,
94
]. In conjunction with NosFY, NosD is thought to serve as an
assembly chaperone or a periplasmic binding protein for the species transported by
NosFY. The emerging picture thus is that NosL is the copper donor at least for the
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