No Laughing Matter: The Unmaking of the Greenhouse Gas Dinitrogen Monoxide by Nitrous Oxide Reductase - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 197

twin-arginine translocation (Tat) pathway, where the entire protein domain can
cross the cytoplasmic membrane in a folded or partially folded state [ 86 ]. However,
if a c -type cytochrome domain is included, as in W. succinogenes NosZ, its
maturation, i.e., the covalent attachment of the heme group to the protein chain
[ 87 ], is strictly dependent on the Sec translocon that transports proteins in their
unfolded state [ 88 ]. In the latter case, all assembly steps of the two copper sites of
N 2 O reductase thus have to take place in the periplasm, after the attachment of the
heme group to the cytochrome domain, while Tat-dependent export of the more
canonical two-domain NosZ would in principle allow to assemble the Cu A and Cu Z
fully or in part in the cytoplasm. Although it is presently assumed that the key steps
of metal center biogenesis do occur in the periplasm in both variants of the nos
operon, the operon architecture itself is different [ 23 ].
Figure 8 The nos operon of Pseudomonas stutzeri . The nosZ gene (Z) is the structural gene for
N 2 O reductase, while the integral membrane protein NosR, encoded by the preceding gene (R), is
required both for the biogenesis of NosZ and for electron transfer to the reductase. NosF (F) and
NosY (Y) form an ABC transporter that is presumed to shuttle sulfur into the periplasm, in a form
that can be incorporated into Cu Z with the help of the nosD gene (D) product. NosL (L) is a
putative copper chaperone that provides Cu(I) for the assembly of both metal sites.
The W. succinogenes nos operon consists of twelve open reading frames, the first
of which encodes the larger version of NosZ with the additional cytochrome
domain. The operon in addition encodes for two periplasmic assembly factors,
NosD and NosL, and ABC Transporter, NosFY, and contains three further open
reading frames ( orfs ) of unknown function. It also holds a putative menaquinol
oxidase, NosGH, and two periplasmic cytochromes, NosC1 and NosC2, with
a putative role in electron transfer to the enzyme [ 23 ]. In contrast, P. stutzeri
possesses a smaller nos cluster that is organized into three transcriptional units
(Figure 8 )[ 89 ]. It commences with the monocistronic nosR gene, encoding a
complex, cofactor-containing membrane protein required both for NosZ maturation
and for N 2 O reduction [ 90 ].
Following nosR , the nosZ gene encoding the actual enzyme is a second
monocistronic transcription unit that includes an N-terminal leader peptide with
the typical twin-arginine motif for Tat-dependent translocation. The following
nosD operon is a four-gene transcription unit that holds the factors required for
periplasmic maturation of Cu Z (and possibly Cu A )[ 89 ]. All four genes, nosDFYL ,
are also present in the W. succinogenes operon, indicating that this is a general
maturation machinery, while NosGH, NosC1 and NosC2 may combine to play the
complex role of the NosR protein in P. stutzeri . As the main focus of the present text
is on P. stutzeri N 2 OR, we will restrict the discussion of maturation factors to this
version of the operon (Figure 8 ).