of an unspecified sulphur compound, has been suggested [69, 70]. A functional
cooperation of NosDYF with the copper chaperone NosL has been discussed and a
function in copper delivery to NosZ was suggested, especially in the context of Cu Z
centre formation [69]. On the other hand, it cannot be excluded that cytoplasmic
ATP hydrolysis is used to drive a so far unknown periplasmic process, similar to the
essential role of the CcmAB complex in periplasmic cytochrome c biogenesis [93].
In this case, NosDYF might work as a motor rather than a transporter. NosD might
interact with NosL in the way that NosD acts as a scaffold for copper centre
assembly facilitated by NosL. Such a process might depend on keeping the copper
atom in a reduced state upon provision of low-potential electrons (see below).
Clade I NGCs from Alpha-, Beta- and Gammaproteobacteria often encode the
two flavoproteins NosR and NosX (note that nosR is often located upstream of nosZ
and that the nosX gene is absent in some cases) [69, 70]. The membrane-bound
flavin mononucleotide (FMN)- and [4Fe-4S] cluster-containing NosR protein might
function in electron transfer to NosZ via its periplasmic flavoprotein domain
(Fig. 3a) [94]. NosR was found to be essential for N 2 O respiration and the phenotype of a corresponding P. stutzeri mutant was in line with a function of NosR in
electron transport to NosZ and/or in the proper assembly of the Cu Z centre (supported by the presence of Cu Z * in NosZ) [94]. Site-directed modification of one or
both CX 3 CP motifs to VX 3 VP indicated that each motif was important for N 2 O
respiration. Thus, it cannot be excluded that the two cytoplasmic iron–sulphur
clusters of NosR are involved in a direct or indirect transmembrane electron
transport to the FMN molecule (although the direct interaction of one of the
[4Fe-4S] clusters with FMN is rather unlikely given the assumed architecture of this
polytopic membrane protein). Nevertheless, this would allow transfer of
low-potential electrons (putatively mediated by ETP red in Fig. 3a) from the cytoplasm to periplasmic NosZ via NosR in a Qcr-independent pathway, and this could
elegantly explain the phenomenon of reductive NosZ activation serving in NosZ
maintenance or repair, as mentioned in Sect. 4. In such a scenario, the Qcr- and
NosR-mediated electron transport pathways were separated and functionally distinct. This view is also in line with experimental evidence from studies with
P. stutzeri and P. aeruginosa [81, 94]. For P. aeruginosa, NosR interaction with
NosZ has been reported [81]. Likewise, NosR might be involved in the
NosDL-dependent incorporation of copper in the process of periplasmic NosZ
maturation.
NosX is a periplasmic flavin adenine dinucleotide (FAD)-binding flavoprotein of
the ApbE family that was shown to be involved in NosR biogenesis [95]. Similar to
the above mentioned nosR mutant, the absence of nosX promoted Cu Z * formation
in NosZ from P. denitrificans, indicative of a functional interaction between NosR
and NosX [96]. In fact, the NosX homologue ApbE from P. stutzeri (an organism
lacking nosX in its NGC) has been shown to act as a flavin donor for NosR [95].
The role of the monohaem cytochrome c NosC, which is encoded occasionally
in clade I NGCs, is unclear. NosC might also play a dedicated and possibly
accessory role in maintaining the Nos system intact. Interactions of NosC with
NosZ and/or NosL are conceivable (Fig. 3a).
198
J. Simon
cooperation of NosDYF with the copper chaperone NosL has been discussed and a
function in copper delivery to NosZ was suggested, especially in the context of Cu Z
centre formation [69]. On the other hand, it cannot be excluded that cytoplasmic
ATP hydrolysis is used to drive a so far unknown periplasmic process, similar to the
essential role of the CcmAB complex in periplasmic cytochrome c biogenesis [93].
In this case, NosDYF might work as a motor rather than a transporter. NosD might
interact with NosL in the way that NosD acts as a scaffold for copper centre
assembly facilitated by NosL. Such a process might depend on keeping the copper
atom in a reduced state upon provision of low-potential electrons (see below).
Clade I NGCs from Alpha-, Beta- and Gammaproteobacteria often encode the
two flavoproteins NosR and NosX (note that nosR is often located upstream of nosZ
and that the nosX gene is absent in some cases) [69, 70]. The membrane-bound
flavin mononucleotide (FMN)- and [4Fe-4S] cluster-containing NosR protein might
function in electron transfer to NosZ via its periplasmic flavoprotein domain
(Fig. 3a) [94]. NosR was found to be essential for N 2 O respiration and the phenotype of a corresponding P. stutzeri mutant was in line with a function of NosR in
electron transport to NosZ and/or in the proper assembly of the Cu Z centre (supported by the presence of Cu Z * in NosZ) [94]. Site-directed modification of one or
both CX 3 CP motifs to VX 3 VP indicated that each motif was important for N 2 O
respiration. Thus, it cannot be excluded that the two cytoplasmic iron–sulphur
clusters of NosR are involved in a direct or indirect transmembrane electron
transport to the FMN molecule (although the direct interaction of one of the
[4Fe-4S] clusters with FMN is rather unlikely given the assumed architecture of this
polytopic membrane protein). Nevertheless, this would allow transfer of
low-potential electrons (putatively mediated by ETP red in Fig. 3a) from the cytoplasm to periplasmic NosZ via NosR in a Qcr-independent pathway, and this could
elegantly explain the phenomenon of reductive NosZ activation serving in NosZ
maintenance or repair, as mentioned in Sect. 4. In such a scenario, the Qcr- and
NosR-mediated electron transport pathways were separated and functionally distinct. This view is also in line with experimental evidence from studies with
P. stutzeri and P. aeruginosa [81, 94]. For P. aeruginosa, NosR interaction with
NosZ has been reported [81]. Likewise, NosR might be involved in the
NosDL-dependent incorporation of copper in the process of periplasmic NosZ
maturation.
NosX is a periplasmic flavin adenine dinucleotide (FAD)-binding flavoprotein of
the ApbE family that was shown to be involved in NosR biogenesis [95]. Similar to
the above mentioned nosR mutant, the absence of nosX promoted Cu Z * formation
in NosZ from P. denitrificans, indicative of a functional interaction between NosR
and NosX [96]. In fact, the NosX homologue ApbE from P. stutzeri (an organism
lacking nosX in its NGC) has been shown to act as a flavin donor for NosR [95].
The role of the monohaem cytochrome c NosC, which is encoded occasionally
in clade I NGCs, is unclear. NosC might also play a dedicated and possibly
accessory role in maintaining the Nos system intact. Interactions of NosC with
NosZ and/or NosL are conceivable (Fig. 3a).
198
J. Simon