thiols such as glutathione. Copper chaperones often use cysteine residues arranged
in a CX 2 C motif as Cu(I) ligands resulting in exceptionally low K D values in the
femto- to zeptomolar (10
–15 to 10
–21 ) range [75].
Unfortunately, knowledge on the maturation of the NosZ Cu A and Cu Z centres is
limited. It is assumed that these centres are synthesized by mutually independent
enzymes at the outside of the cytoplasmic membrane [19, 73, 76, 77]. The Cu A site
of NosZ is supposed to be functionally similar to that of respiratory haem-copper
oxidases, for example, the Cu A centre in subunit II of cytochrome c oxidase [72]. In
both cases, electrons are transferred via Cu A to the respective active sites of N 2 O or
O 2 reduction. The biogenesis of the Cu A site has been investigated in different types
of cytochrome oxidases and involves periplasmic copper chaperones of the
Sco/SenC family [78, 79]. Therefore, it is likely that Sco/SenC-type proteins are
also involved in NosZ Cu A assembly [80]. However, genes encoding such chaperones are generally absent from NGCs.
Most likely, the biogenesis of the Cu Z site involves the copper chaperone NosL,
whose function might depend on the proposed NosDYF complex (Tables 1 and 2).
Depending on the organism, NosL proteins are predicted to be exported by the Sec
or Tat system and some of them might be membrane-anchored lipoproteins.
Interaction of NosL and NosZ was indicated by a proteomic study using Pseudomonas aeruginosa [81]. In P. denitrificans, NosL was recently shown to be
required for NosZ activity and Cu Z (but not Cu A ) assembly only under
copper-limited conditions [77]. This finding was in line with previous reports that
characterized NosL as non-essential for cellular NosZ activity under
copper-sufficent conditions [69, 76, 82]. Thus, a NosL-independent pathway of
NosZ maturation is likely to exist when copper supply is sufficient. This assumption
is also supported by the fact that the nosL gene is absent in some NGCs [28].
A soluble form of P. denitrificans NosL was shown to bind one Cu(I) per protein
with attomolar affinity, whereas Cu(II) did not bind [77]. Therefore, it is tempting to
ask whether copper centre maturation in NosZ needs a Cu(II) to Cu(I) reduction
step and, if yes, whether the required electrons are donated via the same route as in
Cu Z * reactivation). Interestingly, the W. succinogenes NGC encodes three putative
NosL-type copper chaperones (Fig. 2) [83]. Of those, NosL2 is most closely related
to NosL proteins from, for example, Achromobacter cycloclastes and P. denitrificans whose NGCs encode only one NosL-type protein [84, 85]. However, the
individual role of the different NosL-type proteins has not been investigated.
5 The Enzymatic Level, Part II: Electron Transport Routes,
Bioenergetics and Function of Auxiliary Nos Proteins
NosZ or cNosZ are terminal reductases of anaerobic respiration. As such, these
enzymes receive electrons from a membrane-bound and pmf-generating electron
transport chain (ETC). Furthermore, as stated above, electron supply is possibly
involved in copper centre assembly and reductive (re-)activation of NosZ in order
Mitigation of Laughing Gas Emissions …
195
in a CX 2 C motif as Cu(I) ligands resulting in exceptionally low K D values in the
femto- to zeptomolar (10
–15 to 10
–21 ) range [75].
Unfortunately, knowledge on the maturation of the NosZ Cu A and Cu Z centres is
limited. It is assumed that these centres are synthesized by mutually independent
enzymes at the outside of the cytoplasmic membrane [19, 73, 76, 77]. The Cu A site
of NosZ is supposed to be functionally similar to that of respiratory haem-copper
oxidases, for example, the Cu A centre in subunit II of cytochrome c oxidase [72]. In
both cases, electrons are transferred via Cu A to the respective active sites of N 2 O or
O 2 reduction. The biogenesis of the Cu A site has been investigated in different types
of cytochrome oxidases and involves periplasmic copper chaperones of the
Sco/SenC family [78, 79]. Therefore, it is likely that Sco/SenC-type proteins are
also involved in NosZ Cu A assembly [80]. However, genes encoding such chaperones are generally absent from NGCs.
Most likely, the biogenesis of the Cu Z site involves the copper chaperone NosL,
whose function might depend on the proposed NosDYF complex (Tables 1 and 2).
Depending on the organism, NosL proteins are predicted to be exported by the Sec
or Tat system and some of them might be membrane-anchored lipoproteins.
Interaction of NosL and NosZ was indicated by a proteomic study using Pseudomonas aeruginosa [81]. In P. denitrificans, NosL was recently shown to be
required for NosZ activity and Cu Z (but not Cu A ) assembly only under
copper-limited conditions [77]. This finding was in line with previous reports that
characterized NosL as non-essential for cellular NosZ activity under
copper-sufficent conditions [69, 76, 82]. Thus, a NosL-independent pathway of
NosZ maturation is likely to exist when copper supply is sufficient. This assumption
is also supported by the fact that the nosL gene is absent in some NGCs [28].
A soluble form of P. denitrificans NosL was shown to bind one Cu(I) per protein
with attomolar affinity, whereas Cu(II) did not bind [77]. Therefore, it is tempting to
ask whether copper centre maturation in NosZ needs a Cu(II) to Cu(I) reduction
step and, if yes, whether the required electrons are donated via the same route as in
Cu Z * reactivation). Interestingly, the W. succinogenes NGC encodes three putative
NosL-type copper chaperones (Fig. 2) [83]. Of those, NosL2 is most closely related
to NosL proteins from, for example, Achromobacter cycloclastes and P. denitrificans whose NGCs encode only one NosL-type protein [84, 85]. However, the
individual role of the different NosL-type proteins has not been investigated.
5 The Enzymatic Level, Part II: Electron Transport Routes,
Bioenergetics and Function of Auxiliary Nos Proteins
NosZ or cNosZ are terminal reductases of anaerobic respiration. As such, these
enzymes receive electrons from a membrane-bound and pmf-generating electron
transport chain (ETC). Furthermore, as stated above, electron supply is possibly
involved in copper centre assembly and reductive (re-)activation of NosZ in order
Mitigation of Laughing Gas Emissions …
195