Environmental Engineering Reference
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where a soluble nitrous oxide reductase catalyzes the two-electron reduction to N
2
(see equation
1
in Section
1
), using electrons that originate from menaquinol.
Energetically, denitrification is the most efficient respiratory pathway in the
absence of molecular oxygen as a terminal electron acceptor, and consequently
denitrifiers thrive in environments with low oxygen levels and abundant supply of
nitrate [
7
]. Coincidentally, these conditions are met quite precisely in modern
industrial agriculture, where the growth-limiting element nitrogen is provided as
a fertilizer, commonly in the form of nitrate salts. Soil denitrifiers compete with
the assimilating crops for this nutrient, to the effect that approximately half of the
fertilizer dispersed on the fields is returned to the N
2
from which it was produced in
the Haber-Bosch process [
3
].
With a current global fertilizer production of approximately 160 Mt per year, the
overall metabolic flux through the denitrification pathway is therefore substantial.
The second condition for denitrification, an anoxic or microoxic environment, is
met quite readily as well, as the availability of O
2
diminishes drastically already
millimeters or centimeters into the rather stratified soil of a regularly watered field.
Nitrate reduction increases sharply at this oxycline, where traces of O
2
may be
encountered, but can generally be tolerated. The weakest link in the chain of
denitrificatory enzymes, however, is the one catalyzing the final step from N
2
Oto
N
2
, nitrous oxide reductase. Its copper centers show the highest sensitivity, and
while the reaction of the enzyme is presumably coupled to the generation of a
proton motive force [
23
], the organism can tolerate its failure, in particular because
the gaseous substrate N
2
O can be released and will not accumulate to inhibit the
preceding NO reductase. The result is a direct correlation of the increasing use of
nitrogen fertilizers in agriculture and the rise of atmospheric N
2
O released through
incomplete denitrification. While agriculture thus is just one of the factors contri-
buting to the rise of atmospheric levels of nitrous oxide, it is the one most directly
related to human population growth with a direct link to the ongoing industriali-
zation of highly populated countries such as India or China [
16
].
3 Nitrous Oxide Reductase
The enzymatic conversion of N
2
O into the stable products N
2
and water (equation
1
)
is carried out by copper-containing nitrous oxide reductase. After noticing a general
requirement for copper for N
2
O reduction by
Alcaligenes faecalis
[
24
], the enzyme
itself was identified in a dedicated search for copper-containing enzymes in
Pseudomonads [
25
], with the first ortholog to be characterized being the one from
Pseudomonas stutzeri
strain ZoBell (originally
P. perfectomarina
)[
26
].
A variety of other orthologs was characterized over the years and detailed insight
was gained concerning the two metal sites, but it was only in the year 2000 that a
first crystal structure became available for the enzyme from
Pseudomonas nautica
[
27
,
28
] (now
Marinobacter hydrocarbonoclasticus
)
29
] and in 2003 for
Paracoccus denitrificans
[
27
,
30
]. Structural data was later presented for N
2
O
reductase from
Achromobacter cycloclastes
[
31
], and more recently for the
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