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Phil. Trans. R. Soc. B (2012) 367, 1186–1194
doi:10.1098/rstb.2011.0335
Review
Fungal denitrification and nitric oxide
reductase cytochrome P450nor
Hirofumi Shoun*, Shinya Fushinobu, Li Jiang, Sang-Wan Kim
and Takayoshi Wakagi
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although
occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and
have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite
reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite
to nitrous oxide (N2O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases
to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has
the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial
origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene,
modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor
receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N2O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated.
Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a
hybrid N2 or N2O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen
donor (amines and imines). Possible involvement of NirK and P450nor is suggested.
Keywords: denitrification by fungi; co-denitrification; P450nor; mitochondrial anaerobic
respiration; NirK
1. INTRODUCTION
The nitrogen cycle performed by micro-organisms
comprises three processes; nitrogen fixation, nitrification and denitrification. The cycle is very important
for life and global environment, providing nitrogen to
life as nutrition and maintaining homeostasis of the
Earth. Denitrification is the reverse reaction of nitrogen
fixation in the sense that it carries fixed nitrogen back to
the atmosphere. The major source of global nitrous
oxide (N2O) emissions are the microbial activities of
nitrification and denitrification. Therefore, the control
and understanding of microbial denitrification is most
important for reducing N2O emissions. Features of
bacterial-denitrifying systems are well characterized at
a molecular level [1 – 3]. The bacterial-denitrifying
2
system comprises four reducing steps; NO2
3 ! NO2
! NO ! N2O ! N2, each of which is catalysed by a
dissimilatory nitrate reductase (dNar), dissimilatory
nitrite reductase (dNir), nitric oxide reductase (Nor)
and nitrous oxide reductase (Nos), respectively. The
reducing equivalents for these reactions are provided
from the respiratory chain coupling to the synthesis of
adenosine triphosphate (ATP), and thus bacterial
denitrification functions as anaerobic respiration.
Previously, organisms involved in the nitrogen cycle
were thought to be restricted to bacteria (prokaryotes).
About two decades ago, we showed that many fungi
and yeasts (eukaryotes) also exhibit distinct denitrifying
activities [4– 6]. Before our finding, there were many
papers reporting that fungi can evolve a small amount
(at most 15%) of N2O from, in most cases, nitrite,
and thus they may exhibit denitrifying activity [7].
However, these papers only reported the simple observation without providing any evidence that the small
amount of N2O evolution by fungi is a biological reaction. In contrast, we have characterized the denitrifying
system of fungi at the molecular level (identifying
both proteins and genes), mainly employing two
fungal species, Fusarium oxysporum strain MT811
(JCM11502) and Cylindrocarpon tonkinense IFO
(NITE Biological Resource Center; NBRC) 30561.
The most characteristic feature of the fungal-denitrifying system is the involvement of cytochrome P450
(P450) as nitric oxide reductase (P450nor) [8,9].
Since then, many papers from other groups have also
shown that fungal denitrification functions in nature
as a major process in the nitrogen cycle [10 – 13].
* Author for correspondence ([email protected]).
2. FUNGAL-DENITRIFYING SYSTEM
The denitrifying systems of F. oxysporum MT811 (JCM
11502) and C. tonkinense IFO (at present, NBRC)
One contribution of 12 to a Theo Murphy Meeting Issue ‘Nitrous
oxide: the forgotten greenhouse gas’.
1186
This journal is q 2012 The Royal Society
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Review. Fungal denitrification and P450nor
succinate
HCOOH
NADH
Cox
H. Shoun et al.
1187
H2O
complex IV
UQFdh
H– (2e– + H+)
complex II
2H (e– + H+)
NAD+
complex I
CoQ
complex III
dNar
dNir
Cyt. c
O2
(oxygen
respiration)
(denitrification)
NO3–
NO2–
NO
aNar
N 2O
P450nor
NAD(P)H
F. oxysporum MT811:
dNar (NarGHI)
-UQFdh couple
dNir (NirK)
P450nor
Cylindrocarpon
tonkinense IFO 30561:
aNar
dNir (NirK)
P450nor
Figure 1. Mitochondrial-denitrifying system of F. oxysporum MT811 and C. tonkinense sharing the respiratory chain with
oxygen respiration. F. oxysporum contains dNar (NarGHI)–UQFdh couple, NirK (dNir) and P450nor as the terminal
reductases (oxidases). The dNar–UQFdh couple is known to occur among bacteria performing ammonification such as
Escherichia coli, but is not known among denitrifying bacteria. Cylindrocarpon tonkinense also contains NirK and P450nor,
but not dNar. Instead, C. tonkinense can denitrify nitrate by using aNar.
30561 are depicted in figure 1. They comprise the minimal couple, NirK (copper-containing dNir) and
P450nor. The fungal system seems to lack Nos and
thus the final product is N2O. Both systems were
shown to function as the mitochondrial anaerobic
respiration [14]. Fusarium oxysporum MT811 also contains dNar that resembles the bacterial counterpart,
NarGHI. The fungal system of F. oxysporum MT811
is also unique in that dNar is supported by a ubiquinone-dependent formate dehydrogenase (UQFdh)
[15,16]. The couple of dNar and UQFdh from
Escherichia coli is well characterized but not known
among denitrifying bacteria. Formate, the electron
donor for the dNar–UQFdh couple, is provided from
pyruvate by a pyruvate-formate lyase (PfL). We
suggested that the same electron transport system comprising PfL, UQFdh and dNar is also functioning in
F. oxysporum MT811 [17]. The importance of formate
as the electron donor to the fungal-denitrifying system
in natural environments was recently demonstrated
[11]. Fungal denitrification requires a minimal amount
of oxygen supply [18]. Under such conditions oxygen
respiration and denitrification occur simultaneously
(hybrid respiration) [16] in intact mitochondria [18]
(figure 1). Fusarium oxysporum MT811 contains a variety
of strategies for survival under anoxic conditions; they
include not only denitrification but also heterolactic
acid fermentation and ammonia fermentation [19].
In contrast to the involvement of dNar in F. oxysporum, the denitrifying system of C. tonkinense does
not seem to contain dNar. However, it is of extreme
interest that C. tonkinense can denitrify nitrate under
certain conditions employing an assimilatory nitrate
Phil. Trans. R. Soc. B (2012)
reductase (aNar) to reduce nitrate to nitrite [20]. The
assimilatory nitrate-reducing system is ubiquitously distributed among plants and micro-organisms to provide
the nitrogen atoms of nitrate as nutrition for life. Assimilatory and dissimilatory nitrate-reducing systems were
previously thought to function independently of one
another. Therefore, involvement of aNar or assimilatory
nitrate-reducing system (aNar and aNir) in denitrification [20] and ammonia fermentation [18] are the first
instance of use of the assimilatory system for dissimilatory purpose (for producing ATP). In addition to the
difference between the denitrifying system of F. oxysporum and C. tonkinense, their carbon sources are also
different. In F. oxysporum, denitrification is repressed
and heterolactic acid fermentation dominates when glucose is available under anoxic conditions [19]. In
C. tonkinense, denitrification is not repressed by glucose,
but works in parallel with glycolysis via the pentose
phosphate shunt [21]. Nicotinamide adenine dinucleotide phosphate (NADPH)-specific P450nor isozyme
(P450nor2), which is localized in the cytosol, functions
as an electron sink for the pentose phosphate shunt.
The eukaryotic NirK protein and its gene were
firstly isolated from F. oxysporum MT811 [22,23].
Membrane-bound dNir protein was partially purified
from C. tonkinense [24]. The mitochondrial dNir
(C. tonkinense) along with dNar (F. oxysporum) activities were shown to be associated with the respiratory
chain coupling to the synthesis of ATP [14]
(figure 1). This is the first proof of the occurrence of
anaerobic respiration in mitochondria [25]. Recent
genome analyses have revealed the presence of nirK
gene homologues in many genomes of eukaryotes,
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Review. Fungal denitrification and P450nor
(a) 400
aniline + 15NO2–
29N
2
350
(b) 600
15N-aniline
+ NO2–
29N
2
500
250
400
200
300
29N
2
(mmol)
300
150
200
100
100
50
0
40
80 120 160 200 240
incubation time (h)
0
40
80 120 160 200 240
incubation time (h)
Figure 2. Co-denitrification by Fusarium solani. Fusarium solani IFO 9425 was cultured in a 500 ml Erlenmeyer flask containing
150 ml of the medium, which comprised 2 per cent glucose, 5 mM potassium nitrite (KNO2; 15N-labelled or non-labelled),
5 mM aniline (15N-labelled or non-labelled), 0.2 per cent peptone and inorganic salts (pH 7.5), on a rotary shaker at
150 r.p.m., at 308C. The gas phase of flask was replaced with helium to attain anaerobic conditions. Hybrid N2 species
(14N15N) was determined by gas chromatography-mass spectrometry with a Shimadzu GCMS-GP5050 instrument (Shimadzu,
Kyoto, Japan) equipped with a CP-PoraPLOT-Q column (Varian, Palo Alto, CA, USA). Filled circles, F. solani þ; open circles,
F. solani – .
including fungi, protozoa and green algae. We showed
that all of these eukaryotic homologue genes along
with the nirK genes of F. oxysporum and C. tonkinense
[26] form a closely related group (clan) sharing the
same ancestor, in sharp contrast to the random distribution of nirK and nirS (encoding cytochrome cd1 type
dNir) genes among denitrifying proteobacteria [23].
Further, no gene homologous to nirS is found
among fungal genomes. From these results, we proposed the possibility that eukaryotic nirK genes along
with fungal-denitrifying systems originate from the
protomitochondrion (the endosymbiont that gave rise
to the mitochondrion) [23].
The genome analyses have also revealed that not
only nirK homologue genes but also the genes homologous to CYP55 (P450nor) and nap (periplasmic nitrate
reductase) are found in many fungal genomes. Our
BLAST results indicated that out of 72 fungal genomes
19 contained nirK homologues, 16 contained CYP55
and 15 contained nap homologues (26.4 –20.8%).
And many of these genomes contained both nirK
and CYP55. The high ratio of the appearance of
nirK and CYP55 homologues suggests that the denitrifying system comprising NirK and P450nor is widely
distributed among fungi. By contrast, no genome
contained a narGHI homologue, suggesting that
the fungal-denitrifying system that contains NarGHI
like that of F. oxysporum MT811 is minor. The significance of the presence of nap homologues in many
fungal genomes remains to be elucidated.
3. CO-DENITRIFICATION
Fungal denitrification is often accompanied by a unique
phenomenon, co-denitrification, in which a hybrid
N2 or N2O species is formed upon combination of nitrogen atoms from nitrite and other nitrogen compounds
(nitrogen donor) [5,27]. A similar phenomenon was
later found in the anammox reaction [28]. The ratio
of denitrification and co-denitrification varies
Phil. Trans. R. Soc. B (2012)
depending on the conditions (fungal strains and nitrogen donors). The co-denitrification product (N2 or
N2O) varies depending on the redox state of the nitrogen donor. Amines provide N2 [5] whereas imines or
azide form N2O [27] as the co-denitrification product,
as shown below.
15
14
15 14
NO
2 þ R NH2 ! N N
15
14
15 14
NO
2 þ R NHOH ! N NO
P450nor was shown to catalyse the co-denitrification
reaction forming N2O and N2 from NO and azide
[29]. An external electron donor such as nicotinamide
adenine dinucleotide (NADH) is not necessary, indicating that the nitrogen donor also functions as an internal
electron donor to reduce NO. It would therefore appear
that the direct reactant is NO rather than nitrite in the
co-denitrification reaction. In co-denitrification, nitrite
would be reduced to NO by dNir (NirK), followed by
the co-denitrification reaction by P450nor.
Fusarium solani IFO (NBRC) 9425 exhibits
potent co-denitrification activity [5]. Among the
three fungal strains tested (F. oxysporum MT811,
C. tonkinense and F. solani IFO 9425), F. solani exhibited the highest co-denitrification activity against the
nitrogen donor (aniline; figure 2). The recovery of
nitrogen atoms of nitrite and aniline into N2 is high
(usually more than 50%), as shown in figure 2. Inhibitors of NirK (diethyldithiocarbamate and cyanide)
strongly inhibited co-denitrification, suggesting
involvement of NirK in the reaction. The same
product (N2) was formed when nitrite was replaced
with NO (L. Jiang & H. Shoun 2009, unpublished
results), again suggesting that the direct reactant is
NO rather than nitrite. Therefore, the fungaldenitrifying system can produce N2 as the co-denitrification product, although it cannot form N2 by
denitrification. Laughlin & Stevens [10] reported
fungal dominance of denitrification and co-denitrification in a grassland soil. It therefore appears that both
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Review. Fungal denitrification and P450nor
Fusarium_oxysporum_P450nor_CYP55A1
Cylindrocarpon_tonkinense_P450nor1_CYP55A2
Neurospora_crassa_CYP55A6
Cylindrocarpon_tonkinense_P450nor2_CYP55A3
Trichosporon_cutaneum_P450nor_CYP55A4
Aspergillus_oryzae_P450nor_CYP55A5
Streptomyces_avermitilis_PteC_CYP105P1
Streptomyces_avermitilis_PteD_CYP105D6
Streptomyces_griseolus_P450-SU1_CYP105A1
Streptomyces_griseolus_P450-SU2_CYP105B1
Saccharopolyspora_erythraea_P450eryF_CYP107A1
Pseudomonas_putida_P450cam_CYP101A1
Pseudomonas_spp_P450terp_CYP108A1
Saccharomyces_cerevisiae_P450-14DM_CYP51F1
Mycobacterium_tuberculosis_CYP51B1
Homo_sapiens_CYP1A1
Homo_sapiens_CYP2A6
Homo_sapiens_CYP2C9
Persea_americana_CYP71A1
Homo_sapiens_CYP3A4
Parthenium_argentatum_CYP74A2
Candida_maltosa_P450alk_CYP52A3
Rattus_norvegicus_CYP4A10
Bacillus_megaterium_P450-BM3_CYP102A1
Fusarium_oxysporum_P450foxy_CYP505A1
H. Shoun et al.
1189
CYP55
(P450nor)
prokaryotic
eukaryotic
Figure 3. Phylogenetic tree of P450s.
co-denitrification as well as fungal denitrification occur
generally in nature.
4. CYTOCHROME P450nor (FUNGAL NITRIC
OXIDE REDUCTASE)
P450nor was first isolated from F. oxysporum MT811 as
a haem protein possessing lipoxygenase activity and the
properties of P450 [30]. Of course, the real function of
the haem protein as Nor was not then known. The serendipitous finding that the P450 is specifically induced
by nitrate (or nitrite) [31] led us to discover fungal denitrification [4]. Isolation of the gene showed that the
haem protein belongs to the P450 superfamily, with
the family number 55 (CYP55) being identified [32].
Interestingly, in spite of its eukaryotic origin CYP55
exhibits a closer relationship to bacterial P450s than
to eukaryotic P450s (figure 3). The amino acid
sequence of CYP55 shows sequence identities to the
bacterial (actinomycetes) CYP105 members as high
as about 40 per cent. So we suggested that the fungus
had acquired the P450 gene from actinomycetes by
horizontal transfer [32]. Once the fungal denitrification
was found, it was rather easy to find the physiological
function of CYP55 as Nor (P450nor) [8] because
it was involved in denitrification. However, it took
10 years after its isolation to elucidate its physiological
function. Surprisingly, P450nor could receive electrons
directly from NADH. This phenomenon seemed to
oppose the central dogma of physiological electron
transfer, because two electrons of NADH are transferred simultaneously as a hydride ion (H2) and thus
Phil. Trans. R. Soc. B (2012)
a one-electron redox centre such as haem can never
receive the two electrons directly. P450 usually receives
electrons from NAD(P)H via an electron transport
system (redox partner) containing a flavoprotein.
P450 can be classified depending on the type of redox
partner (figure 4). Bacterial and mitochondrial P450s
are supplied with electrons by the couple ferredoxin
reductase and ferredoxin, whereas eukaryotic (microsomal) P450s are supplied by a P450 reductase
containing FAD and FMN. P450nor is an exceptional
P450 [33] that does not require a redox partner
(direct electron transfer from NAD(P)H).
The reaction mechanism of P450nor has been
extensively studied [34,35]. The turnover of the overall reaction, 2NO þ NADH þ Hþ ! N2O þ H2O þ
NADþ, is very rapid: 1000 s21 at 108C, and thus
should be of the order of 105 min21 or more at 258C
[34]. The overall reaction can be divided into three
steps (figure 5). The first substrate (NO) binds to
ferric (Fe3þ) P450nor to form a ferric-NO complex
Fe3þ –NO
is
then
(Fe3þ þ NO ! Fe3þ –NO).
reduced with NADH to form a specific intermediate
(I) with a Soret absorption peak at 444 nm (Fe3þ –
Finally,
I
NO þ NADH þ Hþ ! I þ NADþ).
interacts with the second NO to form N2O (I þ NO
! N2O þ H2O). The chemical entity of I was proposed
to be ferric-hydroxylamine radical complex, as
shown in figure 5 [35]. The reaction mechanism
(figure 5) is also supported by a quantum-chemical
calculation [36].
P450nor is localized to both mitochondria and cytoplasm in the fungal cells. Fusarium oxysporum and
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Review. Fungal denitrification and P450nor
I. bacteria/mitochondria type (ferredoxin–ferredoxin reductase)
ferredoxin
reductase
SH + O2 + 2H+
haem
(P450)
FeS
ferredoxin
FAD
NAD(P)H
SOH + H2O
II. eukaryote/microsome type (P450 reductase)
NAD(P)H
SH + O2 + 2H+
haem
FAD
FMN
SOH + H2O
III. direct transfer (P450nor; NO reductase)
NAD(P)H + H+
2NO
haem
N2O + H2O
Figure 4. Classification of P450 depending on the type of redox partner. Electrons of NAD(P)H are transferred to P450 as
indicated by arrows via each component. P450nor (type III) receives electrons directly from NAD(P)H and thus does not
require such redox partners. Components of the bacterial P450 system are water-soluble. Mitochondrial P450s are membrane-bound (inner membrane), whereas mitochondrial ferredoxin reductase and ferredoxin are soluble in the matrix.
Components of type II (eukaryotic, microsomal type) are membrane-bound. P450nor is water-soluble, localized to both mitochondria and cytosol. SH represents the organic substrate to be hydroxylated by P450 (monooxygenase)-reaction.
(a)
1
431
0.5
absorbance
0.4
444 9
9
0.3
1
0.2
1: 11 ms
2: 19 ms
3: 26 ms
4: 39 ms
5: 54 ms
6: 79 ms
7: 89 ms
8: 99 ms
9: 119 ms
(b) 413nm
Fe3+–H2O
kobs = 40 s–1, 10°C
H2O
NO
H+
0
400 420 440 460 480 500 520 540
wavelength (nm)
NADH
N2O
H–
NO
NAD+
444nm
H
H
0.1
431nm
Fe3+–NO
Fe4+
N
OH
Fe3+
overall reaction: 2NO + 2e– + 2H+
N
OH
N2O + H2O
Figure 5. Reaction mechanism of P450nor. (a) Spectral changes in the bound haem during anaerobic reduction of ferric
(Fe3þ) –NO complex (431 nm species) with NADH to form a specific intermediate, I (444 nm species), observed by rapid
scan analyser [34]. (b) Reaction scheme of P450nor [34]. The structure of I (444 nm species) as an Fe3þ –hydroxylamine
radical complex was proposed by Daiber et al. [35].
C. tonkinense localize P450nor in different manners. Two
P450nor isoforms of F. oxysporum are derived from a
single gene (CYP55A1). P450norA of F. oxysporum is
translated from the first initiation codon of the gene
including the mitochondrial targeting signal, whereas
P450norB is translated from the second initiation
codon below the targeting signal and is thus localized
to cytosol [37,38]. Cylindrocarpon tonkinense contains
two P450nor genes: for P450nor1 (CYP55A2) and
P450nor2 (CYP55A3) [39]. CYP55A2 contains
a sequence for a mitochondria-targeting signal, whereas
CYP55A3 does not. P450nor1 specifically employs
NADH as the electron donor, while P450nor2 prefers
NADPH to NADH, although NADH can afford
sufficient activity [40]. The electron donor specificity
Phil. Trans. R. Soc. B (2012)
depends on the amino acid residues at two positions in
the B0 -helix (73rd and 75th positions in the case of
CYP55A1) [41,42]. Steric hindrance due to side
chains of Ser73 and Ser75 in CYP55A1 excludes the
20 -phosphate moiety of NADPH from the site. In
P450nor2 of C. tonkinense, Ser75 is replaced with Gly,
permitting accommodation of NADPH. Double
mutation at these sites in P450nor of F. oxysporum
(S73G/S75G; GG mutant) markedly improved the
specificity against NADPH [43].
Direct electron transfer from NADH to the haem of
P450nor was conclusively demonstrated by kinetic
analysis [44] and by the determination of a crystal
structure of a P450nor (GG mutant) complexed with
an NADH analogue (nicotinic acid adenine
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Review. Fungal denitrification and P450nor
F-helix
B-helix
NAAD
Glu71
Arg174
Arg64
Asp88
I-helix
NO
Arg292
Cys352
Figure 6. Movement of P450nor on cofactor binding. Superposition of ferric–NO complex (white) and NAAD complex
(black).
dinucleotide; NAAD) [43]. The structure of the
P450nor – NAAD complex is compared with that of
the ferric – NO complex of P450nor [45] in figure 6.
Little difference was observed between the structures
of P450nor in the ferric resting state [46] and in the
ferric – NO complex [45]. By contrast, a remarkable
conformational change in the protein was induced
upon binding of NAAD (figure 6). The entrance
gate of the haem distal pocket is closed. Two Arg residues, Arg64 and Arg174, play a key role in the binding
[41] by putting the pyrophosphate moiety of NAAD
between them. Glu71, Arg64 and Asp88 form a salt
bridge network to stabilize the protein structure
[44,47]. The interaction between Arg64 and Asp88
is broken upon binding of NAAD to destabilize the
protein. This makes a driving force to exclude rapidly
an NADþ molecule from the active site after electron
transfer is finished. One of the propionate side chains
of the haem moves up accompanying the movement
of Arg292, which fixes the nicotinamide ring stereochemically. The conserved Thr residue (Thr243) in
the I-helix interacts with the carboxyl of nicotinic
acid ring to fix it stereochemically. This interaction
of Thr243 together with the propionate of haem
moving upward restricts the conformation of the
nicotinic acid ring so that the pro-R side of C4hydrogens faces the haem, which is consistent with
the pro-R hydrogen-specific hydride transfer [35].
A hydrogen bond network is formed to deliver a
proton from solvent to Ser286 that is located in the
close vicinity of haem [45]. However, the network is
rearranged to form a proton channel upon binding of
NAAD, and the bound NADH (NAAD) which
is itself involved in the network [43] (figure 7),
suggesting that a proton is supplied to the enzymatic
reaction via the proton channel before formation of
the intermediate (444 nm species; in the second step
in figure 5). This is because the hydrogen bond
network containing NAD would be degraded after
the release of NADþ (the last step in figure 5). The
proton supply to form the intermediate is consistent
with the structure of the intermediate (ferric-hydroxyl
amine radical complex).
Phil. Trans. R. Soc. B (2012)
H. Shoun et al.
1191
5. CONCLUDING REMARKS
Fungal denitrification is involved in the nitrogen
cycle in nature as a major pathway. This is supported
by the distribution of nirK (dNir) and CYP55
(P450nor) gene homologues in many fungal genomes
(more than 20%), together with several recent papers
showing the predominance of fungal denitrification
in various environments. Since the final product of
fungal denitrification is N2O, it appears that fungal
denitrification is one of the major sources of N2O
emissions. Acidification of environments, for example,
by acid rain and excess use of ammonia fertilizer,
promotes fungal activity resulting in an increase in
N2O emissions.
Most of fungal-denitrifying systems seem to contain
NirK and P450nor as essential components. These
two genes are the minimum pair to ensure denitrification from nitrite to N2O. Some fungi further use dNar
(Nar GHI type in the case of F. oxysporum MT811)
and/or aNar, which also enable denitrification of
nitrate. NirK and dNar are associated with the mitochondrial respiratory chain, coupled to the synthesis
of ATP. This is the first example of the occurrence of
anaerobic respiration in mitochondria. By contrast,
P450nor and aNar receive electrons directly from
NAD(P)H and thus are not associated with the respiratory chain. Thus, P450nor and aNar function as an
electron sink under anoxic conditions. Thus ATPproducing metabolism, being inefficient in ATP
production, reflects the strategy of fungi for survival
under anoxic conditions, in which preference is for
speed of metabolism over energy efficiency. The significance of the presence of Nap-homologue genes in
many fungal genomes remains to be elucidated.
Eukaryotic nirK and its homologue genes obviously
originate from the same ancestor, possibly the protomitochondrion which harboured NirK-type (but not
NirS-type) dNir [23]. Thus, the fungal and bacterialdenitrifying systems share the same origin. However,
P450nor is unique to the fungal system. It appears
that the mitochondrial-denitrifying system replaced
the original Nor protein with P450nor, whose gene
was initially obtained from bacteria (actinomycetes) by
means of horizontal gene transfer. The prototype
P450 gene would have encoded the usual monooxygenase, whereas fungi would have modulated the gene to
give Nor activity, because P450nor is now not found
among bacteria.
P450 proteins belonging to the P450 superfamily are
among the most diversified enzyme proteins. However,
even among such diversified P450 proteins, the function
of P450nor is peculiar [33]. The function of P450nor is
thus atypical of most diversified P450 proteins. The
mechanism of the stereospecific transfer of H2 from
NADH to the haem of P450nor has been elucidated.
In addition to Nor activity, P450nor will catalyse the
co-denitrification reaction. We have also found that
P450nor exhibits NADH-peroxidase activity (H2O2 þ
NADH þ Hþ ! 2H2O þ NADþ; S. Nakaya & H.
Shoun 2008, unpublished data). P450nor is therefore
a multi-functional detoxifying enzyme. P450nor is also
related to the pathogenicity of a fungus [48].
Co-denitrification is the first process to show the
formation of a hybrid N2 or N2O species [5,27]. It will
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1192
H. Shoun et al.
Review. Fungal denitrification and P450nor
salt bridge side
(a)
Glu71
proton
channel
Ser286
Arg64
(b)
Asp88
proton
channel
Asp393
NADH
channel
Asp393
Ser286
Thr243
hydrophobic side
Figure 7. NADH and proton channels. (a) Perpendicular to and (b) parallel with the haem plane. Water molecules forming the
proton channel are shown as spheres.
depend on the reaction of NO with a nitrogen donor,
which is possibly catalysed by P450nor and thus characteristic of fungal denitrification. Co-denitrification
products (N2 or N2O) vary depending on the redox
state of the nitrogen donor: N2 is formed from amines
and N2O from imines or azide. This suggests that the
nitrogen donors also act as an internal electron donor.
Thus, the co-denitrification process can be considered
to be a kind of Nor reaction employing amines or
imines as an internal electron donor (and nitrogen
donor). The molecular mechanism of co-denitrification
needs further elucidation. The mechanism of co-denitrification by the bacterium Streptomyces antibioticus [49],
in which a very small amount of a hybrid N2 species is
formed, also remains to be elucidated, because
P450nor is not found in bacteria.
This work is the result of collaborations of the author (H.S.)
with many researchers and students of University of
Tsukuba, the University of Tokyo, Riken, University of
Konstanz, Osaka Prefecture University and Chiba University.
This work was supported by Grants-in-aid for scientific
research from the Japan Society for the Promotion of Science
and the Research and Development Programme for New
Bio-Industry Initiatives. We thank the staff of Photon Factory
and SPring-8 for X-ray data collection.
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