(Received for publication, September 1, 1994; and in revised form, November 2, 1994)
From the
A copper-containing nitrite reductase (Cu-NiR) was purified to
homogeneity from the denitrifying fungus Fusarium oxysporum.
The enzyme seemed to consist of two subunits with almost the same M value of 41,800 and contains two atoms of copper
per subunit. The electron paramagnetic resonance spectrum showed that
both type 1 and type 2 copper centers are present in the protein,
whereas the visible absorption spectrum exhibited a sole and strong
absorption maximum at 595 nm, causing a blue but not green color. The
reaction product due to the Cu-NiR was mainly nitric oxide (NO),
whereas a stoichiometric amount of nitrous oxide (N
O) was
formed when cytochrome P-450nor was further added to the assay system.
Therefore, the denitrifying (N
O forming) nitrite reductase
activity that we had detected in the cell-free extract of the
denitrifying cells (Shoun, H., and Tanimoto, T.(1991) J. Biol.
Chem. 266, 11078-11082) could be reconstituted upon
combination of the purified Cu-NiR and P-450nor. The K
for nitrite and specific activity at pH
7.0 were estimated as 49 µM and 447 µmol
NO
min
mg protein
,
respectively. Its activity was strongly inhibited by cyanide, carbon
monoxide, and diethyldithiocarbamate, whereas enormously restored by
the addition of cupric ions. An azurin-like blue copper protein (M
= 15,000) and a cytochrome c were also isolated from the same fungus, both of which together
with cytochrome c of the yeast Saccharomyces cerevisiae were effective in donating electrons to the fungal Cu-NiR. The
result suggested that the physiological electron donor of the Cu-NiR is
the respiratory electron transport system. The intracellular
localization of Cu-NiR was investigated, and it was suggested that the
Cu-NiR localizes in an organelle such as mitochondrion. These findings
showed the identity in many aspects between the fungal nitrite
reductase and bacterial dissimilatory Cu-NiRs. This is the first
isolation of dissimilatory NiR from a eukaryote.
Denitrification is an anaerobic respiratory process in which
nitrogen oxides are reduced to dinitrogen (N) or nitrous
oxide (N
O) coupling with ATP
synthesis(1, 2, 3) . Dissimilatory nitrite
reductases (NiRs) (
)involved in bacterial denitrifying
systems can be divided into two main types based on their prosthetic
groups; one containing dihemes (hemes c and d
) (1, 4, 5) and the
other containing copper atoms. Copper-containing nitrite reductases
(Cu-NiRs) have been isolated from a lot of bacteria, such as Achromobacter cycloclastes(6, 7) , Alcaligenes faecalis strain S-6 (8) , Rhodobacter
sphaeroides sp. denitrificans(9) , Alc.
xylosoxidans subsp. xylosoxidans(10) , Bacillus halodenitrificans(11) , Pseudomonas
aureofaciens(12) , and Hyphomicrobium sp. A
3151(13) . These nitrite reductases, which are localized in the
periplasmic space (1, 14) , are linked to the
respiratory chain and accept electrons from cytochromes c and/or blue copper proteins (azurins) that act as physiological
electron donors(1) .
We recently found that the fungus Fusarium oxysporum has a marked denitrifying activity that
forms NO from nitrate or nitrite under anaerobic
conditions(15) . This was the first unequivocal demonstration
of denitrification by eukaryotic cells, although there had been a few
papers reporting formation of restricted amounts of N
O upon
anaerobic incubation of fungi with nitrite. We could further
demonstrate that many fungi other than F. oxysporum also
exhibit distinct denitrifying activities(16) . We could detect
in the cell-free extract of the denitrifying cells of F. oxysporum nitrite reductase and nitrate reductase (NaR) activities that
formed N
O from nitrite and nitrite from nitrate,
respectively, employing NADH-phenazine methosulfate (PMS) as an
electron donor(15) . We later showed that the latter half of
the nitrite reduction, i.e. the reduction of nitric oxide (NO)
to N
O, depends on a unique nitric oxide reductase (NOR),
cytochrome P-450nor (P-450nor)(17) . It should be of particular
importance and interest whether the fungal denitrification acts as
anaerobic respiration like bacterial systems. P-450nor receives
electrons directly from NADH and thus does not seem to associate with
the respiratory chain(17) . One of the physiological roles of
P-450nor would be a detoxifying mechanism that immediately makes the
hazardous molecule, NO, disappear. In contrast, we suggested a
possibility that NaR and/or NiR of F. oxysporum are associated
with the respiratory chain.
In the present study we purified and characterized NiR of F. oxysporum along with its potential physiological electron donors and reconstituted in vitro the fungal denitrifying process upon combination of purified NiR and P-450nor. The results are indicative of anaerobic respiration associated with the fungal denitrification.
A typical
assay system where NADH-PMS was employed as the electron donor
contained, in a total volume of 5 ml, NiR (0.1-1.3 µg), 2.0
mM sodium nitrite, 20 µM PMS, and 5 mM NADH in 100 mM potassium phosphate buffer (pH 6.2 or 7.0)
in a test tube (space volume, 20 ml). When the assay depended on the
detection of NO as described above, 0.8 µM P-450nor was further added. Before initiating the reaction the
assay system was degassed and the upper space was replaced with helium
and then the tube was sealed with a double butyl rubber stopper. The
reaction was initiated by adding degassed and condensed NADH solution
with a syringe, at 30 °C. Evolved NO or N
O was
determined by GC as reported (15, 16) with a Shimadzu
gas chromatograph GC 12A equipped with a Molecular sieve 5A or a
Porapack Q column. One unit was defined as producing 1 µmol of
NO/min.
When the azurin-like blue copper protein was examined as the electron donor, the reaction was performed in a Thunberg type cuvette. The reaction mixture (2.0 ml) contained NiR, reduced blue protein, 2.0 mM sodium nitrite, and 10 µM P-450nor in the above phosphate buffer. The cuvette was degassed, replaced with helium, and then sealed with a double butyl rubber stopper. The reaction was initiated by adding to the reaction mixture a degassed NiR solution with a syringe and monitored by the difference spectrum of P-450nor as described above. The reference cuvette contained the solution of free ferric P-450nor with the same concentration. The blue protein was purified as below, and its reduced form was prepared by reducing with dithionite followed by immediate desalting and utilization for the assay.
Cytochrome c was examined for its electron-donating activity also in the anaerobic cuvette above by measuring its oxidation caused upon incubation with NiR and nitrite. The blue protein and P-450nor in the reaction mixture (2.0 ml) above was replaced with 10 µM cytochrome c. Cytochrome c of F. oxysporum was obtained in reduced form under the purifying condition. Cytochrome c of the yeast S. cerevisiae was obtained as reduced form from Sigma.
For examining more exact distribution of NiR the cells were disrupted under an isotonic and milder condition, which was attained by replacing among the above components for disruption 10% glycerol with 0.8 M sucrose, and alumina with quartz sand.
After
dialysis against 20 mM malonate buffer containing 5 µM CuSO, the soluble fraction above from 600 g wet weight
of cells was loaded onto an SP-Sepharose column (30
100 mm,
Pharmacia) equilibrated with 20 mM malonate buffer, and eluted
with a linear gradient of 0-100 mM KCl in the same
buffer. Most of NiR activity was collected in a blue-colored fraction
eluted at about 25 mM KCl, whereas 10% of the total activity
was eluted at about 60 mM KCl. The major blue fraction was
concentrated with polyethylene glycol (M
=
20,000) and dialyzed against 20 mM malonate buffer containing
5 µM CuSO
. Then the sample was applied to FPLC
equipped with a Mono S cation-exchanger column (5
50 mm)
equilibrated with 20 mM malonate buffer and eluted with a
linear gradient of 0-40 mM KCl in the buffer. NiR
fraction was concentrated with a Centricon 50 filter (Amicon) and
applied to FPLC equipped with a Superose 12 gel-filtration column (10
300 mm) equilibrated with 50 mM malonate buffer
containing 150 mM KCl, and the collected NiR fraction was used
as purified preparation.
Cytochrome c-549 was strongly absorbed on the SP-Sepharose column and was eluted with a linear gradient of 100-300 mM KCl in 20 mM malonate buffer. The fraction was concentrated and applied to a Superose 12 column equilibrated with 100 mM phosphate buffer (pH 6.7) containing 100 mM KCl and 0.1 mM dithiothreitol. The resulting cytochrome preparation was used for the following experiment.
SDS-PAGE of the purified NiR afforded a single band with M of 41,800 (Fig. 1A), whereas the M
value in the native form was estimated as 83,300
by gel-filtration (data not shown), suggesting that the fungal NiR
consists of two subunits with identical molecular size. We also
succeeded in preparing its crystals (Fig. 1B).
Figure 1:
SDS-PAGE and crystals of purified
copper proteins from F. oxysporum. A, SDS-PAGE. Lanes 1 and 2 were performed respectively on purified
nitrite reductase (lane 1) and purified blue copper protein (lane 2). Arrows indicate respective band of the
purified proteins (10-15 µg). Marker protein kits included: 1, trypsin inhibitor (M = 20,100),
carbonic anhydrase (30,000), ovalbumin (43,000), and bovine serum
albumin (67,000) (Pharmacia); 2, lysozyme (14,400), trypsin
inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000),
and bovine serum albumin (66,200) (Bio-Rad). B, crystals of
nitrite reductase. The purified NiR was concentrated to about 12.5
mg/ml in 20 mM malonate buffer containing 10% glycerol and
stored at 4 °C for 5 days.
Figure 2: Absorption spectra of purified copper proteins. A, nitrite reductase in 20 mM potassium malonate buffer (pH 5.5), 0.33 mg/ml. B, blue copper protein in 50 mM potassium phosphate buffer (pH 6.7), 0.07 mg/ml (from absorbance at 280 nm).
The presence of copper in the fungal NiR was
further confirmed by the electron paramagnetic resonance (EPR)
measurement (Fig. 3). The EPR spectrum was characteristic of
Cu-NiRs that contain both type 1 and type 2 copper
centers(9, 11, 13, 23, 24, 25) ,
with the narrow and sharp hyperfine splitting (A= 6.82 mT, g
= 2.22)
originated from type 1 copper and the broader one derived from type 2
copper (approximate value of g
= 2.32). The
copper content determined by double integration of the EPR spectra was
estimated as 1.91 atoms/subunit. It can be thus concluded that the
fungal NiR contains both type 1 and type 2 coppers with 1 mol each/mol
of subunit.
Figure 3: EPR spectra of nitrite reductase. Conditions: temperature, 10 K; protein concentration, 3.57 mg/ml in 20 mM potassium phosphate buffer (pH 7.0); microwave frequency, 9.13 GHz; microwave power, 1 milliwatt; modulation amplitude, 1 millitesla; modulation frequency, 100 kHz.
Figure 4:
Nitrite reductase activity of the
purified fungal NiR and NO evolution due to the coupling
reaction with P-450nor. NiR was assayed as described under
``Materials and Methods'' at pH 7.0 employing the NADH-PMS
system, in the presence (
,
) or absence (
,
)
of P-450nor. Formed NO (
,
) and N
O (
,
) were determined at each reaction
time.
Formation of a small amount of NO that arises from an
NiR reaction only has been observed also with bacterial NiRs. Jackson et al.(26) proposed a mechanism (for the
N
O evolution) that the product NO rebinds to NiR and reacts
with another nitrite molecule to form N
O. The sticky nature
might also be the case with the fungal NiR.
Reduced blue
protein purified from F. oxysporum acted as an electron donor
of NiR, as shown in Fig. 5. Since the amount of blue protein
available was so small that its reoxidation could not be monitored by
the formed NO with P-450nor (see ``Materials and Methods'').
Similarly, reduced cytochrome c purified from F. oxysporum and cytochrome c of the yeast S.
cerevisiae were both oxidized anaerobically by NiR only when
nitrite was added to the reaction mixture, as shown in Fig. 6.
The fungal NiR also exhibited cytochrome c oxidase activity as
indicated, like bacterial NiRs(1) . The results indicated that
the blue protein as well as cytochrome c could act as electron
donors of the Cu-NiR of F. oxysporum. The absolute reaction
rate of NiR was almost comparable between the systems in Fig. 5B and Fig. 6, although concentrations of
NiR and its electron donor were much lower in the system in Fig. 5B. This indicated that the blue protein is a far
more effective electron donor than cytochrome c.
Figure 5: Reconstitution of nitrite reductase activity with purified, azurin-like blue protein. The fungal NiR was assayed at pH 7.0 employing purified blue protein (reduced form) as the electron donor, and formed NO was detected by the P-450-trapping method. A, the difference spectrum of P-450nor, ferric NO complex minus free ferric form, arose from the NiR-reaction (after 60 min in B) (P-450nor, 10 µM). B, NO formation due to the NiR reaction, detected by the increase of the peak at 434.4 nm in the difference spectrum. NiR, 0.9 µg/2 ml.
Figure 6: Cytochrome c could donate electrons to the fungal nitrite reductase. NiR activity, depending on cytochrome c, was assayed at pH 6.2 as described under ``Materials and Methods.'' The decrease in absorbance at 549 (or 550) nm due to oxidation of cytochrome c was measured. -, complete (anaerobic); - - - -, minus nitrite; - - -, minus nitrite but aerobic. A, cytochrome c isolated from F. oxysporum was used. B, cytochrome c of the yeast S. cerevisiae. NiR, 50 µg. For the anaerobic assay glucose oxidase (0.2 mg), catalase (0.4 mg) (both from Sigma), and 10 mM glucose were supplemented.
A dissimilatory nitrite reductase was isolated for the first
time from a eukaryote, F. oxysporum, in this study. It was
shown to be a copper protein like NiRs typically observed among
denitrifying bacteria. The fungal NiR closely resembled bacterial
Cu-NiRs in many aspects so far as examined such as absorption and EPR
spectra, electron donors, inhibitors, kinetic constants, optimal pH,
and the M value of subunit. Its absorption
spectrum revealed a single band at 595 nm with only a weak shoulder
around 460 nm, showing that NiR of F. oxysporum belongs to
blue copper NiR, like NiRs of Alc. xylosoxidans(10) , P. aureofaciens(12) , and Hyphomicrobium sp.
A3151(13) . NiRs of this type were formerly believed to contain
only type 1 copper center, however, have been proven to contain both
type 1 and type 2 copper
centers(13, 23, 25) , and the fungal NiR has
both, too.
Several bacterial Cu-NiRs have been shown to take
trimeric structures (27, 28, 29) , although
they were formerly considered as dimers on the basis of the M calibration by SDS-PAGE and
gel-filtration(11) . It is possible that this is also the case
with NiR of F. oxysporum. Fortunately, the fungal NiR
crystallized rather easily (Fig. 1), and thus we can expect
crystallographic studies on the NiR in future.
The blue protein of F. oxysporum revealed a strong visible absorption maximum only
at 625 nm, suggesting that it belongs to azurin rather than
pseudoazurin(30) . Purified cytochrome c revealed characteristics of cytochrome c involved in the
respiratory chain. Success in the electron transfer from these
components to the fungal NiR, like in cases of bacterial
NiRs(1) , is highly indicative that the NiR is associated with
the respiratory chain. This is further supported by the result that the
fungal NiR received electrons not only from these small proteins of the
same origin but also from the mitochondrial cytochrome c of
the yeast S. cerevisiae. NiR along with azurin and cytochrome c are usually localized in the periplasmic space in cases of
bacteria and recovered in the soluble fraction upon disruption of
cells. On the basis of the analogy to the bacterial system, it would
appear that these components of the fungus are localized in the space
between outer and inner membranes of mitochondria and recovered in the
soluble fraction when the cells are disrupted under hypotonic
conditions. This is also supported by the preliminary result on
localization of NiR (Table 1).
F. oxysporum performs
the dissimilatory reduction of nitrate up to
NO(15) . It has been shown by the present and
previous studies (15) that the process consists of three steps
as shown below.
It has been shown that similar to bacterial denitrifying systems
distinct reductase is involved in each step. And NiR and NOR of the
fungal system have been characterized as Cu-NiR and P-450,
respectively. Purification of NaR is also underway, and it would appear
that the fungal NaR also resembles the bacterial counterpart. ()It is therefore highly possible that NiR and NaR of the
eukaryote, F. oxysporum, are derived from bacteria. In
contrast, NOR of P-450 type is still not known among bacteria, although
we suggested that P-450nor is originated from bacteria on the basis of
comparison of its deduced primary amino acid sequence with those of
other P-450s(31) . If the fungal NiR and NaR are associated
with the respiratory chain as expected, the fungal denitrifying system
would consist of respiratory enzymes (NaR and NiR) and a
non-respiratory enzyme (P-450nor). However, these components seem to be
induced by a set of same mechanisms in response to nitrate/nitrite and
oxygen tension(15, 18, 32) . We found in the
upstream 5`-flanking region of the P-450nor gene a base sequence
identical to the FNR-motif(32) . So it is of particular
interest to compare the flanking regions between the genes of P-450nor
and other components (NiR, NaR, etc.).
According to the endosymbiotic theory (33) the mitochondrion of eukaryote is derived from a bacterium that had possessed a respiratory ability. And the present day denitrifying bacterium Paracoccus(34) or the alpha group of proteobacteria (33) is regarded as the direct descendant of symbionts, protomitochondria. Further studies on fungal denitrifying systems should contribute to further understandings of such problems as concerning the evolution from prokaryotic to eukaryotic cells.