©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Copper-containing Dissimilatory Nitrite Reductase Involved in the Denitrifying System of the Fungus Fusarium oxysporum(*)

(Received for publication, September 1, 1994; and in revised form, November 2, 1994)

Michiyoshi Kobayashi Hirofumi Shoun (§)

From the Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(r) 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(2)O) was formed when cytochrome P-450nor was further added to the assay system. Therefore, the denitrifying (N(2)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 NObulletminbulletmg 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(r) = 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.


INTRODUCTION

Denitrification is an anaerobic respiratory process in which nitrogen oxides are reduced to dinitrogen (N(2)) or nitrous oxide (N(2)O) coupling with ATP synthesis(1, 2, 3) . Dissimilatory nitrite reductases (NiRs) (^1)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)) (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 N(2)O 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(2)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(2)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(2)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.


MATERIALS AND METHODS

Cultivation

F. oxysporum MT811 was cultivated as described previously (15, 18) with some modifications in a 5-liter Erlenmeyer flask containing 3 liters of the medium. The medium contained 3% glycerol (or soybean oil), 0.2% soybean flour (or peptone), and 10 mM sodium nitrate in 10 mM potassium phosphate (pH 7.5), supplemented with MgSO(4)bullet7H(2)O (0.02%), FeCl(3)bullet6H(2)O (6 ppm), CuSO(4)bullet5H(2)O (1.6 ppm), Na(2)MoO(4)bullet2H(2)O (1.6 ppm), CaCl(2)bullet2H(2)O (1.6 ppm), FeSO(4)bullet7H(2)O (0.6 ppm), and CoCl(2)bullet6H(2)O (0.16 ppm). The seed culture was made in the absence of nitrate and inoculated by 150 ml as reported (15) . The flask was sealed with a cotton plug and agitated at 130 rpm on a rotary shaker for the first 30 h, and at 110 rpm for more 18 h, and then cultured without agitation for more 24 h, at 30 °C. A typical yield of cells from the 3 liters of medium was 45 g in wet weight. When the cells were grown on the soybean oil-peptone medium, the culture flask was continuously agitated at 130 rpm for 4.5 days.

Detection of Nitrite Reductase Activity

Several lines of assay method were employed for detecting NiR activity. The method varied depending on combination of electron donor and the analytical method for determining the reaction product, nitric oxide (NO). As an artificial electron-donating system, NADH-PMS was usually employed. NO could be directly determined by gas chromatography (GC), but more frequently was further reduced to N(2)O by P-450nor and determined by GC. Another method for determining NO depended on the trapping by P-450nor. When P-450nor was added to the assay system, the evolved NO could immediately bind to P-450nor, which resulted in a distinct spectral change in P-450nor, and thus the evolved NO could be determined by the difference spectrum of P-450nor, i.e. ferric NO complex minus free ferric form (Delta at 434.4 nm = 77.6 mMbulletcm)(17) . NO was kept bound to P-450nor without further reduction to N(2)O, if NADH is absent in the system. This method is analogous to the NO-trapping with hemoglobin (19) and applicable to electron-donating systems except NADH-PMS. P-450nor was purified as reported(20) . These assay methods other than the direct determination of the reaction product of NiR, NO, were employed because of the low sensitivity in detecting NO by GC.

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 N(2)O 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(2)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.

Extraction of Nitrite Reductase and Electron Transferring Proteins

For purification of proteins the denitrifying cells were disrupted under a hypotonic condition by grinding with alumina in 20 mM malonate buffer containing 10% glycerol, 5 µM CuSO(4), and protease inhibitors (leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and N-tosyl-L-phenylalanyl chloromethyl ketone; 0.25 mM each). The extract was subjected to centrifugation twice, first at 10,000 times g and then at 150,000 times g, each for 60 min. The resulting supernatant, i.e. the soluble fraction, contained much higher NiR activity than any other fractions and applied to the following purifications of NiR and other components.

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.

Purification of Nitrite Reductase

All purification procedures were performed below 4 °C except that of FPLC (Pharmacia) which was performed at room temperature. Buffers used were potassium malonate (pH 5.5) containing 10% glycerol. NiR was assayed at pH 6.2 employing NADH-PMS as the electron donor and P-450nor for converting NO to N(2)O, respectively, as noted above.

After dialysis against 20 mM malonate buffer containing 5 µM CuSO(4), the soluble fraction above from 600 g wet weight of cells was loaded onto an SP-Sepharose column (30 times 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(r) = 20,000) and dialyzed against 20 mM malonate buffer containing 5 µM CuSO(4). Then the sample was applied to FPLC equipped with a Mono S cation-exchanger column (5 times 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 times 300 mm) equilibrated with 50 mM malonate buffer containing 150 mM KCl, and the collected NiR fraction was used as purified preparation.

Purification of Blue Copper Protein and Cytochrome c

The passed-through fraction from the first SP-Sepharose column above contained another blue protein that had no NiR activity. It was collected and dialyzed against 5 mM potassium phosphate buffer (pH 7.2) containing 10% glycerol, and applied to a DEAE-cellulose column (50 times 150 mm, Whatman DE52) equilibrated with the same buffer. The blue protein passed through the column without adsorption. Then the fraction was applied to a hydroxylapatite column (30 times 70 mm, Wako) equilibrated with 10 mM phosphate buffer (pH 6.7) and eluted with a linear gradient of 10-200 mM phosphate buffer. The eluted blue fraction was concentrated with a Centricon 10 filter and applied to a Superose 12 column equilibrated with 100 mM phosphate buffer (pH 6.7) containing 100 mM KCl. Finally the fraction was dialized and applied to high performance liquid chromatography (Tosoh) equipped with a hydroxylapatite column (HAP BH-08, Bio Tech Research, Kawagoe, Japan) equilibrated with 10 mM phosphate buffer (pH 6.7) and eluted with a linear gradient of 10-200 mM phosphate buffer.

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.

Molecular Weight Determination

The M(r) value of each protein in the native form was determined with gel filtration. The sample and standard proteins were applied to a Superdex 200 column (10 times 300, Pharmacia) equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl and chromatographed with an elution rate of 0.5 ml/min. Standards (Sigma) included beta-amylase (M(r) = 200,000), alcohol dehydrogenase (150,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), and cytochrome c (12, 600). The M(r) value in each subunit form was estimated by SDS-PAGE. Standard protein markers were obtained from Pharmacia and Bio-Rad. The gel was stained with Coomassie Brilliant Blue, and M(r) values were estimated by the method of Weber and Osborn(21) .

Other Analytical Methods

Spectrophotometric measurements were done with a Simadzu UV 2100 spectrophotometer. EPR spectrum was obtained with a JEOL JES-RE 1X spectrometer equipped with an Air Products model LTR-3 Heli-Tran cryostat system. The temperature was monitored and controlled with a Scientific Instruments temperature indicator/controller model 5500-5. Integrated intensities were obtained by comparison with aqueous CuEDTA. Protein was determined according to Lowry et al.(22) .


RESULTS

Distribution of Nitrite Reductase

As observed previously (15) , NADH-PMS-dependent NiR activity could be induced in F. oxysporum only when the cells were incubated with nitrate/nitrite under restricted aeration. This time we examined intracellular distribution of the NiR activity. The cells that had been exposed to the denitrifying condition were disrupted under an isotonic condition (in the presence of 0.8 M sucrose), and the resulting cell-free extract was fractionated by the differential centrifugation and assayed for NiR activity. As shown in Table 1, distribution of the activity depended on the culture medium where the cells were anaerobically incubated with nitrate. When the cells were incubated in the soybean oil-peptone medium a large portion of the activity was recovered in the large particle fraction, whereas most of the activity was recovered in the soluble fraction when the cells were incubated in the glycerol-soybean flour medium. When the cells were disrupted under a hypotonic condition (without osmotic stabilizer), almost all of the activity was recovered in the soluble fraction irrespective of the incubation medium (data not shown). These results are indicative that the NiR of F. oxysporum is a soluble protein but localizes in some organelle, although it cannot be elucidated why the distribution varied depending on the incubation medium.



Purification of Nitrite Reductase

The denitrifying cells of F. oxysporum were disrupted under the hypotonic condition and NiR was purified from the soluble fraction (Table 2). The NiR fraction was separated into two fractions by the SP-Sepharose column chromatography (not shown). The subfraction was also purified in the same manner as the main fraction and shown to exhibit properties almost identical to those of the purified main fraction (data not shown). Occurrence of isoformic Cu-NiRs has been reported also with some bacterial denitrifiers(23) .



SDS-PAGE of the purified NiR afforded a single band with M(r) of 41,800 (Fig. 1A), whereas the M(r) 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(r) = 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.



Spectral Properties

Fig. 2A shows the absorption spectrum of purified NiR, which exhibited in the visible region an absorption maximum at 595 nm ( per monomer = 5.38 mMbulletcm) and two weak shoulders around 470 and 782 nm. The strong absorbance around 595 nm caused a deep blue color. The blue color disappeared upon reduction with dithionite.


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.



Purification and Properties of Azurin-like Copper Protein and Cytochrome c

During the purification of NiR we noticed the presence in the cell-free extract of F. oxysporum of another blue protein and cytochrome c, both of which we also purified as described under ``Materials and Methods,'' since azurins and cytochrome c are physiological electron donors of bacterial NiRs(1) . The blue protein was purified to homogeneity (Fig. 1A) and its M(r) values deduced in both native (gel-filtration) and denatured (SDS-PAGE) forms were almost identical (15,000), indicating its monomeric structure. The absorption spectrum (Fig. 2B) afforded a single band at 625 nm, suggesting that this is also a copper protein. Cytochrome c (c) was purified to the extent so as to give electrophoretically a main band. Its M(r) value was estimated by both SDS-PAGE and gel-filtration as 14,000. It was recovered as a reduced form in the purification buffer containing dithiothreitol and its alpha, beta, and peaks appeared at 549, 521, and 410 nm, respectively (data not shown).

Nitrite Reductase Activity

As shown in Fig. 4, the fungal NiR produced NO and a small amount of N(2)O upon anaerobic incubation with nitrite in the presence of NADH-PMS as an electron donor. When cytochrome P-450nor (P-450nor), NOR derived from the same fungus(15, 17) , was further added to the assay mixture, nitrite was stoichiometrically and immediately converted to N(2)O. Therefore, the N(2)O-forming nitrite reductase activity that we previously detected in the crude extract of the denitrifying cells (15) could be reconstituted upon combination of the purified NiR and P-450nor. NADH is a physiological electron donor of P-450nor(17) , and thus the NADH-PMS system was effective as electron donors for both NiR and P-450nor. The K(m) and specific activity at pH 7.0 under the standard assay condition, where ascorbate-PMS and the P-450-trapping method were employed, were estimated as 49 µM and 447 units/mg, respectively. The optimal pH was below 5.5 (data not shown).


Figure 4: Nitrite reductase activity of the purified fungal NiR and N(2)O 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 (circle, box) or absence (bullet, ) of P-450nor. Formed NO (, box) and N(2)O (circle, bullet) were determined at each reaction time.



Formation of a small amount of N(2)O that arises from an NiR reaction only has been observed also with bacterial NiRs. Jackson et al.(26) proposed a mechanism (for the N(2)O evolution) that the product NO rebinds to NiR and reacts with another nitrite molecule to form N(2)O. The sticky nature might also be the case with the fungal NiR.

Effects of Inhibitors and Chelators on Nitrite Reductase

The fungal NiR was completely inhibited by cyanide and carbon monoxide, as shown in Table 3. Among metal chelators diethyldithiocarbamate, a potent chelator of copper, inhibited the enzyme most strongly. Although EDTA (1 mM) exerted little effect upon such a short time incubation, it (0.1 mM) decreased the activity down to below 10% upon incubation with the enzyme at 4 °C for a few days. The declined activity was, however, completely restored upon incubation with 0.1 mM CuSO(4) for only 10 min at room temperature (data not shown). These results further confirmed that copper is involved in the NiR reaction.



Electron Donors of Nitrite Reductase

Ascorbate-PMS and reduced methylviologen in addition to NADH-PMS were effective as artificial electron donating systems for the fungal NiR, as shown in Table 4. On the other hand, ascorbate-TMPD, ascorbate alone, and NADH alone were poor electron donors. Since ascorbate cannot be an electron donor of P-450nor(17) , the detection method was altered when ascorbate was examined for the assay.



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.




DISCUSSION

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(r) 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(r) 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 N(2)O(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. (^2)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.


FOOTNOTES

*
This work was supported in part by the University of Tsukuba Project Research (A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-53-4603; Fax: 81-298-53-4605.

(^1)
The abbreviations used are: NiR, nitrite reductase; NaR, nitrate reductase; NOR, nitric oxide reductase; P-450; cytochrome P-450; PMS, phenazine methosulfate; TMPD, N,N,N`,N`-tetramethylphenylenediamine; GC, gas chromatography; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

(^2)
A. Takimoto, H. Enjoji, M. Kobayashi, and H. Shoun, unpublished observation.


ACKNOWLEDGEMENTS

We acknowledge Dr. Yasuhiro Isogai of The Institute of Physical and Chemical Research (RIKEN) for his kind help in the EPR measurement.


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