THF. JOURNAI. OF BIOLOGICAL CHISMIS.I.H\ Vol. 251, No. 22, Issue of November 2.5. pp. 699G7000, 1976

Printed rn U.S.A.

A New Oxygenase, Z-Nitropropane Dioxygenase of Hansenula mrakii

ENZYMOLOGIC AND SPECTROPHOTOMETRIC PROPERTIES *

(Received for publication, June 14, 1976)

TOSHIKO KIDO AND KENJI SODA+

From the Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan

TETSUYA SUZUKI AND KOZI ASADA

From the Research Institute for Food Science, Kyoto University, Uji, Kyoto-Fu 611, Japan

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IF0 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000).

Stoichiometrical studies and the results obtained with la02 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively denitrified.

The enzyme, which exhibits absorption maxima at 274, 370,415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe’+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe*+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity.

The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, &hydrox- yquinoline, reducing agents such as 2-mercaptoethanol, and HgCl,. The Michaelis constants are as follows: 2-nitropropane (2.13 x lo-’ M), nitroethane (2.43 x lo-’ M), 3-nitro-2-pentanol (6.8 x 10m3 M),

1-nitropropane (2.56 x lo-’ M), and oxygen (3.03 x 10m4 M, with 2-nitropropane).

Since Little (1, 2) demonstrated the oxidative degradation of damental properties have been shown (5). The enzyme exhibits nitroethane and 2-nitropropane by extracts of Neurospora absorption maxima at 370, 415, and 440 nm and a shoulder at crassa and pea seedlings, respectively, little attention has been 470 nm in the visible region and catalyzes the following reac- paid to the metabolism of nitro compounds. Molina and tion when 2-nitropropane is a substrate. Alexander (3) reported that extracts from the hyphae of a nitrifying strain of Aspergillus flaws produce nitrite and

ZCHFH(NO,)CH, + 0, + ZCH,COCH, + 2HN0,

nitrate from 3-nitropropionate. Recently, we found that 2- In this paper, we present evidence that 2-nitropropane is nitropropane and some other nitroalkanes are oxidatively oxidized by the enzyme with molecular oxygen to yield denitrified by an intracellular enzyme of Hunsenula mrakii and acetone, into which oxygen is incorporated, and also describe other organisms grown in the medium containing the nitro some enzymological and physicochemical properties of this compounds as the sole nitrogen (4). The nitroalkane-oxidizing new oxygenase, 2-nitropropane dioxygenase, with emphasis on enzyme has been purified to homogeneity and some of the fun- the prosthetic groups.

*This work was supported in part by a Grant-in-Aid (021110) for Scientific Research from the Ministry of Education, Science, and

EXPERIMENTAL PROCEDURES

Culture, Japan. Materid-‘*O, gas (‘“0, 95.064 atom %; l7O, 0.404 atom so) was $ To whom correspondence regarding this work should be addressed. purchased from Yeda R & D Co., Rehovoth, Israel. 3.Nitropropionate,

6994

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Properties of 2-Nitropropane Dioxygenase 6995

3.nitro-2.butanol, 2.nitro-1.butanol, and 3.nitro-2.pentanol were pur- chased from Aldrich Chemical Co., Inc., Wis.; Bio-Gel P-150 from Bio-Rad Laboratories, Richmond, Calif.; and Chromosorb 101 from Johns-Manville Research, Denver, Cola. The other chemicals were analytical grade reagents obtained from Nakarai Chemicals, Kyoto, Japan.

Enzyme Preparation-2.Nitropropane dioxygenase was purified to homogeneity from a cell-free extract of Hansenula mrakii (IF0 0895) as described previously (5). The specific activity of the enzyme prepara- tion was approximately 15,500.

Enzyme Assay-2-Nitropropane dioxygenase was assayed by deter- mining nitrite formed. The standard reaction mixture consisted of 50 nmol of 2-nitropropane, 100 nmol of potassium phosphate buffer (pH 8.0), and enzyme in a final volume of 1.0 ml. In a blank the enzyme was replaced by water. Incubation was carried out at 37” for 20 min with shaking, followed by addition of 0.1 ml of glacial acetic acid. After centrifugation, if necessary, 0.1 ml of the solution was used for the determination of nitrite. Nitrite was determined with sulfanilamide and N-(l-naphthyl)ethylenediamine according to the method of Ida and Morita (6). One unit of the enzyme is defined as the amount of enzyme required to form 1 nmol of nitrite per min. Specific activity is expressed as units per mg of protein.

Protein Determination-Protein was determined by measuring the absorbance at 274 nm. The absorbance coefficient of the enzyme (A;‘;,, = 9.80) was used throughout, obtained by absorbance and dry weight determination.

Sedimentation Coefficient and Molecular Weight Measurements- The sedimentation coefficient of enzyme was determined with a Spinco model E ultracentrifuge equipped with a phase plate as a schlieren diaphragm. The top speed of ultracentrifugation was 59,780 rpm (7).

The molecular weight of enzyme was determined by the sedimenta- tion equilibrium method according to the procedure of Van Holde and Baldwin (8). The measurements were made in a Spinco model E ultracentrifuge equipped with Rayleigh interference optics. Multicell operation was employed in order to perform the experiment on four samples of different initial concentrations ranging from 0.067 to 0.119% with the use of An-G rotor and double-sector cells of different side-wedge angles. The rotor was centrifuged at 12,590 rpm at 20”. Interference patterns were recorded at intervals of 1 h to compare and make sure the equilibrium was established. The relationship between the concentration of the enzyme and the fringe shift was determined using the synthetic boundary cell.

The molecular weight was also estimated by gel filtration on a Bio-Gel P-150 column (1 x 70 cm) at 4” according to the method of Andrews (9). The column was standardized with y globulin, asparagi- nase of Escherichia coli (lo), amino acid racemase with low substrate specificity (ll), albumin, ovalbumin, chymotrypsinogen A, myoglobin, cytochrome c, and blue dextran 2000.

The molecular weight of the subunits of enzyme was estimated by sodium lauryl sulfate-polyacrylamide gel electrophoresis according to Weber and Osborn (12).

Amino Acid Composition-Amino acid analyses were performed according to the method of Spackman et al. (13) with a Yanagimoto amino acid analyzer. Samples were hydrolyzed in 6 y HCl under reduced pressure for 24, 48, and 72 h at 110”. Cysteine and cystine were determined as cysteic acid after oxidation of protein with performic acid and hydrolysis (14). Tryptophan and tyrosine were determined spectrophotometrically by the method of Beaven and Holiday (15) with protein samples which had been thoroughly dialyzed against 10 mM acetate buffer (pH 3.5) to remove the prosthetic groups.

Experiments with ‘80,-The isotope incorporation experiments were performed in a 20-ml Thunberg tube equipped with two side arms and a high vacuum stopcock. A Thunberg tube contained, in a total volume of 2 ml, 200 nmol of potassium phosphate buffer (pH 8.0) and enzyme in the main compartment and 100 pmol of 2.nitropropane in the side arm. After the contents of the tube were frozen in liquid N,, the tube was made anaerobic by repeated evacuation and flushing with argon. ‘sO, was introduced into the tube and the temperature of the tube was brought to 37”. The reaction was started by tipping 2.nitropropane into the main compartment and carried out at 37” for 1 h with shaking. The reaction was stopped by the addition of 0.4 ml of 5 mM CuSO,.

Gas Chromatography-Mass Spectrometry-The incorporation of ‘*O, into acetone molecule was verified by gas chromatography-mass spectrometry and mass fragmentography. Assays were performed with a Shimadzu-LKB-9000 gas chromatograph-mass spectrometer. Ioniza-

tion voltage, emission current, and acceleration voltage were 20 eV, 60 PA, and 3.5 kV, respectively. The ion source was kept at 210”. Gas chromatography was performed on a coiled glass column (3 mm, inner diameter x 2.0 m) packed with Chromosorb 101, 80 to 100 mesh. Helium was used as carrier gas at a flow rate of 30 ml/min. Injection port and interface to the mass spectrometer were kept at 140” and 150”, respectively. The column was programmed from 70 to 120” at a rate of S”/min and then to 140’ at a rate of G”/min.

180-labeled acetone was trapped and monitored by mass fragmen- tography using a single ion monitoring system focused at m/e 60. Chart speeds of a recorder for gas chromatogram and an oscillograph recorder for mass fragmentogram were synchronously run at 10 mm/min.

Other Instrumentation-Absorption spectra were taken with a Shimadzu MPS50L recording spectrophotometer. Spectrophotomet- ric assay of the enzyme activity was made with a Hitachi EPO-B spectrophotometer or a Carl Zeiss PMQ II spectrophotometer with a 1.0~cm light path. Oxygen consumption was determined with a Clark type oxygen electrode YSI 4004, Yellow Spring Instruments, Ohio. Fluorescence spectra were recorded with a Shimadzu spectrofluorome- ter type RF 502 with a 1.0.cm light path. Metal analysis was performed with a Hitachi-Perkin-Elmer 303 atomic absorption spectrophotome- ter.

RESULTS

Molecular Weight of Enzyme-In the sedimentation velocity experiments, the sedimentation constant (sza. W) was deter- mined to be 3.43, 3.40, 3.26, and 3.18 S at the enzyme concentrations 0.27, 0.41, 0.48, and 0.55%, respectively. By extrapolation to zero protein concentration the sedimentation coefficient (siO, ,) was calculated to be 3.76 S.

The molecular weight of the enzyme was determined by sedimentation equilibrium method. Enzyme solution at four different concentrations, 0.67, 0.87, 1.07, and 1.19 mg/ml, was centrifuged at 20”. Assuming a partial specific volume of 0.74, a molecular weight of 64,000 was obtained.

A molecular weight of approximately 60,000 was also obtained by the Bio-Gel P-150 gel filtration method.

Structure of Subunit-Incubation of the enzyme with 1% sodium lauryl sulfate or 6 M guanidine . HCl resulted in a gradual and irreversible loss of the activity, which was reduced to one-half its initial value after 50 min at 37” and lost completely after approximately 10 h. The enzyme was dena- tured almost immediately with 8 M urea or thiourea. No activity was recovered after removal of the denaturants by dialysis or Sephadex G-25 gel filtration. These results suggest that the denaturants cause irreversible conformation changes in the enzyme, possibly with dissociation into subunits.

The subunit structure of the enzyme was examined by disc gel electrophoresis. The enzyme was incubated with 1.0% sodium lauryl sulfate and 2-mercaptoethanol in 10 mM sodium phosphate buffer (pH 7.0) at 37” for approximately 12 h. The treated enzyme preparation was subjected to electrophoresis in the presence of 0.1% sodium lauryl sulfate (12). There were two bands of stained protein as shown in Fig. 1. To determine the molecular weights of the polypeptides in the bands, we ran a series of marker proteins treated in the same manner; bovine serum albumin (Mr = 68,000), catalase (Mr = 58,000), alcohol dehydrogenase (Mr = 41,000), n-amino acid oxidase (Mr = 37,000), asparaginase of Escherichia coli (M, = 35,000), chymotrypsinogen A (Mr = 25,000), and ribonuclease (Mr = 13,700). The molecular weights of the subunits were estimated to be approximately 39,000 and 25,000 from a semilogarithmic plot of molecular weight against mobility, suggesting that the enzyme consists of two subunits different in molecular weight.

Amino Acid Composition-The enzyme was dialyzed against water for 48 h. Four samples of 250 pg of the enzyme were lyophilized in acid-washed tubes. One sample was treated with

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6996 Properties of 2-Nitropropane Dioxygenase

TABLE I

Amino acid composition of 2-nitropropane dioxygenase

Experimental details are eiven in the text.

FIG. 1. Sodium lauryl sulfate-disc gel electrophoresis of 2-nitropro- pane dioxygenase. Twenty micrograms of the enzyme was subjected. The direction of migration is from the cathode (top) to the anode.

performic acid. Hydrolysis was performed in 1 ml of 6 N HCl in evacuated and sealed tubes as described above. The hydroly- sates were evaporated to dryness under reduced pressure and subjected to an amino acid analyzer in duplicate. The amino acid composition of the enzyme is given in Table I. All values represent the average of the 24-, 48, and 72-h hydrolysis times except for arginine, valine, leucine, and isoleucine which are given by the 72-h hydrolysis time and for serine and threonine which have been extrapolated to zero hydrolysis time. The number of amide nitrogen was not determined. The enzyme has aspartic acid, glutamic acid, glycine, alanine, and leucine as the plentiful amino acids.

Enzymatic Incorporation of 1802 into Acetone-The enzymatic oxidation of 2-nitropropane was carried out in an ‘8O2 atmosphere and the reaction mixture was analyzed using a Shimadzu-LKB-9000 gas chromatograph-mass spectrometer. Fig. 2 shows the gas chromatogram of the reaction product. The formation of acetone was judged from its retention time. The mass spectrum was taken at the apex of Peak I. The mass spectrum of the acetone peak shows a parent peak of m/e = 60 corresponding to **O-labeled acetone in addition to that of m/e = 58 (Fig. 3). The formation of 180-labeled acetone indicates the incorporation of ‘*OS during the enzymatic oxidation of 2nitropropane. The formation of CH&lBOCHs is partially due to iB02 involved in 95 atom % ‘*OS used and also probably to a slow exchange of I80 between labeled acetone and Hz”0 during the reaction (16).

The mass fragmentogram monitoring [**O]acetone (m/e = 60) is shown in Fig. 4, which was identified by comparison with authentic acetone in retention time of gas chromatograph. The mass fragmentogram of the reaction product obtained under “02 did not show any peak.

These results indicate that molecular oxygen is incorporated into acetone. Thus, the enzyme acts as an oxygenase. When 1-nitropropane and nitroethane were used as substrates, no incorporation of “0 into the products (propionaldehyde and acetaldehyde) was observed, indicating that the exchange of oxygen between the aldehydes and water is far more rapid compared to that between acetone and water.

Amino acid No. of residues No. of proposed

amino acid 24h 48 h 12 h residues

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

38.7 17.5 10.5 65.8 31.2 45.7 60.4 15.9 48.9 46.8

22.7 25.9 27.3 9.7 10.1 9.9

27.5 29.8 31.8 40.1 43.5 45.3 12.8 12.9 13.4 22.4 21.8 22.1

mol amino acidlmol enzymeD

39.9 40.2 18.3 18.1 12.9 14.2 67.3 67.5 30.5 25.1 40.1 36.8 61.5 61.3 15.5 16.3 49.5 51.3 46.5 45.1

40 18 14 67 32 50 61 16 50 46

4 27 10 32 45 13 22 10

a m, = 62,000.

0 10 20 30

RETENTION TIME ( min 1

FIG. 2. Gas chromatogram of acetone (0 produced enzymatically from 2nitropropane (11). A 3-~1 aliquot of the reaction mixture was gas-chromatographed. Partial pressure of “‘0, (95%) was 0.2 atm. Experimental details are given in the text.

Stability of Enzyme-When the enzyme was stored at -20” in 1.0 or 10 mM potassium phosphate buffer (pH 7.0), practi- cally no loss of activity was observed at least for 3 months. When the enzyme was incubated in 10 mM potassium phos- phate buffer (pH 7.0) at various temperatures for 5 min, the remaining activity was 100% of the original activity level at 40”, 51.7% at 45’, 3.4% at 50°, and 2.6% at 55”. The enzyme was found to be relatively stable between pH 6.0 and 8.0.

Effect of pH and Buffers-The pH dependence for 2-nitro- propane dioxygenase was determined in various buffers. The enzyme is active in the alkaline pH region with a maximum at about pH 8.0 (Fig. 5). The activity varied with the kind of buf- fers used and the relative activities in 0.1 M buffers, pH 8.0, were as follows: 100 in potassium phosphate, 48.8 in Tris/HC!l, 46.2 in citric potassium phosphate, and 9.1 in Veronal/HCl.

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Properties of 2-Nitropropane Dioxygenase 6997

Effect of Temperature-The enzyme was assayed at various temperatures. The maximum activity was found at 40”. The reaction velocity increased linearly when the temperature was raised in the range of 20-40” and declined rapidly over 45”. The activation energy calculated according to Arrhenius plot was 14,000 cal/mol.

00 43

80- CH,C-“0’

60- c,-,,c=‘80+ M+ 45 58

40-

60 20- HCs160+

29 HC’laO+ 31

0’ I II I II * I I I . . , I I I 20 30 40 50 60 70

We Substrate specificity of 2.nitropropane diorygenase

FIG. 3. Mass spectrum of Peak I in Fig. 2. Experimental conditions are given in the text.

Nitro compounds Relative activity0

I 0 10 20 30 (min)

FIG. 4. Mass fragmentogram (m/e = 60) of reaction product. Experimental details are given in the text. A, “‘0 experiment; B, I60 experiment.

2oL

f-

FIG. 5. Effect of pH on 2-nitropropane dioxygenase activity. The enzyme activity was measured in the following buffers. 0, potassium phosphate; A, citric-potassium phosphate; 0, Tris/HCl; A, glycine/ KOH; 0, Veronal/HCl.

Substrate Specificity-The ability of the enzyme to catalyze oxygenation of various nitroalkanes was examined (Table II). In addition to 2-nitropropane, nitroethane and 3-nitro-2-pen- tanol were the preferred substrates, whereas nitromethane and 2-nitro-l-butanol were oxidized slightly or not at all. These substrates were converted into nitrite and the corresponding carbonyl compounds.

Kinetics-Fig. 6 shows a double reciprocal plot for the relationship between reaction velocity and substrate concen- tration. The Michaelis constant for 2-nitropropane estimated under the standard conditions was 2.13 x lo-’ M (&rue Z).

2-Nitropropane was little oxidized under anaerobic condi- tions. The K, for oxygen was determined to be 3.03 x lo-’ M

(Fig. 6, Curve ZZ).

The K, values for other substrates also were determined as follows: nitroethane, 2.43 x lo-* M; 3-nitro-2-pentanol, 6.8 x 10m3 M; and l-nitropropane, 2.56 x lo- * M.

hhibitors-The various compounds were investigated for their inhibitory effects on the enzyme activity (Table III). The

TABLE II

2.Nitropropane 100

Nitroethane 88.7 3-Nitro-2-pentanol 40.6 1-Nitropropane 23.4 3-Nitro-2.butanol 13.8 3-Nitropropionate 11.7 2-Nitro-I-butanol 2.7 Nitromethane 0

“Relative activities were calculated from the values obtained at the following optimum pH for each substrate. 2-Nitropropane, 3.nitro-2- pentanol, 1-nitropropane, 3.nitro-2-butanol, and 2-nitro-1.butanol (pH 8.0); 3-nitropropionate (pH 7.5); and nitroethane (pH 7.0). When nitromethane was used, the activity was determined at pH 7.0, 7.5, and 8.0.

0.5 1.0 0.1

0.05 sn

FIG. 6. Effect of concentrations of 2-nitropropane (0 and oxygen (U) on 2-nitropropane dioxygenase activity. The activity was deter- mined in the reaction mixtures containing various concentrations of 2-nitropropane and oxygen under the standard conditions. Various oxygen concentrations were obtained by replacing oxygen in the reaction mixture with argon. The reaction was started by the addition of enzyme. The reactions under various oxygen concentrations were followed polarographically. S,, concentrations of 2.nitropropane, 10 mM; s,,, concentrations of oxygen, 0.1 mM; V,, nitrite formed, nmol/ min; V,,, oxygen consumed, 0.1 rmol/min.

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Properties of 2-Nitropropane Dioxygenase 6998

TABLE III

Effect of inhibitors

The standard reaction mixture was used except that the mixture contained an inhibitor listed. The concentration of inhibitors was 1 mM. The reaction was started by addition of enzyme.

Addition Relative activity

Tiron 0 Oxine 17.5 EDTA 87.0 TPTZ 111.1 a,oc’-Dipyridyl 128.3 o-Phenanthroline 126.9 Cysteine 0 Z-Mercaptoethanol 28.2 Glutathione 0 NaHSO, 15.1 p-Chloromercuribenzoate 63.0 N-Ethylmaleimide 62.0 Iodoacetate 92.0 WX 2.2 None 100

enzyme was strongly inhibited by tiron’ and oxine, while EDTA did not inhibit the enzyme significantly. TPTZ, 01, a’-dipyridyl, and o-phenanthroline were activators rather than

inhibitors. The addition of thiol compounds such as cysteine and glutathione resulted in a marked decrease in the enzyme activity.

Among the thiol inhibitors tested p-chloromercuribenzoate and N-ethylmaleimide, and HgCl, acted as moderate and potent inhibitors, respectively, although iodoacetate was almost ineffective. Nitromethane inhibited noncompetitively the enzymatic oxygenation of 2-nitropropane.

Prosthetic Groups

2-Nitropropane dioxygenase exhibited absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm (Fig. 7, Curve I), suggesting the occurrence of flavin nucleotide and also non-heme iron as prosthetic groups (5).

Fluorescence Spectrum-The fluorescence spectrum of the enzyme was measured in 10 mM potassium phosphate (pH 7.0). The enzyme emitted fluorescent light with a maximum at 520 nm upon irradiation at 370, 440, and 470 nm. When fluores- cence emission was monitored at 520 nm, excitation spectra exhibited maxima at 380 and 450 nm (Fig. 8). The fluorescence is characteristic of a flavoprotein.

Identification of FAD-The identification and determina- tion of flavins in the enzyme were carried out according to Udenfriend (17). The lyophilized enzyme (0.450 mg) was treated with 0.5 ml of 10% trichloroacetic acid followed by centrifugation. The supernatant solution neutralized with 0.5 M K,HPO, activated apo-n-amino acid oxidase. Thus, the flavin moiety was identified as FAD.

FAD Content-To the enzyme solution (0.59 mg/0.5 ml) was added 0.5 ml of 10% trichloroacetic acid and the mixture was centrifuged. After the supernatant solution was allowed to stand at room temperature overnight, 0.4 ml of the solution was neutralized with 0.4 ml of 0.5 M K,HPO, and was filled up to 1.0 ml with water. The intensity of fluorescence of the

1 The abbreviations used are: tiron, pyrocatechol-3,5-disulfonate disodium salt; oxine, 8-hydroxyquinoline; TPTZ, 2,4,6-tripyridyl-s- triazine.

1 UI 350 400 450

WAVELENGTH ( nm 1

FIG. 7. Reduction of FAD of 2-nitropropane dioxygenase by 2.nitro- propane. A Thunberg-type cuvette contained the enzyme (1 mg) in 10 mM potassium phosphate buffer (pH 8.0) in the main chamber and 0.1 nmol of 2.nitropropane in the side arm in a total volume of 1.5 ml. After evacuation, 2-nitropropane was tipped into the enzyme solution and the incubation was carried out at 25” for 5 min (II). Oxygen was introduced into the reaction mixture after reduction (110. I, the absorption spectrum of the native enzyme.

WAVELENGTH (nml

FIG. 8. Excitation spectra of 2-nitropropane dioxygenase (0 and FAD (10. The spectra of the enzyme (17 pM) and FAD (2 x 10m5 M) analyzed at 520 nm were measured in 10 mM potassium phosphate buffer (pH 7.0) at 25”. Intensity is given as uncorrected values.

solution at 520 nm, with excitation at 450 nm, was measured and the amount of flavin was determined on the basis of fluorescence of authentic FAD. An average value of 9.22 nmol of FAD/O.59 mg of protein was obtained. The result indicates that the enzyme contains 0.95 mol of FAD/mol of enzyme protein.

An attempt was made to dissociate the bound FAD from the protein moiety. To the enzyme solution was added ammonium sulfate to 70% saturation, and the mixture was acidified to pH 2.0 by the cautious addition of 1 N HCl. After 2 h the solution was brought to 90% saturation with ammonium sulfate. The resultant precipitate was dissolved in a small volume of 10 mM

potassium phosphate buffer (pH 7.0) and dialyzed against the same buffer. The enzyme thus treated showed only an absorp- tion peak at 274 nm and was not reconstituted by incubation with Fad and potassium ferricyanide. When the enzyme was acidified to pH 3.0 and treated in the same way, the prosthetic groups did not dissociate from the protein and almost full activity remained.

Labile Sulfur and Heme-When the enzyme was examined by the method of King and Morris (18), no significant amount

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Properties of 2-Nitropropane Dioxygenase 6999

of labile sulfur was detected. When 2.1 M pyridine in 0.075 N

NaOH was added to the dithionite-reduced enzyme, the absorption spectrum corresponding to the pyridine hemo- chrome (19) was not observed.

Metal Analysis of Enzyme-An atomic absorption analysis for metals was conducted with the enzyme dialyzed thoroughly against 0.1 mM potassium phosphate buffer (pH 7.0) for 48 h. The enzyme (2.548 mg/ml) was digested with 0.3 ml of 60% perchloric acid in a micro-Kjeldahl flask until the contents became colorless and iron was determined. The result was corrected for the blank which contained 1.37 ml of the dialysate and 0.3 ml of 60% perchloric acid. The enzyme (9.52 nmol) contained 10.4 nanoatoms of iron, indicating the occurrence of 1.09 g atom of iron/m01 of enzyme.

Spectrophotometric Behavior

The absorption spectrum of the enzyme in the visible region is characterized by maxima at 370,415, and 440 nm and a shoul- der at 470 nm (Fig. 7, Curve Z).

The enzyme-bound FAD was reduced by 2-nitropropane under anaerobic conditions and absorption bands at 370, 440, and 470 nm disappeared (Fig. 7, Curve ZZ). The addition of oxygen caused reoxidation of the reduced enzyme (Fig. 7, Curve III).

A spectral change was also observed under the anaerobic conditions by the addition of nitromethane, which is not a substrate (Fig. 9). The reduced enzyme was not influenced by the addition of oxygen, but dialysis under stirring caused the reappearance of only an absorption peak at 415 nm correspond- ing to the bound Fe3+ (&rue IV).

The anaerobic addition of sodium dithionite also led to the immediate disappearance of the absorption peaks in the visible region, indicating the reduction of enzyme-bound FAD and also Fe3+ (Fig. 10). When the enzyme was thoroughly dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immedi- ately after the reduction, the absorption spectrum similar to that of the native enzyme reappeared with concomitant restoration of approximately 80% of the enzyme activity (Table IV). However, the activity of the enzyme treated with sodium dithionite at 25“ for a long period (e.g. 1 h) was not recovered

0.2 ( I

by dialysis and the spectral change to the native form was not observed, suggesting the denaturation of enzyme.

DISCUSSION

The initial step of nitroalkanes metabolism in Hansenula mrakii has been demonstrated to be an oxidative denitrifica- tion to yield nitrite and the corresponding carbonyl com- pounds, and nitrite is further metabolized to ammonia by nitrite reductase (4). We have purified the nitroalkane-oxidiz- ing enzyme to homogeneity from the cells grown in a medium containing nitroethane as the inducer (5). The stoichiometry of the reaction suggested that the enzyme is an oxygenase rather than an oxidase. From the present results obtained with “0, and 2-nitropropane and the stoichiometry, it is evident that 2 atoms of oxygen molecule are incorporated into the carbonyl groups of 2 molecules of acetone formed. Dioxygenases are de- fined as enzymes catalyzing reactions in which both atoms of molecular oxygen are incorporated into substrates (20). Thus, the nitroalkane-oxidizing enzyme is designated “2-nitropro- pane dioxygenase” and classified in E.C. class 1.13.11. Diox- ygenases acting on one and two oxygen acceptor substrates are referred to as intramolecular and intermolecular dioxygenase, respectively (21). According to this terminology, 2-nitropro- pane dioxygenase is an intermolecular dioxygenase and is unique in incorporating 2 atoms of oxygen molecule into 2 molecules of the same acceptor. Furthermore, the enzyme is the first oxygenase to be isolated from yeasts and to be shown to act upon a nitro group. The characterization of prosthetic group of the enzyme shows that 2-nitropropane dioxygenase is a non-heme iron flavoprotein containing 1 mol of FAD and 1 g

02 , I

WAVELENGTH ( n m 1

FIG. 9. Reduction of 2.nitropropane dioxygenase by nitromethane. A Thunberg-type cuvette contained the enzyme (1.2 mg) in 10 mM potassium phosphate buffer (pH 8.0) in the main chamber and 0.1 pm01 of nitromethane in the side arm in a total volume of 1.5 ml. After evacuation, nitromethane was tipped into enzyme solution and the incubation was carried out at 25” for 5 min (10. Oxygen was introduced into the reaction mixture (110, and then the enzyme was dialyzed (IV). I, the spectrum of the native enzyme.

350 403 450 x0

WAVELENGTH (nm I

FIG. 10. Effect of sodium dithionite on the spectrum of 2-nitropro- pane dioxygenase. Enzyme solution (0.1%) in 10 mu potassium phosphate buffer (pH 7.0) was used. The spectra were taken before (I) and after (10 the addition of sodium dithionite (0.1 mg) under anaerobic conditions and after dialysis of the dithionite-reduced enzyme against 10 mM potassium phosphate buffer (pH 7.0) (110.

TABLE IV

Effect of sodium dithionite on 2.nitropropane diorygenase actiuity

Enzyme preparations Relative activity

Native enzyme 100 Reduced enzyme I” 79.6 Reduced enzyme II* 0

a The enzyme was dialyzed immediately after the addition of sodium dithionite to the enzyme.

*The enzyme was dialyzed after incubation of the enzyme with sodium dithionite for 1 h.

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7000 Properties of 2-Nitropropane Dioxygenase

atom of iron/mol of enzyme but no labile sulfur. Attempts were

made to dissociate reversibly the prosthetic groups from the protein moiety without success.

2-Nitropropane, the preferred substrate, and several other aliphatic nitro compounds are oxidized more or less by

2-nitropropane dioxygenase, but nitromethane is inert. The enzyme is characterized by its high Michaelis constants (2.13 x lo-* M for 2-nitropropane and 3.03 x 1O-4 M for oxygen). Other oxygenases studied so far have generally a relatively strong affinity for the substrates, e.g. lysine monooxygenase: K,, for lysine and oxygen, 1.45 x 10 ~’ and 1.0 x 10e4 M, re- spectively (22); pyrocatechase, 4 x 1O-.6 M for pyrocatechol and 2.0 x 10m5 M for oxygen (23); and gentisate 1,2-dioxy-

genase, 7.1 x 10e6 M for gentisate (24). When the substrate nitro compounds are incubated with the

enzyme under anaerobic conditions, the bound FAD is reduced although the absorption peak corresponding to Fe3+ is not influenced. On the contrary, dithionite reduces both the bound

FAD and Fe3+. The enzyme reduced by the substrates or dithionite is reoxidized by oxygen. These findings suggest that the substrates react first with the FAD to be dehydrogenated, and the FAD is regenerated by oxygen, although the function of iron in the catalytic reaction has not been elucidated. The anaerobic addition of nitromethane, which is not a substrate, to the enzyme also results in the reduction of both the enzyme-bound FAD and Fez+. When oxygen is introduced to the reduced enzyme, the bound FAD is not reoxidized, but Fe2+ is. Furthermore, nitromethane inhibits noncompetitively the denitrification of 2-nitropropane. Further investigation is needed to shed light on the mechanism of inhibitory action by nitromethane.

The enzyme is significantly inhibited by tiron and oxine, whereas some other chelating agents for divalent or trivalent metals such as EDTA and a,cu’-dipyridyl act as weak inhibitors or activators. The inhibition by tiron and oxine is probably not due to chelation of iron bound to the enzyme. Thiol compounds are as effective inhibitors as tiron and oxine, although thiol reagents also inhibit the enzyme to some extent. Recently we

have obtained evidence for the participation of superoxide anion as an intermediate in the reaction.’ It has been reported that tiron, oxine, and thiols trap superoxide anion effectively

*T. Kido, K. Soda, and K. Asada, manuscript in preparation.

(25, 26). Therefore, it is likely that the inhibition by these inhibitors results from the removal of superoxide anion.

Achnowledgments-We thank Dr. S. Yamamoto, Dr. M. Nozaki, Dr. Y. Ishimura, Dr. K. Yano, and Dr. T. Yamamoto for their helpful advice.

REFERENCES

1. Little, H. N. (1951) J. Biol. Chem. 193, 347-358 2. Little, H. N. (1957) J. Biol. Chem. 229, 231-238 3. Molina, J. A. E., and Alexander, M. (1971) J. Bacterial. 105,

489-493 4. Kido, T., Yamamoto, T., and Soda, K. (1975) Arch. Microbial.

106, 165-169 5. Kido, T., Yamamoto, T., and Soda, K. (1976) J. Bacterial. 126,

1261-1265 6. Ida, S.. and Morita. Y. (1973) Plant and Cell Phvsiol. 14.661-671 7. Schachman, H. (1957) Methods Enzymol. 4, 32-71 ’ 8. Van Holde. K. E.. and Baldwin. R. L. (1958) J. Phvs. Chen. 62. ”

734-743 9. Andrews, P. (1964) Biochem. J. 91, 222-233

10. Nakamura, N., Morikawa, Y., Fujio, T., and Tanaka, M. (1971) Agr. Biol. Chem. 35, 219-225

11. Soda, K., and Osumi, T. (1969) Biochem. Biophys. Res. Commun. 35, 363-368

12. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 13. Spackman, D. H.. Stein, W. H.. and Moore. S. (1958) Anal. Chem.

-30, 1190-1206 14. Moore, S. (1963) J. Biol. Chem. 238, 235-237 15. Beaven, G. H., and Holiday, E. R. (1952) Ado. Protein Chew 7,

319-386 16. Breslow, R. (1974) Organic Reaction Mechanisms, p. 185, W. A.

Benjamin, Inc., New York 17. Udenfriend, S. (1962) Fluorescence Assay in Biology and Medi-

cine, p. 241, Academic Press, New York 18. King, T. E., and Morris, R. 0. (1967) Methods Enzymol. 10,

634-637 19. Paul, K. G., Theorell, H., and Akerson, A. (1953) Acta Chem.

Stand. 7, 1284-1287 20. Hayaishi, 0. (1974) Molecular Mechanisms of Oxygen Actiuation,

p. 6, Academic Press, New York 21. Hayaishi, 0. (1974) Molecular Mechanisms of Oxygen Actiuation,

p. 7, Academic Press, New York 22. Takeda, H., Yamamoto, S., Kojima, Y., and Hayaishi, 0. (1969) J.

Biol. Chem. 244, 293552941 23. Hayaishi, O., Katagiri, M., and Rothberg, S. (1957) J. Biol. Chem.

229, 905-920 24. Crawford, R. L., Hutton, S. W., and Chapman, P. J. (1975) J.

Bacterial. 121, 794-799 25. Miller, R. W., and Rapp, U. (1973) J. Biol. Chem. 248,6084-6090 26. Asada, K., and Kanematsu, S. (1976) Agr. Biol. Chem. 40, 1891-

1892

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T Kido, K Soda, T Suzuki and K Asadaand spectrophotometric properties.

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic

1976, 251:6994-7000.J. Biol. Chem. 

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