Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

Bacterial strains and growth conditions. Acidovorax sp. (formerly Pseudomonas sp.) strain JS42 (20) was used for the isolation of oxygenase_2NT and ferredoxin_2NT. DH5α(pDTG800), which carries the ntdAaAbAcAd genes from JS42 on pUC18 (29), was also used for the purification of these proteins using the same method. Escherichia coli JM109(DE3)(pDTG871) was used for the isolation of reductase_2NT. The expression clone pDTG871 was constructed by amplification of the gene encoding reductase_2NT (ntdAa) from plasmid pDTG800 (29) using PCR and the following primers: 5′-ntdAa, 5′-GATCCATATGGAACTGGTAGTAGAACCCCTC-3′, and 3′-ntdAa, 5′-GATCAAGCTTCAGACGCCGCTGGGATAGAACGC-3′. The 1-kb fragment was cloned into XcmI-digested pTAV1 (4). The resulting plasmid, pDTG870, and vector pT7-7 (40) were digested with NdeI and HindIII. Insertion of ntdAa into pT7-7 gave plasmid pDTG871, in which ntdAa is under the control of the T7 promoter. E. coli DH5α(pDTG927), which carries the nbzAaAbAcAd genes from JS765 in pUC18 (24), was used for the isolation of oxygenase_NBZ. Ferredoxin_NBZ and reductase_NBZ, which are identical in sequence to ferredoxin_2NT and reductase_2NT, respectively, were also purified from E. coli DH5α(pDTG927) using the methods described below.

Strain JS42 was maintained on plates containing 1% Bacto tryptone, 0.5% yeast extract, and 1.8% Bacto agar (Becton Dickinson, Sparks, MD). For enzyme purification, cells were grown at 30°C in 10 liters of the above medium using a Biostat B fermentor (B. Braun Biotech Inc., Allentown, PA). Cells were grown to late log phase (optical density at 660 nm [OD₆₆₀] of ∼2) and harvested by centrifugation at 14,000 × g at 4°C for 60 min. Cell pellets were resuspended in an equal volume (wt/vol) of MEGD buffer (50 mM 2-[N-morpholino]ethanesulfonic acid [MES]) (pH 6.8, 5% ethanol, 5% glycerol, and 1 mM sodium dithiothreitol) and frozen at −70°C until required.

JM109(DE3)(pDTG871), DH5α(pDTG800), and DH5α(pDTG927) were maintained on mineral salts medium (MSB) (37) plates containing 1.8% Bacto agar, 10 mM glucose, 1 mM thiamine, and 150 μg/ml ampicillin. Reductase_2NT could be purified from DH5α(pDTG800), DH5α(pDTG927), or JS42, but much higher levels of protein were obtained from JM109(DE3)(pDTG871). For reductase_2NT purification, JM109(DE3)(pDTG871) cells were grown at 28°C in 10 liters of Terrific Broth (Life Technologies, Rockville, MD) containing 150 μg/ml ampicillin using a Biostat B fermentor. Cells were grown to early log phase (OD₆₆₀ = 0.5 to 0.7), at which time isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the medium (100 μM final concentration). Cells were incubated for an additional 3 h and then harvested by centrifugation (14,000 × g at 4°C for 15 min). For oxygenase_2NT and ferredoxin_2NT purification, DH5α(pDTG800) cells were grown at 28°C in 10 liters of 0.5× MSB containing 10 mM glucose, 1 mM thiamine, and 200 μg/ml ampicillin. Cells were harvested by centrifugation at 14,000 × g at 4°C for 15 min when the OD₆₆₀ reached approximately 5. Cell pellets were resuspended in an equal volume (wt/vol) of BTGD buffer (50 mM Bis-Tris [pH 6.8], 5% glycerol, 1 mM sodium dithiothreitol) and stored frozen at −70°C until required. For oxygenase_NBZ purification, DH5α(pDTG927) was grown in MSB containing 10 mM glucose, 1 mM thiamine, and 150 μg/ml ampicillin. Large-scale cultures (10 liters) were grown in a Biostat B fermentor at 37°C with air supplied at a rate of 1 to 2 liters/min and agitation maintained at 400 rpm. When the culture reached mid-exponential phase (turbidity at 660 nm = 0.6 to 0.8), the temperature was reduced to 28°C and IPTG was added to a final concentration of 150 μM. After 4 h, cells were harvested by centrifugation. Cell paste (70 g) was stored at −70°C. Prior to cell disruption, frozen cell paste was thawed and resuspended in 70 ml BTGD buffer.

Enzyme purification.All purification procedures were performed at 4°C using an automated fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA). Chromatography columns and column resins were from Amersham Biosciences, Piscataway, NJ, except for the ceramic hydroxyapatite (Bio-Rad, Hercules, CA). Cell extracts were prepared by allowing frozen cell suspensions to thaw on ice. DNase I was added to a final concentration of 0.01 mg/ml. Cell suspensions were passed through a chilled French pressure cell, maintaining an internal cell pressure of approximately 20,000 lb/in². Cell debris and membranes were removed by centrifugation at 145,000 × g for 60 min at 6°C. The resulting cell extracts were used immediately for enzyme purification. All purified proteins were frozen in liquid nitrogen and stored at −70°C.

Purification of reductase_2NT.Cell extract (3,595 mg of protein) prepared from E. coli JM109(DE3)(pDTG871) was applied to an XK50/30 chromatography column with a bed volume of approximately 500 ml of Q-Sepharose FF that had been preequilibrated with 1,500 ml of BTDG buffer. Unbound proteins were eluted with 2 column volumes of buffer at a flow rate of 2.0 ml/min. Bound proteins were eluted at a flow rate of 2.0 ml/min with a linear gradient from 0 to 0.5 M KCl (total gradient volume of 1,500 ml). Fractions exhibiting reductase_2NT activity were pooled and concentrated using an Amicon ultrafiltration system equipped with a YM30 membrane. Ammonium sulfate was added to the protein solution to a final concentration of 1.0 M, incubated for 2 h, and centrifuged for 30 min at 14,000 × g. The supernatant was loaded (0.5 ml/min) onto an XK26/40 column containing 100 ml (bed volume) of phenyl-Sepharose preequilibrated with 500 ml BTGD buffer containing 1.0 M ammonium sulfate. Unbound protein was eluted at a flow rate of 0.5 ml/min with 250 ml BTGD buffer containing 1.0 M ammonium sulfate. Fractions containing reductase_2NT were pooled and concentrated as described above. Ammonium sulfate was removed from the concentrated protein by buffer exchange with 1 mM potassium phosphate buffer, pH 6.8, using an Amicon ultrafiltration system equipped with a YM30 membrane. The reductase preparation was then loaded onto an XK16/70 column containing 100 ml (bed volume) of ceramic hydroxyapatite that had been preequilibrated with 250 ml 1 mM potassium phosphate buffer, pH 6.8. Unbound protein was eluted with 100 ml of 1 mM potassium phosphate buffer, pH 6.8, at a flow rate of 1 ml/min. Bound proteins were eluted with a linear gradient from 1 mM to 50 mM potassium phosphate buffer, pH 6.8 (total gradient volume of 1,000 ml), at a flow rate of 1 ml/min. Fractions exhibiting reductase_2NT activity were pooled, concentrated, and exchanged into 50 mM MES buffer, pH 6.8, as described above.

Purification of ferredoxin_2NT.Cell extract (4,340 mg of protein) prepared from cells of JS42 was applied to an XK50/30 column containing approximately 500 ml (bed volume) of Q-Sepharose FF that had been preequilibrated with 1,500 ml of MEGD buffer. Unbound proteins were eluted with 500 ml of the same buffer at a flow rate of 2.0 ml/min. Bound proteins were eluted with a linear gradient from 0 to 0.65 M KCl (total gradient volume of 1,520 ml) at a flow rate of 2.0 ml/min. Fractions exhibiting ferredoxin_2NT activity were pooled and concentrated by ultrafiltration with a YM10 membrane. Ammonium sulfate was added to the concentrated protein solution to give a final concentration of 1.5 M. After 2 hours, the solution was centrifuged for 30 min at 14,000 × g. The supernatant solution was applied to an XK26/40 column containing 100 ml (bed volume) of octyl-Sepharose that had been preequilibrated with 500 ml of MEGD buffer containing 1.5 M ammonium sulfate. Unbound protein was eluted at a flow rate of 0.5 ml/min with 250 ml MEGD buffer containing 1.5 M ammonium sulfate. Fractions containing ferredoxin_2NT were pooled, concentrated, and exchanged into 50 mM MES buffer, pH 6.8, as described above.

Purification of oxygenase_2NT.Cell extract (1,728 mg of protein) was applied to an XK50/30 column containing approximately 500 ml (bed volume) of a Q-Sepharose FF column, preequilibrated with 2,000 ml of MEGD buffer, pH 6.5. Unbound proteins were eluted from the column with 500 ml of the same buffer at a rate of 2.0 ml/min. Bound proteins were eluted with a linear gradient from 0 to 0.5 M KCl in MEGD buffer at the same flow rate. Fractions exhibiting oxygenase_2NT activity were pooled and concentrated under nitrogen, by ultrafiltration, using a YM100 membrane. The concentrated fraction was brought to 1.2 M with ammonium sulfate. After 2 h, the precipitate was removed by centrifugation at 14,000 × g for 30 min. The supernatant was applied to an XK26/40 column containing 150 ml (bed volume) of butyl-Sepharose. Unbound proteins were eluted at a flow rate of 2.0 ml/min with 1.2 M ammonium sulfate in MEGD buffer. Bound proteins were eluted with a linear gradient from 1.2 to 0 M ammonium sulfate at the same flow rate. Fractions exhibiting oxygenase_2NT activity were combined and concentrated, and the buffer was exchanged to 1 mM KPO₄, pH 6.8, by ultrafiltration with a YM100 membrane. The concentrated protein solution was applied to an XK16/70 column containing 100 ml (bed volume) of hydroxyapatite. The column was washed with 100 ml of 50 mM phosphate buffer (pH 6.8), and bound proteins were eluted from the column by a linear gradient of 50 to 500 mM phosphate buffer, pH 6.5, at a flow rate of 2 ml/min. Fractions containing oxygenase_2NT were pooled and concentrated by ultrafiltration. The final buffer exchange was conducted with 50 mM MES, pH 6.8.

Purification of oxygenase_NBZ.Cell extract (3,864 mg of protein) was applied to an XK50/30 column containing approximately 500 ml (bed volume) of Q-Sepharose FF previously equilibrated with BTGD buffer. The unbound protein was eluted with 500 ml of BTGD buffer at a flow rate of 2.0 ml/min. Fractions containing NBDO activity eluted with the unbound protein, indicating that oxygenase_NBZ did not bind to Q-Sepharose. Bound protein was eluted with a 1,500-ml linear KCl gradient (0 to 0.6 M) in BTGD buffer. Fractions containing the reductase and ferredoxin components were pooled and later purified by the methods described above for reductase_2NT and ferredoxin_2NT. Fractions containing NBDO activity were pooled and concentrated by ultrafiltration over an Aminco YM100 membrane. Ammonium sulfate was added to give a final concentration of 0.8 M. After the precipitate was removed by centrifugation, the supernatant was applied to an XK26/40 column containing 150 ml (bed volume) of butyl-Sepharose equilibrated with BTGD buffer containing 0.8 M ammonium sulfate. Unbound proteins were eluted at a flow rate of 2.0 ml/min with 150 ml 0.8 M ammonium sulfate in MEGD buffer. Bound protein was eluted with a linear gradient from 0.8 M to 0.2 M ammonium sulfate (gradient volume, 240 ml). Fractions containing oxygenase_NBZ were pooled and concentrated by ultrafiltration. The buffer was exchanged to 50 mM MES, pH 6.8.

Enzyme assays.Oxygenase_2NT and ferredoxin_2NT activities were determined by measuring nitrite released from 2NT as described previously (1). Reaction mixtures (1.0 ml) contained 50 mM MES buffer (pH 6.8), 0.4 mM NADH, 0.2 mM ferrous ammonium sulfate, 1.0 mM 2NT, and appropriate amounts of the purified 2NTDO components. NBDO activity was determined by the measurement of nitrite released from nitrobenzene in the same way. The reaction mixtures contained 50 mM MES buffer (pH 6.8); 0.2 mM NADH; 0.2 mM ferrous ammonium sulfate; 0.1 mM nitrobenzene; and appropriate amounts of purified reductase_2NT, ferredoxin_2NT, and oxygenase_NBZ.

The assay for reductase_2NT activity was based on the NADH:cytochrome c oxidoreductase assay (41). Reactions were carried out in 1.0 ml Bis-Tris (50 mM, pH 7.0) and contained 50 μM horse heart cytochrome c (Sigma), 150 μM NADH, and an appropriate amount of reductase_2NT. Activity was determined by monitoring the increase in absorbance at 550 nm. Addition of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), or ferredoxin_2NT did not significantly stimulate activity (data not shown). An extinction coefficient of 21,000 M⁻¹ cm⁻¹ for reduced minus oxidized cytochrome c was used in calculating the activity (41). Assays with the alternative electron acceptors ferricyanide, 2,6-dichlorophenol indophenol, and nitroblue tetrazolium were carried out as previously described (38).

Flavin determination.Identification and quantification of the flavin present in reductase_2NT was determined by high-pressure liquid chromatography (HPLC) as previously described (35) and from the absorbance of the FAD (ε₄₆₀ = 11,300 M⁻¹ cm⁻¹) (43). After thermal denaturation of the protein at 100°C for 5 min, protein was removed by centrifugation at 10,000 × g for 5 min at 4°C, and the supernatant was analyzed. HPLC analyses were performed with a Waters Associates HPLC system (600E solvent delivery system, U-6K injector, model 910 photodiode array detector, and Millennium Chromatography Manager software). Separations were carried out on a Beckman Ultrasphere reverse-phase column (4.6 mm by 25 cm) with a mobile phase of methanol-water (20:80) at a flow rate of 1 ml/min.

Kinetics.Apparent K_m values were determined polarographically. Reactions were carried out at 30°C in air-saturated 50 mM MES buffer (pH 6.8) in a total volume of 1 ml in a Clark-type oxygen electrode (Rank Brothers, Cambridge, England). Mixtures contained 0.5 mM NADH, 0.1 mM ferrous ammonium sulfate, 0.28 μM reductase_2NT, 0.77 μM ferredoxin_2NT, 0.27 μM oxygenase_2NT, and various concentrations of 2NT or naphthalene. Stock solutions of 2NT and naphthalene were 25 mM in a methanol-water mixture (40:60). Rates were corrected for the small background consumption of oxygen, which was 10% of the signal for the lowest substrate concentration. The apparent K_m values were determined by nonlinear fitting using the program SigmaPlot 5.0.

Spectroscopy.Electron paramagnetic resonance (EPR) spectra of each of the purified proteins were recorded at 77 K in a Bruker model ESP 300 spectrometer (ESR Facility, University of Iowa) as isolated (oxidized) and following reduction with an excess of sodium dithionite. The settings were as follows: 5-mW microwave power, 3,650-G centerfield, 9.29-GHz microwave modulation frequency, 42-s sweep time, and 1.0 × 10⁵ receiver gain. The absorbance spectrum of each protein was recorded under an argon atmosphere on a Beckman DU7500 or an Aminco DW-2000 spectrophotometer as isolated and during reduction with NADH and catalytic amounts of the required electron transfer proteins.

Iron and acid-labile sulfide.Iron and acid-labile sulfide were determined by published methods (2, 44).

Protein concentrations.Protein concentrations were determined by the method of Bradford (5) using bovine serum albumin as the standard.

Molecular weight determinations.The native molecular weights of reductase_2NT, ferredoxin_2NT, and oxygenase_2NT were determined by gel filtration using an XK16/70 column containing 100 ml (bed volume) of Sephacryl 3000. MES buffer (50 mM; pH 6.8) containing 150 mM NaCl was used as the mobile phase at a flow rate of 0.5 ml/min. The column was calibrated with ferritin (44,000), bovine serum albumin (158,000), aldolase (67,000), and ferredoxin_NAP (11,000) (18). The native molecular weight of oxygenase_NBZ in BTGD buffer was determined by dynamic light scattering at 6°C (16) using a Dynapro instrument from Protein Solutions. The subunit molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (23) on 12.5% gels with low-molecular-weight standards (Bio-Rad Laboratories, Hercules, CA). Samples were boiled for 1 min immediately prior to loading gels.

N-terminal amino acid sequence analysis.The N-terminal amino acid sequences of reductase_2NT, ferredoxin_2NT, and the α and β subunits of oxygenase_2NT and oxygenase_NBZ were determined by Edman degradation on an automated sequencer (Applied Biosystems, Foster City, CA) at the University of Iowa College of Medicine Molecular Analysis Facility after SDS-PAGE and electroblotting of the purified proteins onto a polyvinylidene difluoride membrane (ProBlott; Applied Biosystems).

Crystallization.Initial crystallization screenings were carried out with the Crystal Screens I and II kits (Hampton Research, Riverside, CA) (3). Oxygenase_2NT (20 mg/ml) was crystallized using the hanging and sitting drop vapor-diffusion methods (3) after buffer exchange into 50 mM MES buffer (pH 6.8). The crystallization drops, containing 2 μl protein solution and 2 μl precipitant solution, were equilibrated against 0.75 to 1.0 ml of precipitant solution. Oxygenase_NBZ (8 to 10 mg/ml) was crystallized using the sitting drop vapor-diffusion method (3) after buffer exchange into 50 mM MES buffer (pH 6.8). The crystallization drops, containing 1.5 μl protein solution and 1.5 μl precipitant solution, were equilibrated against 1 ml of precipitant solution. Details of optimized crystallization conditions for oxygenase_2NT and oxygenase_NBZ are given in Results. Extensive crystal screenings with reductase_2NT and ferredoxin_2NT did not result in the production of diffraction-quality crystals.

Data collection and processing.Crystal data collections were performed on beamlines ID14-4, at the European Synchrotron Radiation Facility, Grenoble, France. Data were collected at 100 K on crystals flash-cooled in liquid nitrogen after a 10- to 30-s soak in a cryoprotection solution, which consisted of the reservoir solution containing 25% (vol/vol) ethylene glycol. The data were indexed, integrated, scaled, and merged using Mosflm (32) and Scala (8).

Purification of 2NTDO and NBDO components.The purification and properties of the reductase, ferredoxin, and oxygenase components of the 2NTDO system are reported below. The 2NTDO components are designated reductase_2NT, ferredoxin_2NT, and oxygenase_2NT and correspond respectively to components B, C, and A reported previously (1). The purification and properties of oxygenase_NBZ of the NBDO are also reported. Recombinant reductase_NBZ and ferredoxin_NBZ were also purified and characterized. Their properties were identical to those of reductase_2NT and ferredoxin_2NT (data not shown). This result was expected because the genes encoding the two sets of electron transfer proteins (ntdAa/nbzAa; ntdAb/nbzAb) are identical in sequence (24, 29).

Reductase_2NT.Reductase_2NT was purified by a three-step procedure utilizing ion-exchange, hydrophobic interaction, and hydroxyapatite chromatography (Table 1). The purified enzyme gave a single band when analyzed by SDS-PAGE (Fig. 2, lane 3). The molecular mass of reductase_2NT determined by gel filtration and SDS-PAGE indicated that reductase_2NT consists of a single polypeptide chain, and the size and N-terminal amino acid sequence corresponded to the sequence deduced from the ntdAa gene (Table 2). NADH was the preferred electron donor for reductase_2NT. NADPH, at the same concentration, gave only 37% of the activity observed with NADH. This preference for NADH is similar to that of reductase_NAP, which was only 39% as active with NADPH (19). In addition to cytochrome c, the reductase_2NT catalyzed the NADH-dependent reduction of ferricyanide and 2,6-dichlorophenol indophenol (Table 3).

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FIG. 2.

SDS-PAGE analysis of purified 2NTDO and NBDO components. Lane 2, ferredoxin_2NT; lane 3, reductase_2NT; lane 4, oxygenase_2NT; lane 5, oxygenase_NBZ. Each lane was loaded with 5 μg of protein. Lane 1, molecular mass standards (numbers at left are molecular masses in kDa). Protein was stained with Coomassie brilliant blue R250.

Solutions of reductase_2NT were deep orange in color and gave absorption maxima at 271, 344, 398, and 460 nm with shoulders at 490 and 540 nm (Fig. 3A) (Table 2). After reductase_2NT was boiled and the precipitated protein was removed by centrifugation, the yellow supernatant solution had the same absorption spectrum as that of FAD (absorption maxima at 376 and 446 nm). The identity of FAD was confirmed by HPLC analysis (retention time [Rt], 12.60 ± 0.16 min). FMN (Rt, 8.50 ± 0.05 min) was not detected. The FAD content of reductase_2NT was determined by HPLC and from the absorbance of the FAD released by heat treatment (ε₄₆₀ = 11.30 mM⁻¹ cm⁻¹) (43), which gave values of approximately 1 mol of FAD/mol reductase_2NT, respectively (Table 2). Addition of FAD or FMN to assay mixtures did not stimulate activity, indicating that the flavin site was fully occupied in the purified preparation. The iron and acid-labile sulfur contents of reductase_2NT indicated the presence of a [2Fe-2S] center, and this was confirmed by EPR studies (Table 2). Partial reduction of reductase_2NT gave a spectrum with g values of 2.03, 2.003, and 1.93 (Table 2). The signal at g = 2.003, which disappears upon full reduction of the reductase to leave a spectrum similar to that of a plant-type [2Fe-2S] ferredoxin, is indicative of a neutral flavin semiquinone (26).

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FIG. 3.

Anaerobic titration of protein components with NADH. Each cuvette and its contents were made anaerobic by alternately evacuating and flushing with argon for 15 min each. (A) Anaerobic titration of reductase_2NT. The cuvette contained reductase_2NT (36 nmol) in 1.0 ml of 50 mM MES buffer, pH 6.8. The absorbance spectrum of the oxidized reductase (curve 1) was then titrated with an anaerobic solution of NADH. The inset shows the decrease in absorbance at 460 nm versus the amount of NADH added. (B) Anaerobic reduction of ferredoxin_2NT by NADH in the presence of reductase_2NT. The cuvette contained, in a final volume of 1.0 ml of 50 mM MES buffer (pH 6.8), ferredoxin_2NT (12 nmol) and catalytic amounts of partially purified reductase_2NT. The absorbance spectrum of the oxidized ferredoxin (curve 1) was titrated with an anaerobic solution of NADH. The inset shows the decrease in absorption at 456 nm after successive additions of NADH. (C) Anaerobic reduction of oxygenase_2NT by NADH in the presence of reductase_2NT and ferredoxin_2NT. The cuvette contained, in a final volume of 1.0 ml of 50 mM MES buffer (pH 6.8), oxygenase_2NT (50 nmol) and catalytic amounts of reductase_2NT and ferredoxin_2NT. The absorbance spectrum of the oxidized oxygenase_2NT (curve 1) was then titrated with an anaerobic solution of NADH. The inset shows the decrease in absorption at 454 nm after successive additions of NADH. (D) Anaerobic reduction of oxygenase_NBZ by NADH in the presence of reductase_2NT and ferredoxin_2NT. The cuvette contained, in a final volume of 0.6 ml of 50 mM MES buffer (pH 6.8), oxygenase_NBZ (40.1 nmol) and catalytic amounts of reductase_2NT and ferredoxin_2NT. The absorbance spectrum of the oxidized oxygenase_NBZ (curve 1) was then titrated with an anaerobic solution of NADH. The inset shows the decrease in absorption at 460 nm after successive additions of NADH.

The results of the anaerobic titration of reductase_2NT with NADH showed that a decrease in absorbance at 398 and 460 nm is accompanied by a small initial increase in absorbance between 550 and 650 nm (Fig. 3A). The inset shows that the increase in absorbance at 600 nm peaked at two electron equivalents, indicating the reduction of the [2Fe-2S] center and the concomitant formation of a neutral flavin semiquinone (13). The absorbance at 600 nm decreased with the addition of one further electron equivalent to give the fully reduced reductase. The decrease in absorbance at 460 nm (Fig. 3A) also shows that reductase_2NT can accommodate three electrons.

Ferredoxin_2NT.Ferredoxin_2NT was purified to homogeneity from JS42 cells and from DH5α(pDTG800) cells by a two-step procedure utilizing ion-exchange and hydrophobic interaction chromatography. Data shown in Table 1 are from a purification using JS42 cells. The purified protein gave a single band when analyzed by SDS-PAGE (Fig. 2, lane 2). The molecular mass of ferredoxin_2NT determined by gel filtration and SDS-PAGE (Table 2) corresponded to the deduced molecular mass derived from the sequence of the ntdAb gene (29), and the N-terminal amino acid sequence of ferredoxin_2NT was identical to the amino acid sequence predicted from the nucleotide sequence of ntdAb (29) with the exception of the absence of a N-terminal methionine residue. The spectroscopic properties of ferredoxin_2NT (Table 2) indicated the presence of a Rieske [2Fe-2S] cluster (34). Supporting evidence was provided by iron and acid-labile sulfur analyses (Table 2). The midpoint reduction potential of ferredoxin_2NT was −145 mV when measured against the standard hydrogen electrode (L. Eltis, personal communication).

The results of the anaerobic reduction of ferredoxin_2NT by NADH in the presence of catalytic amounts of reductase_2NT are shown in Fig. 3B. The inset in the figure shows that 0.5 mol of NADH is required to reduce 1.0 mol of ferredoxin_2NT, confirming the role of ferredoxin_2NT as a single electron acceptor/donor in the 2NTDO system.

Purified ferredoxin_NAP from the naphthalene dioxygenase system in Pseudomonas sp. strain NCIB 9816-4 was able to effectively substitute for ferredoxin_2NT in the standard nitrite release assay with saturating amounts of reductase_2NT and oxygenase_2NT present (Table 4). In contrast, ferredoxin_BPH from the biphenyl dioxygenase system in Burkholderia sp. strain LB400 (17) and ferredoxin_TOL (39) from the toluene dioxygenase system in Pseudomonas putida F1 were not useful substitutes for ferredoxin_2NT (Table 4).

Oxygenase_2NT.Oxygenase_2NT was purified to homogeneity as shown in Table 1. SDS-PAGE revealed the presence of two subunits, designated α and β, with molecular masses (Fig. 2, lane 4) (Table 2) similar to the values reported previously for partially purified component A (1) and to the deduced sequences of the ntdAc and ntdAd genes (29). Gel filtration chromatography gave a molecular mass of 210 kDa, indicating that native oxygenase_2NT is an α₃β₃ hexamer. The N-terminal amino acid sequences of the α and β subunits of oxygenase_2NT (Table 2) were identical to those predicted from the nucleotide sequences of the ntdAc and ntdAd genes (29) with the exception of the absence of a methionine residue at the start of the α subunit. Spectroscopic analyses and iron and acid-labile sulfur contents of oxygenase_2NT (Fig. 3C) (Table 2) indicated the presence of a Rieske [2Fe-2S] center. Figure 3C shows the reduction of oxygenase_2NT when titrated anaerobically with NADH in the presence of catalytic amounts of reductase_2NT and ferredoxin_2NT. The inset shows that 0.5 mol of NADH is required to reduce each αβ heterodimer of oxygenase_2NT.

Reaction mixtures containing 0.5 mM NADH, 0.28 μM reductase_2NT, 0.77 μM ferredoxin_2NT, 0.27 μM oxygenase_2NT, and various amounts of either 2NT or naphthalene (both good substrates for 2NTDO) (30) were used to determine the apparent Michaelis constants for these substrates. Reactions with 2NT and naphthalene were fully coupled to oxygen and NADH consumption. The apparent K_m for 2NT under these conditions was 20 ± 7 μM, and that for naphthalene was estimated to be 121 ± 36 μM. This number is only an estimate, because it appears that the saturating concentration is above the solubility limit of naphthalene (approximately 250 μM). The apparent turnover numbers for 2NT and naphthalene were very similar (2.3 and 2.4 s⁻¹, respectively), and the specificity constants were 7.0 ± 0.5 μM⁻¹ min⁻¹ for 2NT and 1.2 ± 0.4 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate.

Oxygenase_NBZ.Oxygenase_NBZ was purified to homogeneity as shown in Table 1. SDS-PAGE revealed the presence of two subunits designated α and β (Fig. 2, lane 5) (Table 2), which are consistent with those deduced from the DNA sequence (24). The native molecular mass of oxygenase_NBZ was determined to be 215,900 by dynamic light scattering, indicating an α₃β₃ subunit configuration. The N-terminal amino acid sequences of the α and β subunits of oxygenase_NBZ (Table 2) are identical to those predicted from the nucleotide sequences of the nbzAc and nbzAd genes (24) with the exception of the absence of a methionine residue at the start of the α subunit.

The physical properties of oxygenase_NBZ (Table 2) are consistent with the presence of a Rieske [2Fe-2S] cluster. Figure 3D shows the reduction of oxygenase_NBZ when titrated anaerobically with NADH in the presence of catalytic amounts of reductase_2NT and ferredoxin_2NT. Complete reduction resulted in the loss of absorption at 460 and 560 nm and the appearance of new absorption maxima at 380 and 525 nm. The inset shows that 0.5 mol of NADH is required to reduce each αβ heterodimer of oxygenase_NBZ.

Oxygenase crystallization.Oxygenase_2NT crystallized as shards in 1.5 M ammonium sulfate, 100 mM MES (pH 6.8), or 100 mM HEPES (pH 6.0) at 4 to 8°C. Crystals appeared in 3 to 5 days. In order to obtain large enough crystals for data collection the crystallization drops were streak-seeded after 1 day with material from crushed 2NTDO crystals grown under the same conditions. Crystals appeared within 1 day of seeding (Fig. 4A). The oxygenase_2NT crystals diffracted to 3.2 Å and belonged to the monoclinic space group C2 with cell dimensions a = 233.2 Å, b = 179.9 Å, c = 223.8 Å, and β = 139.4°. The protein had one α₃β₃ heterohexamer in the asymmetric unit, corresponding to a solvent content of 82%.

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FIG. 4.

Photomicrographs of oxygenase crystals. (A) Oxygenase_2NT; (B) oxygenase_NBZ hollow rods; (C) oxygenase_NBZ hexagonal plates and tulip-shaped crystals (enlarged in inset).

Initially, oxygenase_NBZ crystallized as hollow rods (Fig. 4B) in 12% (wt/vol) polyethylene glycol 8000, 100 mM Tris buffer (pH 7.5) at 9°C. These crystallization conditions were optimized to 4 to 8% (wt/vol) polyethylene glycol 8000, 5 mM NiCl₂ or 5 mM NiSO₄, 100 mM MES (pH 6.0), or 100 mM HEPES (pH 6.5) at 15 to 18°C. Hexagonal plates (0.15 × 0.15 × 0.05 mm; Fig. 4C) and tulip-shaped crystals (0.15 × 0.15 × 0.5 mm [Fig. 4C and inset]) were obtained within 3 to 6 h. The tulip-shaped crystals diffracted to 1.2 Å and belonged to the hexagonal space group P6₃ with cell dimensions a = b = 121.6 Å and c = 84.4 Å. The protein had one αβ heterodimer in the asymmetric unit, corresponding to a solvent content of 50%.