INTRODUCTION

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Cr(VI) (chromate) is a widespread industrial and nuclear waste. At the Department of Energy (DOE) sites, for example, it is the second most common heavy metal contaminant, ranging in concentration between 0.008 to 173 µM in groundwater and 98 nM to 76 mM in soil and sediments (15). Since soil water is stored in small capillary spaces, the latter may represent very high concentrations. Chromate is toxic, mutagenic, and probably carcinogenic (20, 22). It is also soluble and therefore does not remain confined to the site of initial contamination. Several bacteria possess chromate reductase activity that can convert chromate to Cr(III), which is much less toxic and less soluble, and thus reduction by these enzymes affords a means of chromate bioremediation.

Field studies have established that biostimulation of indigenous (wild-type) bacteria is an effective means of removing environmental pollutants (10, 11). In this method, nutrients are added to the environments, such as aquifers, to stimulate the growth of indigenous bacteria. Although this enhances the transforming ability of these bacteria, making them more effective agents of bioremediation, it also results in the generation of a large amount of biomass, which confines effective bioremediation to a narrow zone (10). Moreover, most contaminated sites, especially the DOE sites, contain several pollutants (15). The classical biostimulation approach (10) is likely to be of limited use at such sites, since individual enzymes as well as bacteria capable of remediating a given contaminant are inhibited by the presence of the other pollutants (19).

There is increasing recognition, therefore, that bioremediation can benefit from the application of molecular and bioengineering approaches, and an element of the DOE Natural and Accelerated Bioremediation Research Program is aimed at this objective. Thus, engineering of bacteria by using appropriate promoters can minimize the nutrient need and biomass production (8, 9). Furthermore, protein bioengineering can increase the ability, for example of chromate reductases, to function in the presence of other contaminants (19).

To improve bacterial chromate remediation by these approaches, it is necessary to clone the genes that encode chromate reductase activities. Once the promoters and other elements that regulate these genes are known, rational approaches can be devised to improve gene expression under in situ conditions. Cloned genes will also permit facile production of large quantities of pure enzymes using modern molecular methods. Easy availability of pure enzymes will permit detailed investigations of their kinetic and inhibition properties, leading to the identification of targets for improvement; it will also permit determination of high-resolution structure of these enzymes so that the desired improvements can be effectively attempted.

A chromate reductase activity was purified from Pseudomonas ambigua by Suzuki et al. in 1992 (18), and our initial aim was to study this enzyme. However, neither the enzyme nor the bacterium (which is not included in the American Type Culture Collection) was made available to us. Our resulting studies with the common environmental bacterium Pseudomonas putida led to the identification of a new chromate reductase activity in this bacterium that has not been reported previously (4, 18, 21). We describe here the purification to homogeneity and kinetic properties of this novel enzyme; a future report will deal with the molecular regulation of the encoding gene and the physiological role of the enzyme.

While this work was in progress, Suzuki et al. deposited the sequence of the gene encoding their enzyme in GenBank (accession number D83142). Our studies using PCR showed that this gene is absent from P. putida; this is supported by the fact that no homologue to the gene reported by Suzuki et al. is found in the genomic sequence recently put on the World Wide Web by The Institute of Genomic Research.

	MATERIALS AND METHODS

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Chromate reductase assays. Chromate reductase activity was determined by measuring the decrease in chromate concentration during enzyme assays. Chromate was quantified, as described previously (2), by adding to the reaction mixture H₂SO₄ and 1,5-diphenylcarbazide to final concentrations of 0.1 M and 0.01%, respectively, and measuring the absorbance at 540 nm (A₅₄₀), using a calibration curve relating chromate concentration (0 to 20 µM) to A₅₄₀.

Chromate reductase purification. P. putida MK1 (6) (identical to strain ATCC 12633 except for resistance to rifampin) was grown in Luria-Bertani medium (500 ml of medium in 2-liter flasks at 30°C, shaken at 250 rpm) without chromate to an A₆₆₀ of 2.5 to 3.0. The entire procedure was carried out twice in independent runs, using 20-liter cultures each time. Purification was carried out at 4°C. Following harvesting by centrifugation (10,000 × g for 30 min) and washing in T50 buffer, the cells were suspended in 320 ml of T50 buffer (A₆₆₀, 80 to 100) and disrupted by passage through a French pressure cell (16,000 lb/in²). The lysate was centrifuged (13,500 × g for 30 min) to remove unbroken cells and debris, resulting in crude extract. This extract was further centrifuged (150,000 × g for 90 min) to remove membrane-associated material, generating the soluble extract. Measurements showed that chromate reductase activity was present exclusively in the soluble extract.

Analytical techniques. The protein concentration was measured by a Bio-Rad assay kit, using bovine serum albumin as the standard. In the FPLC column eluent, protein was monitored by A₂₈₀ measurement. PAGE (7) under denaturing conditions was performed with 12 and 4% acrylamide resolving and stacking gels, respectively. A MiniProtean II electrophoresis cell (Bio-Rad) was used, and the gels were stained with Coomassie brilliant blue R-250 or with silver (3).

XANES spectroscopy. X-ray absorption spectroscopy provides information on the electronic and structural state of an element (17). In the X-ray absorption near-edge-structure (XANES) spectrum, the stable oxidation states of chromium, Cr(VI) and Cr(III), can be distinguished by the pronounced pre-edge feature of the former (5). X-ray absorption spectra were collected on beamline 4-3 at the Stanford Synchrotron Radiation Laboratory run under dedicated conditions at 3 GeV. The ring intensity varied from ca. 100 mA directly after a fill to ca. 40 mA before a fill; energy selection was accomplished using a double monochromator composed of Si(220) crystals. The beam passing through the monochromator was detuned (75% at 6,000 eV) to minimize higher-order harmonics and had a vertical spread of 1 mm. Incident and transmitted X-ray intensities were recorded in 15-cm-long ionization chambers. The fluorescence intensity was used as the absorption measure for unknown samples and was established using a 13-element Ge solid-state detector (1). The spectrometer was calibrated to element Cr (foil), with the inflection point of the metal set to 5,989 eV. Standards of sodium chromate and Cr₂O₃ were used as reference for end-point oxidation states. Spectra were collected from 100 eV below the edge to 200 eV above, using 0.25-eV steps across the pre- and main-edge regions. For kinetic experiments, the total scan time was minimized so as to acquire successive spectra every 3.1 min. The enzyme reaction was started off-line by adding chromate (final concentration, 1.5 mM). The reaction mixture was then transferred to a 0.5-cm² Teflon cell, which was sealed with Kapton film, and placed in the spectrometer. The lapse time between starting the reaction and performing the scan was 1.1 min.

Chemicals. NADH and NADPH were purchased from Boehringer Mannheim (Indianapolis, Ind.). Tris, ammonium sulfate, 1,5-diphenylcarbazide, silver nitrate, -mercaptoethanol, and Coomassie brilliant blue R-250 were obtained from Sigma (St. Louis, Mo.). The polyvinylidene difluoride membrane was purchased from Bio-Rad Laboratories (Hercules, Calif.). The column chromatography materials were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). All other chemicals, of analytical grade, were from standard suppliers.

	RESULTS

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Purification of chromate reductase. The method and purification parameters are summarized in Table 1. The soluble extract had a 2.5-fold-higher chromate reductase specific activity than the crude extract did. Also, its total reductase activity was almost twice that of the crude extract (Table 1), suggesting that some crude extract component(s) inhibited the enzyme. That the crude extract contains activity-modifying compounds was also found by Suzuki et al. (18) for P. ambigua. The soluble extract was subjected to stepwise ammonium sulfate precipitation, and the highest reductase specific activity fraction (55 to 70% ammonium sulfate saturation) was applied to a DEAE Sepharose CL-6B column.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Anion-exchange chromatography of the ammonium sulfate fraction (see Results) on a DEAE-Sepharose CL-6B column. The column was preequilibrated with 50 mM Tris-HCl buffer (pH 7.0). The inset shows an SDS-PAGE gel prepared from the specified fractions (numbers above the lanes). The arrowhead indicates the 20-kDa protein band whose intensity corresponds to chromate reductase activity in individual fractions.

View larger version (45K): [in this window] [in a new window]   FIG. 2.   (A) Chromatofocusing using a Polybuffer exchanger 94 column of fractions 23 to 31 containing the chromate reductase activity obtained from ion-exchange chromatography. The column was eluted as described in Materials and Methods, and the chromate reductase activity of each fraction was measured. (B) SDS-PAGE (12% polyacrylamide) analysis of fractions obtained from chromatofocusing (Fig. 2A). The fractions are identified by numbers above the lanes; the unnumbered lane represents the pooled active fractions obtained from anion-exchange chromatography (Fig. 1). A 100-µl volume of each fraction was concentrated 10-fold before loading. The arrowhead shows the position of the presumptive chromate reductase band.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   FPLC of active fractions (fractions 27 to 31) from the chromatofocusing column (Fig. 2A) using a Superose 12 HR 10/30 column. These fractions were concentrated and analyzed. Note the presence of a single sharp peak of chromate reductase activity.

View larger version (76K): [in this window] [in a new window]   FIG. 4.   Silver stain of SDS-PAGE of proteins at various stages of purification. Lanes: 1, crude extract (0.5 µg of protein loaded on the gel); 2, soluble extract (0.5 µg); 3, ammonium sulfate fraction (0.5 µg); 4, DEAE-Sepharose active fractions (0.3 µg); 5, Polybuffer exchanger 94 active fractions (0.2 µg); 6, gel filtration active fractions (0.1 µg). The arrowhead indicates the position of the chromate reductase.

Optimum reaction conditions and stability. A ca. 200-fold-purified chromate reductase preparation (active fractions obtained at the DEAE-Sepharose CL-6B step [Table 1]) was used in these and subsequent studies. The enzyme absolutely required NADH or NADPH for activity, and both were equally effective (data not shown). The reductase became sharply more active as the reaction temperature increased up to 80°C and then showed a sharp decline as the temperature was increased further (Fig. 5). Nonenzymatic conversion of chromate became significant only above ca. 70°C.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Effect of reaction temperature on chromate reductase activity. Net enzymatic reaction rates were obtained by subtracting nonenzymatic reduction from reduction in the presence of enzyme and NADH. The assay was conducted at pH 5.0, using 50 mM citric acid-NaOH buffer (pH 5.0).

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Residual enzyme activity after incubation at the indicated temperatures for the indicated duration. The assay was conducted at 50°C using 50 mM citric acid-NaOH buffer (pH 5.0).

Enzyme kinetics. The apparent K_m value obtained from the Lineweaver-Burk plot was 374 µM CrO₄²; the V_max was 1.72 µmol/min/mg of protein (data not shown). Inhibition of the enzyme activity by selected metals and anions was also determined. Cd²⁺ and Zn²⁺, when supplied as chloride salts, had little effect at concentrations up to 10 mM, and AsO₄³ was also not inhibitory (Table 2). CdSO₄, however, produced significant inhibition at 1 mM and above, suggesting that the SO₄² was inhibitory. Since CuSO₄ was no more inhibitory than CdSO₄, Cu²⁺ was also noninhibitory. Kinetic measurements, using CdSO₄ concentrations of 0 to 9 mM (at 0.1 to 0.5 mM chromate concentrations), showed that sulfate inhibited the enzyme noncompetitively (K_i  11 mM [data not shown]).

End product of chromate conversion. As stated in Materials and Methods, the end product was determined by XANES; chromium was speciated using the spectral analysis and calibration procedure of Patterson et al. (13). The fraction of Cr(VI) was calculated by dividing the height of the Cr(VI) pre-edge peak by the total atomic absorption; that of Cr(III) was calculated from the difference between the amount of chromium represented by the pre-edge peak and the total absorption jump (Fig. 7 and 8). Good agreement (r²  0.988) was found between the known and calculated amounts of Cr(VI) in standards by using this method.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   XANES spectra of chromium at successive times after chromate was reacted with chromate reductase. The reduction in the pre-edge intensity relative to the main-edge intensity with time denotes the progressive transformation of Cr(VI) to Cr(III).

View larger version (27K): [in this window] [in a new window]   FIG. 8.   The isolated pre-edge feature of the Cr-XANES spectra illustrating diminished intensity with increased reaction time. (Inset) Fraction of Cr(VI) in the sample as a function of time determined from the ratio of the pre-edge to main-edge intensity.

	DISCUSSION

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Only a single peak of chromate reductase activity was detected at the final (FPLC) stage of purification, and the silver-stained gels of active fractions at this stage showed only a single band (Fig. 4). This is the first report of purification to homogeneity of a bacterial chromate reductase. Ishibashi et al. (4) could purify the enzyme from P. putida PRS 2000 only partially, while Suzuki et al. (18) achieved a 38-fold purification of chromate reductase from P. ambigua. From the N-terminal and internal amino acid sequence of our purified enzyme and the sequence data of P. putida obtained from The Institute of Genomic Research, we have determined the sequence of our chromate reductase-encoding gene, which confirms that the enzyme is indeed a novel protein and embodies features of other reductases (C. H. Park, M. Keyhan, and A. Matin, unpublished data).

That the chromate reductase purified in this study from P. putida MK1 is a new enzyme is supported by its properties. It is soluble, while that of Enterobacter cloacae (21) is membrane bound; although the P. putida PRS2000 and P. ambigua reductases are also soluble (4, 18), they exhibit different properties from that of the MK1 enzyme. For example, the MK1 enzyme is optimally active at 80°C, but the optimal reaction temperature of the P. ambigua enzyme is 50°C; the MK1 enzyme has a pH optimum of 5.0, while P. ambigua and PRS2000 enzymes have pH optima of 8.6 and between 6.5 and 7.5, respectively. Similarly, the K_m values of the three enzymes are different: 370 µM for MK1 versus 13 and 40 µM for the P. ambigua and PRS2000 enzymes, respectively. The MK1 enzyme is inhibited by sulfate, while the PRS2000 enzyme is not. The elution patterns of the MK1 and PRS2000 enzymes from anion-exchange columns also point to differences between them, with the pI value of the former being higher than 7.0 (Fig. 1) and that of the latter being lower than 7.0 (4).

The MK1 enzyme gave a monomer molecular mass on SDS-PAGE of 20 kDa and a molecular mass for the native protein on gel filtration of ca. 50 kDa. Therefore, it is not known whether the enzyme is a dimer or a trimer. The P. ambigua chromate reductase exhibited a similar behavior, with a molecular mass of 25 kDa on SDS-PAGE and a mass of 65 kDa on gel filtration (18). Proteins can exhibit nonproportionate movement upon SDS-PAGE and gel filtration for several reasons. One possibility is that both reductases possess intrasubunit disulfide cross-linkages that, by producing altered conformation and Stokes radii, influence protein movement in gels differently (16).

The N-terminal amino acid of the MK1 enzyme is not methionine (Park et al., unpublished), raising the possibility that it is a periplasmic protein. Most periplasmic proteins are synthesized with a leader sequence that is cleaved off as they cross the inner membrane (14). Since chromate can easily pass through the membranes and in the cytoplasm is undoubtedly toxic to the bacterium (20), it would be advantageous to the cell to have chromate detoxification capability localized close to the cell surface. However, although the growth of MK1 is not affected by chromate up to a concentration of 0.8 mM (Park et al., unpublished), it remains to be determined if the reductase contributes to this resistance. Anaerobic growth of P. putida MK1 was not supported by chromate (Park et al., unpublished).

The question of the role that chromate reductases play in bacterial physiology has not been explored. The E. cloacae enzyme might be involved in anaerobic respiration, with chromate as the electron acceptor (21), but no definite role for the soluble reductases has been established. In Pseudomonas fluorescens, chromate reductase activity was equally strong in chromate-resistant and -sensitive strains, suggesting that this activity did not confer protection against chromate toxicity (12). Ishibashi et al. (4) suggested that chromate reduction might be a vicarious property of reductases with different primary roles. The fact that we found an additional peak of chromate reductase activity upon ion-exchange chromatography, as did Suzuki et al. (18), may be consistent with this possibility.

However, whether or not the soluble chromate reductases have an actual physiological purpose of detoxifying Cr(VI), such activities can still be useful in bioremediation. Indeed, cometabolic processes account for many successful bioremediation approaches. For example, methane monooxygenase of methanotrophs has been exploited to bioremediate trichloroethylene, even though the physiological role of this enzyme is to oxidize methane (10). The soluble chromate reductases can also be similarly useful in cometabolic chromate remediation. The fact that the optimal activity of our enzyme occurs at a high temperature does not detract from its potential usefulness in chromate remediation. It is not unusual for even essential enzymes to exhibit maximum activity in vitro under conditions different from the optimal growth conditions of an organism; possibly their maximal activity in vivo more closely matches the organism's optimal growth conditions (7a). Similarly, the P. putida enzyme studied here still exhibits significant activity at 30°C, even though it is less than 1/10 the maximal activity; this may be amenable to further enhancement by protein engineering. Further, the gene could be cloned into a thermophile, and in an immobilized enzyme-bioreactor-based remediation system, its thermotolerant product can have a decisive advantage. These considerations also apply to the chromate reductase purified by Suzuki et al. (18), which has a temperature optimum of 50°C; the optimal temperatures for other chromate reductases reported so far remain to be determined.

Systematic studies are needed to determine the real nature of activities so far identified as chromate reductases; it is pertinent to note in this connection that we have found chromate reductase activity in enzymes characterized in completely different contexts (Park et al., unpublished). A future communication will deal with the cloning and molecular regulation of the gene encoding the enzyme purified in this study, the effect of a knockout mutation of this gene on P. putida physiology, and comparative molecular features of the various enzymes that exhibit chromate reductase activity. This information will greatly facilitate the use of protein and genetic engineering to enhance the chromate remediation potential of P. putida.