J Biol Chem, Vol. 274, Issue 29, 20287-20292, July 16, 1999

Purification and Molecular Characterization of ortho-Chlorophenol Reductive Dehalogenase, a Key Enzyme of Halorespiration in Desulfitobacterium dehalogenans^*

Bram A. van de Pas^§, Hauke Smidt^¶, Wilfred R. Hagen, John van der Oost, Gosse Schraa, Alfons J. M. Stams, and Willem M. de Vos

From the  Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands and the  Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, NL-6703 HA Wageningen, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a V_max of 28 units/mg protein and a K_m of 20 µM. The pH and temperature optimum were 8.2 and 52 °C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E_m) = 440 mV), one [3Fe-4S] cluster (E_m = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an E_m of 370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Anaerobic bacteria that are able to conserve metabolic energy from the dechlorination of chlorinated compounds have gained a lot of attention because of their role in bioremediation of contaminated sites and the novel respiration pathways they possess (1). Halorespiring bacteria have been found within the groups of low G + C Gram-positives, green nonsulfur bacteria, and - and - proteobacteria. These bacteria can use chloroalkenes, e.g. tetrachloroethene (PCE)¹ and trichloroethene (TCE) or chloroaromatic compounds such as chlorophenols or 3-chlorobenzoate as the terminal electron acceptor.

The halorespiratory pathway of anaerobic PCE degradation has been studied in some detail. A key enzyme in this respiratory pathway is the PCE reductive dehalogenase, which catalyzes the reductive removal of a chlorine atom from PCE and TCE. A 58-kDa PCE reductive dehalogenase was purified from Dehalospirillum multivorans, which contains cobalamin and probably two iron-sulfur clusters (2). Cloning and sequencing of the corresponding pceA gene revealed the presence of an additional open reading frame, pceB, being cotranscribed with pceA and coding for an 8-kDa membrane-spanning protein (3). The PCE reductive dehalogenases isolated from Dehalobacter restrictus (60 kDa) and Desulfitobacterium frappieri strain PCE-S (65 kDa) resemble the enzyme from Dehalospirillum multivorans with respect to cofactor content and catalytic properties (4, 5). EPR analysis of the D. restrictus enzyme confirmed the presence of cobalamin and two [4Fe-4S] clusters. All chloro alkene reductive dehalogenases characterized up to now are monomeric and either membrane-bound or membrane-associated.

Enzymes involved in chloroaryl respiration have been studied in Desulfomonile tiedjei and Desulfitobacterium species (6-8). However, no further molecular characterization of these enzymes was reported.

We investigated ortho-chlorophenol dechlorination in Desulfitobacterium dehalogenans. This organism is able to couple the reductive dechlorination of different ortho-chlorinated phenolic compounds to growth with lactate, pyruvate, formate, or hydrogen as electron donor (9, 10). Comparison of biomass yields on pyruvate and different electron acceptors indicated that chlorophenol dechlorination in D. dehalogenans is an energy-yielding process (11). This study for the first time describes the purification and characterization of the catalytic subunit of the ortho-chlorophenol reductive dehalogenase (o-CP dehalogenase) from Desulfitobacterium dehalogenans. Its redox properties were studied by EPR spectroscopy, and the corresponding cprA gene was cloned and characterized, revealing structural resemblance with haloalkene reductive dehalogenases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains, Plasmids, and Growth Conditions-- D. dehalogenans strain JW/IU-DC1 (DSM 9161) was cultivated under anaerobic conditions (100% N₂ gas phase) in 25-liter vessels containing 20 liters of basal medium as described by Neumann et al. (12), supplemented with 0.2% yeast extract, 20 mM lactate sodium salt, 20 mM 3-chloro-4-hydroxyphenyl acetate, 50 mM NaHCO₃, and trace elements and vitamin solution as recommended by the German Collection of Microorganisms. The 20-liter cultures were incubated at 37 °C for 2 days. After 1 day of incubation, 250 ml of 2 M NaOH was added to the culture to avoid acidification of the medium.

Escherichia coli XL1-Blue (Stratagene) was used as a host for cloning vectors. The strain was grown in Luria Bertani medium at 37 °C, and ampicillin was added at 100 µg/ml when appropriate. The cloning vectors pUC18 and pUC19 were purchased from Amersham Pharmacia Biotech, and pMON38201 was obtained from Monsanto.

Preparation of Cell Extracts-- Late exponential phase cultures of D. dehalogenans were harvested by continuous flow centrifugation at 16,000 × g (Biofuge 28RS, Heraeus Sepatech), which yielded 1.6 g of concentrated cells/liter of culture. The concentrated cells were stored at 20 °C. 8 g of cells was resuspended in 8 ml of buffer 1, consisting of 100 mM potassium phosphate (KP_i), pH 7.5, and 2.5 mM dithiothreitol. A few crystals of DNase I were added to the cell suspension. Cells were broken by sonication (Vibra, Sonic Materials Inc.) under anaerobic conditions. The cell debris was removed by centrifugation for 5 min at 20,000 × g. The supernatant was incubated for 10 min in the presence of 0.5 M KCl and 0.02% Triton X-100 and then separated into a membrane fraction and a soluble fraction by centrifugation for 90 min at 140,000 × g and 4 °C. The membrane fraction was resuspended in 8 ml of buffer 1 supplemented with 1% Triton X-100 and 20% glycerol and incubated for 60 min under anaerobic conditions at 4 °C. The insoluble fraction was removed from this preparation by centrifugation for 60 min at 140,000 × g and 4 °C. The solubilized enzyme fraction was stored under a N₂ gas phase at 4 °C.

Column Chromatography-- All chromatographic steps were performed by fast protein liquid chromatography (Amersham Pharmacia Biotech) in an anaerobic chamber with N₂/H₂ (95%/5%) gas phase. The Triton X-100 concentration of the sample was raised to 3% before it was applied to a column to prevent protein aggregation. The solubilized enzyme preparation was loaded on a Q-Sepharose column (2.2 × 8.9 cm) (Amersham Pharmacia Biotech) equilibrated with buffer A (50 mM KP_i, pH 6.0, 0.1% (w/v) Triton X-100, 20% glycerol, and 1 mM dithiothreitol). The column was eluted with a 75-ml linear gradient from 0 to 300 mM NaCl in buffer A at a flow of 2.5 ml/min. The o-CP dehalogenase activity was eluted at a NaCl concentration of approximately 180 mM. Fractions containing the highest dechlorinating activity were pooled and diluted with an equal volume of buffer A. The sample was applied on a Mono Q column (Amersham Pharmacia Biotech) equilibrated with buffer A. The enzyme was eluted with a 40-ml linear gradient from 0 to 400 mM NaCl in buffer A and a flow rate of 1.0 ml/min at a NaCl concentration of 180 mM.

Combined fractions containing dechlorinating activity were mixed with an equal volume of buffer B (50 mM Tris-HCl, pH 7.8, 0.1% w/v Triton X-100, 20% glycerol, and 1 mM dithiothreitol) and applied on a Mono Q column equilibrated with the same buffer. The enzyme activity was eluted with a 40-ml linear gradient from 0 to 400 mM NaCl in buffer B and a flow rate of 1.0 ml/min at a NaCl concentration of 280 mM.

Enzyme Assay-- Chlorophenol reductive dehalogenase activity was assayed in stoppered 1-cm cuvettes at 30 °C and pH 7.8 by photometric recording of the oxidation of titanium(III) citrate reduced methyl viologen at 578 nm (₅₇₈ = 9.7 mM¹·cm¹) as described by Schumacher and Holliger (13). The assay mixture contained 0.3 mM methyl viologen and had an initial absorption at 578 nm of 2.6. The assay was started by the addition of 20 µl of 50 mM Cl-OHPA to give a final concentration of 1 mM Cl-OHPA. One unit is defined as the amount of enzyme that catalyzed the reduction of 1 µmol of chlorinated substrate or the oxidation of 2 µmol of reduced methyl viologen per minute. The same specific activity was obtained whether methyl viologen oxidation, Cl-OHPA disappearance, or 4-hydroxyphenyl acetate appearance was followed. The protein content of the samples was determined according to Bradford (14) with bovine serum albumin as a standard.

Kinetic Parameters-- The pH optimum was determined in a 200 mM Tris-maleate buffer ranging from pH 5.5 to 9.0. Michaelis-Menten constants were determined from Lineweaver-Burk representations of data obtained by determining the initial rate of Cl-OHPA reduction under the assay conditions described above and using 5 µM to 10 mM substrate in the cuvette.

Composition of o-CP dehalogenase-- The molecular mass of the denatured protein was determined by SDS-polyacrylamide gel electrophoresis according to Laemmli (15). A low molecular weight marker (Bio-Rad) was used as reference. The gels were stained with Coomassie Brilliant Blue R-250. The concentration of acid labile sulfur of three individual samples was determined according to Rabinowitz (16). The iron and cobalt content of three independent enzyme preparations was measured by inductively coupled plasma mass spectrometry (Elan 6000, Perkin-Elmer). The protein concentration of the inductively coupled plasma mass spectrometry samples was determined by measuring the absorbance changes in the Rose Bengal binding assay as described by Elliot et al. (17) with bovine serum albumin as a standard. A correction factor was determined with purified o-CP dehalogenase to compare the Rose Bengal protein determination and the Bradford protein determination. A correction factor of 1.10 was applied for the Rose Bengal determinations.

Cobalamin and Iron-Sulfur Cluster Analysis by EPR-- EPR spectra were recorded on a Bruker 200 D spectrometer with cryogenics, peripheral equipment, and data acquisition as described previously (18). The protein concentration of the EPR samples was 0.4 mg/ml in buffer B. The enzyme was completely reduced in 45 min by deazaflavin/EDTA-mediated light reduction as described by Massey and Hemmerich (19). Deazaflavin was synthesized according to Janda and Hemmerich (20).

N-terminal Amino Acid Sequence-- Purified enzyme was transferred from a 12% SDS-polyacrylamide gel onto a polyvinylidene difluoride membrane (Immobilon polyvinylidene difluoride, Millipore Corp.) by blotting with a Trans-Blot SD semidry transferring cell (Bio-Rad). Blotting was carried out at 14 V for 2 h using a transfer buffer containing 48 mM Tris, 39 mM glycine, and 20% methanol, pH 9.1. The transferred protein was stained with Coomassie Brilliant Blue R-250. The N-terminal amino acid sequence of the blotted protein was determined as described by Schiltz et al. (21).

DNA Isolation, Manipulation, and Oligonucleotides-- Chromosomal DNA from D. dehalogenans was isolated as follows. Protoplasts were prepared from 12 ml of culture (A₆₀₀ = 0.4) as described by van Asseldonk et al. (22), recovered at 13,000 × g for 2 min, and resuspended in 100 µl of THMS buffer (30 mM Tris-HCl, pH 8.0, and 3 mM MgCl₂ in 25% sucrose). After the addition of 400 µl of 50 mM Tris-HCl (pH 8.0), containing 5 mM EDTA, 50 mM NaCl, and 0.5% SDS, chromosomal DNA was purified through successive steps of phenol/chloroform extraction and recovered by ethanol precipitation.

Plasmid DNA was isolated from E. coli by using the alkaline lysis method, and standard DNA manipulations were performed according to established procedures (23) and the manufacturers' instructions. Enzymes were purchased from Life Technologies, Inc., Roche Molecular Biochemicals, or New England Biolabs. Oligonucleotides and [-³²P]dATP were obtained from Life Technologies and Amersham Pharmacia Biotech, respectively. Prehybridization and hybridization were performed at 65 and 50 °C, respectively. Posthybridization washes were conducted at 40 °C.

Oligonucleotides used in this study were BG 444 (5'-GCI GA(A/G) ACI ATG AA(C/T) TA(C/T) GTI CCI GGI CCI ACI AA(C/T) GCI GCI (A/T)(C/G)I AA(A/G) (C/T)TI GGI CCI GT-3', nucleotides 644-703), BG 458 (5'-GCC GGA GCC TTG ATC GC-3', nucleotides 427-411), and BG 475 (5'-GGC AGG TCT GGG AGA ATT G-3', nucleotides 1366-1384). In order to restrict the extent of degeneration for BG 444, inosine (I) was used at 3- or 4-fold degenerated positions.

DNA Amplification by Inverse PCR-- Inverse PCR (24) was performed as follows. Chromosomal DNA was digested with HincII and ligated at a concentration of 0.5 ng/µl. 5 ng of self-ligated DNA was used as the template in a 25-µl PCR reaction containing the following: 2 ng/µl each primer; 2.25 mM MgCl₂; 200 µM dATP, dCTP, dGTP, and dTTP; and 1 unit of Expand^TM Long Template enzyme mixture (Roche Molecular Biochemicals). The DNA was amplified using the GeneAmp^® PCR System 2400 (Perkin-Elmer). After preheating to 94 °C for 2 min, 35 cycles were performed, consisting of denaturation at 94 °C for 20 s, primer annealing at 50 °C for 30 s, and elongation at 68 °C for 3 min. After 10 cycles, the elongation time was extended with 20 s/cycle. A final extension of 7 min at 68 °C was included. PCR products were purified from agarose gel by Gene Clean (Bio 101) and cloned into pMON38201 cut with XcmI.

DNA Sequencing and Sequence Analysis-- DNA sequencing was performed using a Li-Cor DNA sequencer 4000L. Plasmid DNA used for sequencing reactions was purified with the QIAprep Spin Miniprep kit (Qiagen GmbH). Reactions were performed using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit (Amersham Pharmacia Biotech). Infrared labeled oligonucleotides were purchased from MWG Biotech. Sequence similarity searches and alignments were performed using the BLAST 2.0 program (25) (NCBI) and the programs Clustal X and GeneDoc (26),² respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification and Characterization of o-CP Dehalogenase-- o-Chlorophenol reductive dehalogenase was purified under strict anaerobic conditions from the membrane fraction of D. dehalogenans grown on lactate and 3-chloro-4-hydroxyphenylacetate (Table I). The specific activity increased 90-fold upon purification and amounted to 28 units/mg protein with reduced methyl viologen as an artificial electron donor. The purified enzyme had a pH and temperature optimum of 8.1 and 52 °C, respectively. At 30 °C, the enzyme showed Michaelis-Menten kinetics for Cl-OHPA. The K_m for this chlorinated substrate was determined to be 20 µM at a methyl viologen concentration of 0.3 mM. Cl-OHPA showed no inhibitory effect up to 10 mM, which was the highest concentration used. Several halogenated compounds were tested as possible alternative substrates for o-CP dehalogenase. Activity of o-CP dehalogenase was observed with 2-CP, 2,3-dichlorophenol (2,3-DCP), 2,4-DCP, 2,6-DCP, and pentachlorophenol as substrate (Table II). 3-CP, 4-CP, and 2,5-DCP were not dechlorinated. Additionally, 2-bromo-4-chlorophenol, but not 2-fluoro-4-chlorophenol, could be dehalogenated. This confirms that reductive dehalogenation is the reaction mechanism of o-CP dehalogenase, since bromide and chloride are more readily reductively removed than fluoride. No activity was measured with PCE or TCE, indicating that chlorinated aliphatics do not serve as a substrate for the o-CP dehalogenase.

SDS-polyacrylamide gel electrophoresis analysis of the purified enzyme preparation revealed one band of approximately 48 kDa (Fig. 1). An accurate determination of the native size of the enzyme was not possible due to the high concentration of detergent needed to prevent protein aggregation (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   12% SDS-polyacrylamide gel electrophoresis with the purified ortho-chlorophenol reductive dehalogenase of D. dehalogenans (5 µg) in lane 1. Molecular size markers are shown in lane 2. The arrow indicates the purified protein band. The gel was stained with Coomassie Brilliant Blue R-250.

The analysis of metals revealed the presence of 0.7 ± 0.1 mol of cobalt and 7 ± 1.4 mol of iron atoms per mol of monomer. Acid-labile sulfur analysis showed 9.9 ± 1.2 mol of sulfur atoms/mol of monomer. We conclude from these results and the EPR data (see below) that 1 cobalamin and 2 iron-sulfur clusters are present per mol of enzyme.

N-terminal Sequence, Cloning, and Sequencing of the cprA Locus-- The N-terminal amino acid sequence of the o-CP dehalogenase purified from D. dehalogenans was determined and revealed the sequence NH₂-AETMNYVPGPTNARSKLRPVHDFA. A 59-bp 256-fold degenerated oligonucleotide (BG 444) was designed based on the sequence of the first 20 N-terminal amino acids. Southern blot analysis of EcoRI-HindIII-digested chromosomal DNA of D. dehalogenans revealed a 2.7-kilobase fragment that hybridized strongly to radiolabeled BG 444. This fragment was cloned in E. coli using EcoRI-HindIII-digested pUC18, resulting in pLUW910. Sequence analysis of the HindIII-HincII 1.8-kilobase fragment of pLUW910 revealed the determined N-terminal amino acids immediately downstream of the HindIII site, indicating that pLUW 910 lacks the translation start of the gene of interest. Therefore, the divergent primer pair BG 458/BG 475 was used to specifically amplify the pLUW910 upstream flanking fragment in an inverse PCR from HincII-digested chromosomal DNA. To ensure determination of the correct nucleotide sequence, three independently obtained PCR products were cloned yielding pLUW912a-c. From these, HincII deletion clones were prepared, giving the corresponding pLUW913a-c. Fig. 2 shows a restriction map of the DNA region cloned and sequenced.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Restriction map of the D. dehalogenans genomic cpr region. The vertical arrows mark DNA restriction sites. The horizontal bars indicate fragments, cloned either in pUC 18 or pMON38201. The horizontal arrows indicate open reading frames. Oligonucleotides used in this study are shown. The 32 C-terminal amino acids of ORF X show some similarity with the C-terminal part of GroEL-type chaperonines. ORF1 exhibits no significant similarities with known proteins.

Organization of the cprA Locus-- Sequence analysis revealed the presence of two closely linked open reading frames, namely cprB (nucleotides 194-505) and cprA (nucleotides 518-1861). A third open reading frame, ORF1, starts at nucleotide 1958. Preceding each of the three open reading frames, potential Shine Dalgarno sequences were found (data not shown).

The predicted gene product of cprA is a polypeptide of 447 amino acids with a molecular mass of 49,720 Da. The first 42 N-terminal residues of CprA comprise a leader sequence that is cleaved off upon maturation of the protein, leaving a mature 405-amino acid polypeptide with a calculated molecular mass of 45,305 Da. The leader sequence contains an RR motif characteristic for a large number of mainly periplasmic proteins binding different redox cofactors (28). These twin arginine signal sequences (consensus (S/T)RRXFLK) are thought to play a major role in the maturation and translocation of such proteins. As all twin arginine signal sequences, the CprA leader sequence shows the structural characteristics of standard Sec signal sequences. Furthermore, the established cleavage site -VANAAETM- follows the "1/3 rule" of von Heijne (29).

The D. dehalogenans CprA sequence reveals the presence of an extended cluster of cysteine residues (Cys³³⁰-Cys³⁸⁷, Fig. 3). The first group of four cysteines Cys³³⁰-Cys³⁴⁰ is identical to the consensus sequence of bacterial ferredoxin type clusters (CXXCXXCXXXCP; Ref. 30), including the conserved proline at position 341. The second cluster shows the same conserved residues (Cys³⁸⁰-Pro³⁸⁸) but lacks the first cysteine. The B₁₂ binding motif DXHXXG-(41)-SxL-(26-28)-GG, as it was determined for a subset of B₁₂-dependent enzymes (31), is not present in CprA.

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Primary sequence alignment for the ortho-chlorophenol reductive dehalogenase from D. dehalogenans (CprA) and the PCE dehalogenase from D. multivorans. The alignment was performed using the programs Clustal X and GeneDoc (26).2 The light gray boxes mark identical residues. The dark gray boxes show residues from the twin arginine consensus motif. Residues highlighted in black indicate the conserved iron-sulfur cluster binding motifs. CprA, ortho-chlorophenol reductase from D. dehalogenans (GenBankTM accession number AF115542); PceA, PCE dehalogenase from D. multivorans (GenBankTM accession number AF022812).

Upstream of cprA, a second potential gene, cprB, was found, that could encode a 103-amino acid polypeptide with a calculated molecular mass of 11,517 Da. The predicted cprB gene product does not exhibit significant similarities with any known proteins present in the data bases. A hydrophilicity plot indicates the presence of three membrane-spanning helices (Fig. 4). Following the positive-inside rule for integral membrane proteins (32), the N terminus of this polypeptide is predicted to point outward, whereas the C-terminal part is located at the cytoplasmic face of the membrane. CprB and cprA are separated by only 12 nucleotides. Neither transcription termination nor initiation signals are present between the two genes. Preliminary experiments suggest co-transcription of both genes (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Hydrophilicity plot and charge distribution for CprB. The hydrophilicity plot was determined according to the method of Kyte and Doolittle (27). The analysis was performed using the program Protean from the DNAstar software package.

Cobalamin Involved in Electron Transfer in o-CP Dehalogenase-- Cobalt in biological systems occurs in oxidation states 3+, 2+, and 1+. Only the Co²⁺ 3d⁷-system is half-integer spin and, therefore, readily detectable in EPR spectroscopy. In cobalamin, the Co²⁺ is low spin S = 1/2. The EPR of D. dehalogenans o-CP dehalogenase, as isolated, exhibits a signal characteristic for Cob(II)alamin in the base-off form and a weak, near isotropic, S = 1/2 signal around g = 2 indicative of [3Fe-4S] (see below).

Previously, it was found that full chemical reduction of another reductive dehalogenase, the PCE reductase from D. restrictus, could not be achieved with dithionite (4). Therefore, we used the light-induced strongly reducing system of deazaflavin plus EDTA. Prolonged illumination resulted in a clear EPR spectrum that is dominated by a signal with g values of 2.05, 1.93, and 1.87, typical for reduced [2Fe-2S] or [4Fe-4S] clusters (Fig. 5, trace A). The signal rapidly broadens above 20 K, which indicates that its origin is a [4Fe-4S]¹⁺ cluster. Cob(II)alamin in the base-on form is present as a minor component in trace A, while the base-off form of Cob(II)alamin is fully reduced.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   EPR spectra of D. dehalogenans o-CP dehalogenase. Trace A, the [4Fe-4S] signal from enzyme fully reduced by illumination with visible light for 50 min in the presence of 20 µM deazaflavin and 2 mM EDTA. Base-on Cob(II)alamin can be detected as a minor component in trace A. Trace B, the base-off Cob(II)alamin signal from enzyme reoxidized by 0.5-min anaerobic incubation with 2 mM Cl-OHPA. Trace C, the [3Fe-4S] signal from enzyme fully oxidized by anaerobic incubation with 2 mM potassium ferricyanide for 5 min. EPR conditions were as follows: microwave frequency, 9.41 GHz; microwave power 5 milliwatts (trace A, 0.8 milliwatts); modulation frequency, 100 kHz; modulation amplitude, 0.63 millitesla; temperature, 9.5 K (trace A), 30 K (trace B), 15 K (trace C).

When the enzyme is anaerobically hand-mixed with the substrate Cl-OHPA and immediately frozen in liquid nitrogen (i.e. a reaction time of ~0.5 min), another spectrum is obtained (Fig. 5, trace B). This is the signal of the base-off form of Cob(II)alamin (4). The signal is essentially identical to that obtained from enzyme as isolated. In a control experiment where water, flushed with nitrogen gas, was added to a reduced o-CP dehalogenase sample, no base-off cobalamin signal developed. The addition of an excess of ferricyanide did not affect the signal, and this indicated an unusually high oxidation potential for the Co(II/III) couple, as previously found for the D. restrictus dehalogenase (4). Estimation of the spin-Hamiltonian parameters by simulation gives g values of 1.99, 2.35, and 2.35 and cobalt hyperfine (I = 7/2) values of 14, 7.5, and 7.5 millitesla. These values are close to those found for the dehalogenase from D. restrictus (4). The simulation indicates furthermore that the spectrum contains a minor second component, namely a base-on form of Cob(II)alamin; this form is also detectable as a minor component in trace A.

Upon incubation with excess potassium ferricyanide, the Co²⁺ signal is still present at maximal amplitude, but it is now hardly discernible, since the gain has been reduced for the observation of a near isotropic signal around g = 2 typical for a [3Fe-4S]¹⁺ cluster (Fig. 5, trace C). The broad peak at low field is the g_z from excess [Fe(CN)₆]³⁺.

All three signals, the [4Fe-4S]¹⁺ signal, the Cob(II)alamin signal, and the [3Fe-4S]¹⁺ signal, integrate to approximately the same value, corresponding to a spin count close to 1 spin per 48-kDa monomer.

The signals behave as expected in reductive (dithionite) and oxidative (ferricyanide) bulk redox titrations in the presence of a mixture of redox mediators (Fig. 6); in an oxidative titration, the signal ascribed to a [3Fe-4S] cluster appears with an oxidation potential of E_m,7.8= +70 mV; in a reductive titration the Co²⁺ signal disappears with a reduction potential of E_m,7.8= 370 mV; and the signal from the [4Fe-4S] cluster appears with E_m,7.8= 440 mV. The E_m values for Co(II) and [4Fe-4S] are similar to those found for the D. restrictus dehalogenase. However, that enzyme contains two [4Fe-4S] clusters (4). The EPR of the present D. dehalogenans enzyme strongly suggests the presence of one [4Fe-4S] and one [3Fe-4S] cluster, consistent with sequence analysis (see above).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   EPR-monitored redox titration of the metal centers in D. dehalogenans o-CP dehalogenase. +, [3Fe-4S]1+; , Cob(II)alamin; , [4Fe-4S]1+. Starting from a redox potential of 130 mV, the sample was reduced by substoichiometric additions of dithionite and oxidized by substoichiometric additions of ferricyanide, both in the presence of a mixture of redox mediators covering the full potential axis. Amplitudes are given as a percentage of maximal signal intensities. The latter correspond to enzyme fully oxidized by excess ferricyanide or enzyme fully reduced by light/deazaflavin/EDTA. These extreme forms have undefined potentials and are presented as points on the vertical borders. EPR conditions were as in Fig. 5. The solid traces are fits to the Nernst equation assuming single electron transfer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ortho-Chlorophenol reductive dehalogenase is the terminal reductase involved in the halorespiratory chain of D. dehalogenans. Here we describe the purification and molecular characterization of this key enzyme and its gene cprA. This membrane-associated enzyme mediates the electron transfer from a yet unidentified electron donor to the halogenated substrate. The substrate spectrum of the purified enzyme was similar to that reported for resting cells, indicating that a single enzyme is involved in dehalogenation of ortho-halogenated phenols (Ref. 10, Table II).

The purified o-CP dehalogenase contains one [4Fe-4S] cluster, one [3Fe-4S] cluster, and one cobalamin per monomer. The presence of two iron-sulfur clusters was confirmed by the identification of one ferredoxin-like and one truncated iron-sulfur cluster binding motif (Fig. 3) in the sequence of CprA. These iron-sulfur clusters might be involved in the electron transfer to the active site that contains the cobalamin. The primary sequence alignment of CprA with PceA, the PCE reductive dehalogenase of D. multivorans (3), revealed a rather high degree of similarity on the amino acid level in the C-terminal part of both enzymes (Fig. 3). In PceA, the same two iron-sulfur cluster binding motifs are present, indicating a conserved mode of intramolecular transport of electrons. Both reductive dehalogenases probably differ in iron-sulfur cluster contents from the PCE reductase isolated from D. restrictus, where two [4Fe-4S] clusters were identified (4). In the case of the 47-kDa Cl-OHPA reductive dehalogenase of the closely related Desulfitobacterium hafniense, the presence of three iron-sulfur clusters has been reported (8). However, more sequence information on both the enzymes from D. restrictus and D. hafniense is not yet available.

The formation of Co(II) in base-off conformation upon the addition of Cl-OHPA to light-reduced o-CP dehalogenase confirms the involvement of the cobalamin in the dechlorination reaction. PCE reductase from D. restrictus, which converts PCE via TCE to 1,2-cis-dichloroethene, also contains cobalamin (E_m = 350 mV) in its base-off conformation (4). A similar mechanism could account for both chlorophenol and PCE dechlorination, although PCE is not a substrate for o-CP dehalogenase and D. restrictus is not capable of dechlorinating chlorophenols.

The cprA gene encodes a proprotein, in which the mature polypeptides is proceeded by a twin arginine-type signal sequence characteristic for periplasmic enzymes containing complex redox cofactors. A similar leader sequence is present in the pceA gene product. For both dehalogenases, it has been proposed by dye-mediated activity measurements in intact and broken cells that the dehalogenating activities are located at the inner face of the cytoplasmic membrane (data not shown; Ref. 3). The only other twin arginine enzyme with similar contradictory results concerns the E. coli Me₂SO reductase (28, 33). Additional experiments will be required to solve the topology of these enzymes.

Elucidation of the nucleotide sequences upstream and downstream of cprA revealed the presence of a second potential gene, cprB. The hydrophobic gene product, CprB, might have a role in anchoring the catalytic subunit of the o-CP reductive dehalogenase to the cytoplasmic membrane. A similar function has been proposed for PceB in D. multivorans (3).

Although CprA and PceA exhibit highly conserved boxes, both primary sequences lack the consensus sequence for the binding of the corrinoid cofactor conserved among several methylcobalamin-dependent methyltransferases and mutases (31).

The role of cobalamin in the reductive dehalogenases from chlorophenol and PCE-degrading organisms is of special interest, since it does not mediate the "usual rearrangement" or alkyl transfer but an elimination reaction (31). Two models have been proposed for the reaction mechanism of PCE reductive dehalogenation. One model involves the formation of a Co(III)-chloroethene carbon-metal bond (2), whereas the second model postulates the formation of a chloroethene radical (4). However, neither of these intermediates has been demonstrated unequivocally for PCE reductive dehalogenases. Based on our data, it is not possible to determine which model applies for ortho-chlorophenol reductive dehalogenase from D. dehalogenans. On one hand, an essential intermediate in the first model, Cob(III)alamin, was not formed upon oxidation of the enzyme. On the other hand, there was no radical formation upon the addition of substrate to the reduced enzyme. The latter could be due to the slow reaction time, which makes it difficult to detect a reactive compound such as a phenol radical. Additional experiments are required in which the supposed radical would be stabilized.

The similarities between the o-chlorophenol reductive dehalogenase of D. dehalogenans and the PCE reductive dehalogenases of Dehalospirillum multivorans and Dehalobacter restrictus on both mechanistic and structural properties as well as their primary sequences suggest that these enzymes constitute a novel class of corrinoid-containing reductases.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF115542.

^§ To whom correspondence should be addressed. Tel.: 31-0-317483741; Fax: 31-0-317483829; E-mail: Bram.vandepas@ algemeen.micr.wau.nl.

^¶ Supported by the Studienstiftung des Deutschen Volkes.

² K. B. Nicholas and H. B. J. Nicholas, unpublished communication.

	ABBREVIATIONS

The abbreviations used are: PCE, tetrachloroethene; TCE, trichloroethene; Cl-OHPA, 3-chloro-4-hydroxyphenyl acetate; CP, chlorophenol; DCP, dichlorophenol; PCR, polymerase chain reaction; ORF, open reading frame; o-CP dehalogenase, ortho-chlorophenol reductive dehalogenase.

	REFERENCES

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