Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS

Article abstract of DOI:10.1021/ja511653d

Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe³⁺, and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

Full text of DOI:10.1021/ja511653d

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Article
Biochemical and EPR-spectroscopic Investigation into
Heterologously Expressed Vinyl Chloride Reductive
Dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS
Anutthaman Parthasarathy, Troy A. Stich, Svenja T. Lohner,
Ann Lesnefsky, R. David Britt, and Alfred M. Spormann
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja511653d • Publication Date (Web): 16 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Biochemical and EPR-spectroscopic Investigation into Heterologously Expressed Vinyl
Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS
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Anutthaman Parthasarathy , Troy A. Stich , Svenja T. Lohner , Ann Lesnefsky , R. David Britt ,
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and Alfred M. Spormann
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Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford
University, Stanford, CA,
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Department of Chemistry, University of California Davis, Davis, CA
*
Corresponding author’s address: James H. Clark Center, 318 Campus Drive, Stanford
University, Stanford, CA
Phone: 650.723.3668, email: spormann@stanford.edu
ABSTRACT: Reductive dehalogenases play a critical role in the microbial detoxification of
aquifers contaminated with chloroethenes and chlorethanes by catalysing the reductive
elimination of a halogen. We report here the first heterologous production of vinyl chloride
reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was
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reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe , and
sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA
catalyzing vinyl chloride reduction with Ti(III)ꢀcitrate as reductant and methyl viologen as
mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain
VS. VcrA was also found to catalyze a novel reaction, the environmentally important
dihaloelimination of 1,2ꢀdichloroethane to ethene. Electron paramagnetic resonance (EPR)
spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the
presence of Cob(II)alamin. Addition of Ti(III)ꢀcitrate resulted in the appearance of a new signal
characteristic of a reduced [4Fe−4S] cluster and the disappearance of the Cob(II)alamin signal.
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UVꢀvis absorption spectroscopy of Ti(III)citrateꢀtreated samples revealed the formation of two
new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Feꢀ
4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Feꢀ4S]
cluster reduces Co(II) to Co(I) of the enzymeꢀbound cobalamin. Vinyl chloride reduction to
ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a
vinyl radical as a potential reaction intermediate.
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INTRODUCTION
Chloroethenes, such as tetrachloroethene (PCE), trichloroethene (TCE), the dichloroethenes
(DCE), vinyl chloride (VC), as well as the chloroethanes 1,1,2 trichloroethane (TCA), and 1,2
dichloroethane (DCA) are the most prevalent groundwater contaminants in developed countries
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including the United States . Key for the microbial degradation of such chlorinated aliphatic
hydrocarbons to harmless organic compounds are catabolic reductive dehalogenases (Rdh). Rdhs
are highly oxygenꢀsensitive enzymes and catalyze the reductive elimination of a halide. The
catalytically active subunit RdhA contains a corrinoid and two [4Feꢀ4S] clusters and is typically
associated with the outer leaflet of the cytoplasmic membrane presumably via the small integral
membrane protein RdhB. Rdhs are unique among the FeS cluster/corrinoid enzymes in that they
catalyse a net reduction reaction directly by the cobalamin without the intervention of other
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organic cofactor radicals .
Current understanding of Rdh is limited to results from studies with only a few purified enzymes
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PceA , CprA , TceA and VcrA . These studies indicated a relatively high degree of
substrate specificity of each Rdh. More recently, dihaloeliminating reductive dehalogenases were
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identified, resulting in the formation of chloroethenes from chloroethanes
. Thus, the
degradation of both classes of organohalogens intersect via a mosaic metabolic networks of
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diverse Rdhs . Whether such dihaloeliminating activities are due to a single, monoꢀ and
dihaloeliminating Rdh or the concerted action of two specific Rdhs has not been revealed.
However, dihaloelimination is of critical importance for successfully engineering bioremediation
and avoiding accumulation of more toxic intermediates such as vinyl chloride.
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Reductive dehalogenation is a catabolic process coupling energy conservation with a
dehalogenation reaction. It is found primarily in the anaerobic Dehalococcoides-,
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Sulfurospirillumꢀ, and Dehalobacterꢀtype microorganisms. In Dehalobacter restrictus ,
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Desulfomonile tiedjei and Sulfurospirillum multivorans , reductive dehalogenation is one of
many possible modes of energy conservation. In contrast, Dehalococcoides sp. are highly nicheꢀ
specialized and can only conserve energy by reductive dehalogenation. Dehalococcoides Rdhs
are unique and distinctive in that the electrons for chloroethene reduction are derived obligatorily
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from H . Hydrogen is presumably oxidized by a HupL hydrogenase, which appears to form a
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membraneꢀbound complex facing the outside of the cell with an Rdh (Fig S1) . How the
electrons are transferred to an Rdh, and how Dehalococcoides spp. conserve metabolic energy
during this unique respiratory process, are unknown.
The development of a molecular understanding of Dehalococcoidesꢀtype Rdhs, in particular the
study of the reaction mechanism has been hampered by the slow doubling time of the
microorganisms, the low biomass yield, and the oxygen sensitivity of native Rdhs. Numerous
unsuccessful attempts have been conducted by different research groups to heterologously
produce active Rdhs. Only recently two reductive dehalogenases were heterologously expressed
in the background of a dehalogenating strain: reductive dehalogenase (PceA) of
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Desulfitobacterium hafniense strainY51 was overexpressed in Shimwellia blattae
and the
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NpRdhA of Nitratireductor pacificus in Bacillus megaterium . We report here the first
successful heterologous overproduction in E. coli of a Dehalococcoidesꢀtype Rdh, its
reconstitution to an active form, as well as its initial biochemical characterization.
EXPERIMENTAL SECTION
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Plasmid construction: Standard protocols were used for all genetic work. Promega kits were
used for isolation and purification of DNA. Vector pLICꢀHMK (a gift from Dr. James Berger,
University of California, Berkeley) was used as the plasmid backbone. The vcrA gene (vinyl
chloride reductase A) from Dehalococcoides VS (NCBI Gene ID: 8658217,
NC_013552.1:complement(1187299ꢀ1188753)) devoid of the first 35 amino acids of the 5’
region coding for the TATꢀsignal peptide was amplified from the genomic DNA of
Dehalococcoides mccartyi strain VS using 5’ꢀ TAC TTC CAA TCC AAT GCA
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GACATTGATGAACTTGTTTCAAGC – 3’ as the upstream primer and 5’ – T TAT CCA CTT
CCA ATG TTA TTA AGTACCGGGATCAAAGCC – 3’ as the downstream primer, and
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inserted into pLICꢀHMK using ligation independent cloning or LIC
to create an IPTG
inducible Nꢀterminal, 6xꢀHistidine, Maltose Binding Protein (MBP), TEV protease cleavage site
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vcrA fusion gene
. Fermentas LIC qualified T4 DNA polymerase was used to perform the
LIC reaction. The fidelity of the entire gene fusion was verified by DNA sequencing. This
plasmid was denoted NNHMꢀVcrA for Nꢀterminus, No TAT signal sequence, 6xꢀHistidine Tag,
Maltose binding protein vcrA gene fusion. For simplicity we address the NNHMꢀVcrA protein
in this manuscript as VcrAꢀMBP with the understanding that it is the fusion protein, as described
here: refer to Fig S2.
Heterologous expression of VcrA-MBP in E. coli: pLICꢀHMK NNHMꢀVcrA was transformed
into chemically competent cells of E. coli BL21 DE3. 5 ml cultures of this strain were grown
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overnight at 37°C in nonꢀinducing minimal medium (MDG medium ) supplemented with 2 g/L
glucose and 100 ꢁg/ml of kanamycin. The overnight cultures were then inoculated into LB
medium supplemented with 0.2 % (w/v) of glucose and 100 ꢁg/ml of Kanamycin up to an initial
OD600 of 0.1. After the cultures had reached an OD600 of 0.5 at 37°C, expression of the plasmid
was induced upon addition of 1 mM IPTG final concentration. Following 3 h of induction, the
cells were harvested by centrifugation at 6000 × g at 4°C for 20 min on a Beckman Coulter
Avanti Jꢀ20 XP centrifuge. The overproduction was verified by SDS PAGE. The bulk of the
overproduced protein was found in the insoluble fraction (data not shown).
Purification of VcrA-MBP by affinity chromatography: 3 g of NiꢀIDA or NiꢀTED bonded to
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silica
for HisꢀTag affinity chromatography (Protino® NiꢀIDA Resin & Protino ® NiꢀTED
Resin, Macherey Nagel) was added as slurry in distilled water to a 20 ml fritted column
(diameter 2 cm) and allowed to pack by gravity to about 8 ml volume. Gentle suction was
applied at the bottom of the column through a vacuum line to drain the aqueous phase. Dry
columns were stored at room temperature until use.
All subsequent operations were carried out in a Coy anoxic chamber (95%N , 5%H ). All
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buffers, filters, centrifuge tubes and columns were incubated at least overnight in the anoxic
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chamber prior to use. Buffers and solutions were filtered through a sintered glass filter layered
with Whatman GF/C 55 mm glass fibre filters and were subsequently subjected to 4 cycles of
degassing and purging with nitrogen (99.99%). In the anoxic chamber 2 mM 2ꢀmercaptoethanol
final concentration was added to all solutions and buffers.
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About 3 g wet weight of induced cells carrying pLICꢀHMK NNHMꢀVcrA were reꢀsuspended in
ca. 30 ml of Buffer A (50 mM potassium phosphate pH 8.0 containing 0.5 M NaCl, 20% (v/v)
glycerol and 1 mM PMSF as a protease inhibitor). After sonication in the anoxic chamber at 70%
power for 15 min (30 s pulse followed by 30 s cooling) at 4°C, the suspension was centrifuged
for 30 min at 4°C at 30,000 × g on a Beckman Coulter Avanti Jꢀ20 XP centrifuge. The
supernatant was discarded and the pellet reꢀsuspended in 15 ml of denaturing buffer (Buffer A +
8 M urea). Following another round of sonication at 70% power for 15 min (30 s pulse followed
by 30 s cooling) at 4°C, the suspension was stirred on ice for 60 min to solubilize the pellet.
Another centrifugation for 30 min at 4°C at 10,000 × g was performed to remove any remaining
insoluble material.
The supernatant containing denatured VcrAꢀMBP was loaded onto the NiꢀIDA column, which
had been washed with 5 column volumes of Buffer A and equilibrated with 3 column volumes of
denaturing buffer. VcrAꢀMBP elution was achieved with 3 column volumes of 250 mM
imidazole in the denaturing buffer. The eluted fractions were collected and analysed separately.
The samples were desalted with PDꢀ10 columns (GE Healthcare) which had been previously
washed with several volumes of anoxic buffer, to remove urea and imidazole, and eluted with
Buffer
A
containing 0.2%
CHAPS
(3ꢀ[(3ꢀcholamidopropyl)dimethylammonio]ꢀ1ꢀ
propanesulfonate) as detergent. The eluted fractions were pooled and allowed to refold in the
refolding buffer (50 mM Tris pH 8.0 containing 0.5 M NaCl, 20% (v/v) glycerol, 0.2% CHAPS
and 1 mM PMSF (phenylmethylsulfonyl fluoride) as a protease inhibitor and 2 mM 2ꢀ
mercaptoethanol) for 1 h at room temperature and then concentrated via Amicon Ultracel 50 K
centrifugal filters (EMD Millipore) at 6000 × g on a Clay Adams Compact II centrifuge. The
concentrated protein was mixed with a minimum volume of the refolding buffer and frozen at ꢀ
80°C until further use. The purity of VcrAꢀMBP was verified by SDSꢀPAGE (Fig 1) and peptide
mass fingerprinting of the corresponding SDS gel band.
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Cofactor reconstitution: Anoxic solutions of 200 mM DTT, 100 mM FeCl and 30 mM
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Na S.9H O were prepared in degassed 100 mM TrisꢀHCl pH 7.4 purged with 99.99% N prior to
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the cofactor reconstitution. In order to initiate the reconstitution of the ironꢀsulfur clusters of
VcrA or VcrAꢀMBP, the protein was reduced by the addition of 5 mM DTT, and the solution
was incubated at room temperature for 30 min to be completely reduced. The ironꢀsulfur clusters
were subsequently reconstituted by adding FeCl at 5 mol excess over protein and Na S at 10
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mol excess over protein to the mixture, which was then incubated at room temperature for 90
min. Precipitated protein and iron sulfide particles were removed by centrifugation at 5,000 × g
for 5 min. Finally, adenosylꢀ or hydroxocobalamin was added to a final concentration of 10
mg/ml, and the mixture was incubated at room temperature for 30 min. Unbound iron sulfide and
cobalamin were removed by passage through PDꢀ10 columns (GE Healthcare). The samples
were filtered through 0.2 μm Thermo Scientific Nalgene® Syringe filters, eluted with the
refolding buffer, concentrated to ca. 20 mg/ml and stored at ꢀ80°C until further use. The
reconstitution of the enzyme could be performed with any one of the following B derivatives –
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adenosylcobalamin, hydroxocobalamin, dicyanocobinamide or aquahydroxocobinamide,
although only the first two were used for trials involving activity assays.
TEV-protease digestion and purification of untagged VcrA: Samples of purified but
unassembled VcrAꢀMBP apoprotein were digested anaerobically with manufacturerꢀ
recommended amounts of ProꢀTEV (Promega) protease (which contains a HQ tag allowing for
Hisꢀtagged affinity chromatography) in 50 mM Tris HCl pH 8.0 free of protease inhibitors for 3
h at 20°C to remove the MBP & His tags. The digest was passed over a NiꢀIDA column which
retained ProꢀTEV protease via its HQ tag as well as the HisꢀMBP tag of VcrA. The untagged
VcrA (defined here and described as ‘VcrA’ as opposed to the parent ‘VcrAꢀMBP’) was
collected from the flow through and confirmed pure by PAGE (Fig. S3); 3 mg of VcrAꢀMBP
yielded 0.8 mg of untagged VcrA.
SDS-PAGE and protein determination: Samples for SDSꢀPAGE and protein quantification
were prepared by using Zeba® Micro Spin desalting columns 7 MWCO (Thermo Scientific)
following the manufacturer’s protocol and eluted in distilled water. Further processing for SDSꢀ
PAGE was performed according to the NuPage® Novex system (Invitrogen). NuPage® Novex
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.0 mm BisTris 10ꢀ12% acrylamide precast gels (Invitrogen) were used for the PAGE analysis.
The SeeBlue Plus2® Prestained Standard (Invitrogen) was used as a molecular weight marker.
Protein concentrations were determined by the BCA assay using the Micro BCA® Protein Assay
Kit (Thermo Scientific), using Bovine Serum Albumin as standard.
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Metal analysis: For metal analysis, concentrated enzyme (either VcrAꢀMBP or VcrA) was
desalted, eluted in distilled water, digested with 5 % HCl and filtered to remove the precipitated
protein. Nonꢀheme iron in the protein was quantified with Ferene (3ꢀ(2ꢀpyridyl)ꢀ5,6ꢀbis (5ꢀsulfoꢀ
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2ꢀfuryl)ꢀ1,2,4ꢀtriazine, disodium trihydrate)
. For the calibration curve, 2ꢀ20 ꢁM standards
were prepared freshly from a stock solution containing 0.2 mM (NH ) Fe(SO ) .6H O. The iron
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standards, the water reference and the proteinꢀcontaining solutions were each filled with H O to
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a final volume of 100 ꢁl. The solutions were mixed with 100 μL 1% HCl (v/v) and incubated at
80 ºC for 10 min. After cooling to room temperature 500 ꢁl ammonium acetate 7.5% (w/v), 100
ꢁ
l ascorbic acid 4% (w/v), 100 ꢁl of SDS 2.5% (w/v) and 100 ꢁl of Ferene 1.5% (w/v) were
sequentially added into each sample with vortex mixing. The Eppendorf tubes containing the
reaction mixtures were centrifuged at 13,000 × g for 10 min. The supernatant was used for
measuring the UVꢀVIS absorbance at 593 nm. The iron content of the protein solution was
calculated from the calibration curve of the absorbance.
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Cobalt content was analysed by ICPꢀMS
. Solutions of 5 ml were prepared for the cobalt
standards and samples of unknown concentration were prepared in clean unused plastic BD tubes
in 5% HCl. The calibration curve was from 0.1 to 5000 parts per billion. The samples were
analysed on a Thermo XSeries II ICPꢀMS, with a 60 s rinse of 3% nitric acid between samples
and an uptake time of 60 s for each sample. 3 readings were collected and the average was
reported. Internal standards of cobalt (2.5 ppm and 5 ppm) were used; scandium and yttrium
were used for corrections.
GC methods: Chloroꢀethenes and ethanes were quantified by gas chromatography using an
Agilent Technologies 6890 Network GC System instrument equipped with a flame ionisation
detector (FID) and an HP 624 Special Analysis column (capillary 30.0 m × 530 μm × 300 μm).
The injector was maintained at 200°C, and the FID was operated at 280°C. Helium was used as
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carrier gas (99.9% purity) at a pressure of 4.86 psi and a flow rate of 5.1 ml/min. The
temperature program was 8.6 min at 100° C with no ramping.
VcrA activity assays: All enzyme activity experiments were performed in triplicates in anoxic,
sealed crimped serum vials of 10 ml volume stoppered with butyl rubber stoppers, containing 6
ml of the aqueous phase and 4 ml of headspace. 0.1 ml headspace samples were injected into the
GC for analyzes. Standards of ethene were prepared by diluting a commercial 1.01% standard
(Supelco) into serum vials, assuming an ideal gas volume of 22.4 L per mole. For the chlorinated
compounds, commercial standards of 2 mg/ml dissolved in methanol (Supelco) were diluted into
the serum vials.
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Henry’s
constants
KH
at
20°C
from
the
US
EPA
website
(http://www.epa.gov/athens/learn2model/partꢀtwo/onsite/esthenry.html) were used to calculate
cc
the partition of 1,1ꢀDCE, VC and 1,2ꢀDCA between gas and aqueous phases. For ethene, a KH
30
of 7.6 was used . Calibration curves obtained by correlating peak area and the number of moles
were used for calculation of the number of moles observed in the enzymatic assays.
A typical assay mixture in an anoxic 10 ml serum vial contained 0.2ꢀ1 mg/ml of fully
reconstituted VcrA, 1 mM Ti (III) citrate and 0.4 mM methyl viologen as electron donor and
mediator, respectively, in 50 mM TrisꢀHCl pH 8.5. The reaction was started by injecting a
defined volume of aqueous 1,1ꢀDCE from a saturated aqueous solution of approximately 26 mM.
For vinyl chloride a methanolic solution of 32 mM was used; for 1,2ꢀDCA, an aqueous stock
solution of 100 mM was used. The incubation time was generally 60 min; the 1,1ꢀDCE
concentrations and the incubation times were varied during different experiments. For
cobalaminꢀonly measurements, the same mixture was used; enzyme was omitted and 2 mg/ml
cobalamin was added. After allowing the reaction to proceed for a specified time, the reaction
was stopped by the addition of 0.1 ml of concentrated HCl.
Peptide Mass Fingerprinting (PMF): Mass mapping was performed at the PAN Center,
Stanford University using a 4700 Proteomics Analyzer that features tandem (MS/MS) timeꢀofꢀ
flight (TOF) optics to provide peptide structural information, in addition to high accuracy MS
data. The data showed 25ꢀ60 ppm accuracy in mass mapping experiments without the need for
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internal calibration. The laser repetition rate was 200 Hz. Sample preparation for PMF was
carried out as follows: 45 mM dithiothreitol (Sigma) in 10 mM ammonium bicarbonate (pH 7.8)
was added to polyacrylamide gel slices and then incubated for 30 min at 55°C. The solution was
changed to 100 mM acrylamide (BioꢀRad) in 10 mM ammonium bicarbonate and incubated 1 h
at room temperature. The ammonium bicarbonate solution was removed, and 0.5 ml of 10 mM
ammonium bicarbonate and 50% acetonitrile was added to the gel and incubated for 30 min. The
gel slice was then dried to completion. A small volume (2 to 10 ꢁl) of 10 mM ammonium
bicarbonate containing 4 to 20 pmol of trypsin (Promega) was added to the slice. The gel was
then saturated with 10 mM ammonium bicarbonate buffer. Buffer was added continuously over 2
h until the gel was swollen and covered with buffer. The gel was then incubated with trypsin
overnight at 37°C.
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Peptides were extracted onto Ziptips (Millipore) from an aliquot of the solution and then washed
with 0.1% trifluoroacetic acid (Applied Biosystems) and eluted directly to a matrixꢀassisted laser
desorption ionization (MALDI) plate with 0.5 ꢁl of 50% acetonitrile and 0.1% trifluoroacetic
acid. The eluate was partially dried, and then 0.5 ꢁl of alphaꢀcyanoꢀ4ꢀhydrozycinnamic acid
(Agilent) (5 mg/ml) was added. MALDIꢀmass spectrometry (MS) was performed using the
reflector mode to obtain monoꢀisotopic peptide masses and tandem MS. The results were then
used to search protein and genomic databases with the Mascot software. Further details are given
3
1
in Bechtel et al .
EPR spectroscopy: Samples for Xꢀband EPR spectroscopy were prepared in 3.8 mm thin walled
precision quartz EPR sample tubes (Wilmad Lab Glass) by freezing anaerobicallyꢀprepared,
buffered protein solutions with 20% (v/v) of 4 M sucrose as a glassing agent in liquid nitrogen
and capping the tubes immediately. The frozen samples were stored in liquid nitrogen.
All EPR spectra were measured at the CalEPR Center at the University of California, Davis.
Continuousꢀwave (CW) Xꢀband spectra were acquired with an Eꢀ500 spectrometer (Bruker,
Billerica, MA) under nonꢀsaturating slowꢀpassage conditions using a SuperꢀHigh Q resonator
(ER 4122SHQE). Cryogenic temperatures were achieved and maintained using an Oxford
Instruments ESR900 liquid helium cryostat in conjunction with an Oxford Instruments ITC503
temperature and gasꢀflow controller. Spectral simulations were performed with Matlab using the
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EasySpin 4.0 toolbox . Quantitation of the concentration of S = 1/2 spin systems in the vicinity
of g = 2 concentration within a sample was achieved by comparing the double integral of the
EPR intensity to that of a 308 ꢁM Cu(II) in 100 mM EDTA (EDTA = ethylenediaminetetraacetic
3
3
acid) at pH 7 spinꢀstandard . All data were corrected for differences in incident microwave
power, acquisition temperature, and cavity quality factor. All concentrations are given in ꢁM.
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Detection of Co(I) and Co(II) cobalamins by electronic absorption spectroscopy: Enzymeꢀ
bound Cob(I)alamin was detected in VcrAꢀMBP by the UVꢀVis absorption at 385 nm (388 nm
for VcrA) and 555 nm (550 nm for VcrA) and estimated by the measured extinction coefficients
ꢀ1
ꢀ1
ꢀ1
ꢀ1
of 22 mM cm at 385 or 388 nm and 9 mM cm at 550 or 555 nm. For the detection of the
ꢀ1
ꢀ1
enzymeꢀbound Cob(II)alamin, the absorbance at 475 nm was monitored (ε = 9.47 mM cm at
3
4
477 nm
). The spectra were measured on an Ultrospec 2100 Pro® UVꢀvisible
spectrophotometer (Amersham Biosciences) in cuvettes of 1 cm path length.
Synthesis and purification of aquahydroxocobinamide [(H O)OHCbi]: 10 mg of (CN) Cbi
2
2
or dicyanocobinamide (Sigma Aldrich) whose UVꢀvisible spectrum was identical to the reported
3
5,36
spectrum
, was dissolved in 3 ml anaerobic 0.1 M NaOH, and small aliquots of sodium
borohydride pellets were added every 5 minutes, until a molar excess of about 10ꢀfold of
borohydride over (CN) Cbi was reached. The reaction proceeded for about 1.5 h until the
2
mixture was tranferred into a beaker and quenched by the addition of acidified water. The final
pH was adjusted to 3 and the mixture was purified over a 1 g Sep Pak C reverse phase column
1
8
(Waters). The column was preꢀconditioned prior to sample loading by washing once with 100%
methanol and five times thereafter with 0.1% (v/v) TFA (trifluoroacetic acid). After the sample
had passed through the column, salts were removed by washing with 0.1% TFA for five column
volumes. This was followed by elution with 100% methanol, and the collected orange fractions
were evaporated overnight at room temperature. The collected product (6 mg) was identified as
(
H O)OHCbi by its UVꢀvisible absorption spectrum which was identical to the spectrum
2
3
5,36
reported in the literature
.
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RESULTS AND DISCUSSION
Overproduction, reconstitution, and specific activities: 3ꢀ5 mg of MBPꢀtagged VcrAꢀMBP
was produced from 3 g of cells (wet weight). VcrA, as isolated from Dehalococcoides mccartyi
strain VS, was shown previously to contain molar ratios of Fe and S consistent with a
stoichiometry of two [4Feꢀ4S] clusters as well as one corrinoid cofactor per enzyme; the
monomeric enzyme is membraneꢀassociated putatively via a small integral membrane protein
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1
0,16
(VcrB) facing the outside
(see Fig S1). Heterologous overproduction of VcrAꢀMBP from
plasmid pLICꢀHMK resulted in the formation of inclusion bodies, and all VcrAꢀMBP was found
in the insoluble fraction (data not shown). No significant soluble VcrAꢀMBP was observed upon
increasing the induction time beyond 3 h, or by varying the growth medium between several
rich (LB, Standard I, TB) or minimal media containing glucose, the growth temperatures from
16ꢀ37°C, the host E. coli strain (BL21, BL21 DE3, BL21pLys) or the inducer concentration
between 50ꢀ1000 µM (data not shown). Therefore, VcrAꢀMBP was purified from inclusion
bodies under denaturing conditions to apparent homogeneity using Hisꢀtag affinity
chromatography. Using a NiꢀIDA matrix column, nearly homogenous protein was obtained (Fig
1). Subsequently, the denatured protein was desalted in the presence of 20% glycerol and 0.2%
CHAPS followed by refolding and reconstitution of the cobalamin and ironꢀsulfur cluster
cofactors. Excess cofactors were removed, and metal analysis (via ICPꢀMS and
spectrophotometric estimation) was performed, indicating a 1ꢀ1.4 mol Co and 8ꢀ12 mol nonꢀ
heme iron ratio per mol purified VcrAꢀMBP. When reconstituted VcrA (see below) was
subjected to the same analysis, a Co content of 0.8ꢀ1.1 mol and a nonꢀheme iron content of 7ꢀ10
mol per mol of purified enzyme was found.
Heterologously produced and cofactorꢀreconstituted VcrAꢀMBP catalysed the reduction of vinyl
chloride, 1,1ꢀDCE, cis and trans 1,2ꢀDCE at high specific activities (Table 1). Vinyl chloride
ꢀ
1
ꢀ1
was reductively dehalogenated to ethene at a Vmax of 1050 nmol*min *[mg protein] and a K
M
of 10 µM, which is very similar to the specific activity of the purified, native VcrA (990
ꢀ1
ꢀ1
10
nmol*min *[mg protein] , for which a K was not reported
.
Also, 1,1ꢀDCE was
M
ꢀ1
ꢀ1
dehalogenated to vinyl chloride and ethene at a Vmax of about 295 nmol*min *[mg protein]
K = 50 µM), (Table 1, Fig 2a). 1,1ꢀDCE undergoes a twoꢀstep conversion into ethene via vinyl
(
M
chloride as intermediate and does not follow MichaelisꢀMenten behavior below 10 µM (data not
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ꢀ1
ꢀ1 10
shown). 1,1ꢀDCE in native VcrA proceeded at a Vmax of 390 nmol*min *[mg protein]
. When
testing as an abiotic control coenzyme B at equivalent molar concentration but without VcrA in
1
2
the assay, a slow rate of vinyl chloride reduction of 100 nmol/min was observed (data not
shown). For 1,1ꢀDCE dehalogenation, the maximum abiotic dechlorination rates were about 25
nmol/min, which is less than 10% of the enzymatic rate, at B concentrations comparable to
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those present in the enzyme.
Interestingly, VcrAꢀMBP was also found to mediate the dihaloelimination of 1,2ꢀDCA to ethene
(
Table 1; Fig 2b). This is a novel activity and was not previously observed. The maximum
ꢀ1
specific activity of complete 1,2ꢀDCA dehalogenation by VcrAꢀMBP was 450 nmol*min *[mg
ꢀ1
protein] (Table 1). However, the K of 1300 μM is 2 orders of magnitude higher than for vinyl
M
chloride or 1,1ꢀDCE, suggesting that 1,2ꢀDCA may not be a physiological substrate in situ for
VcrA. This extended substrate specificity of VcrA has implications for chlorinated solventꢀ
contaminated sites, which contain both chloroethanes and chloroethenes. Dehalococcoides VS
could be expected to consume 1,2ꢀDCA faster than vinyl chloride, 1,1ꢀ or the 1,2ꢀDCE isomers,
but only if the initial concentration of the former is much higher than those of the chloroethenes
and higher than those encountered in most natural environments. It is unclear whether 1,2ꢀDCA
dihaloelimination is also coupled to energy conservation in vivo. However, 1,2ꢀDCA is also far
more waterꢀsoluble (up to 100 mM) than the chloroethenes; so the actual dechlorination rates in
the environment may be different from what the MichaelisꢀMenten parameters predict.
Methyl viologen-independent dehalogenation: Consistent with previous in vitro
3
7,38
dehalogenation studies
, the presence of methyl viologen for reductive dehalogenation activity
of VcrAꢀMBP was not essential, although a methyl viologenꢀinduced acceleration of the reaction
rates was observed. Methyl viologenꢀindependent dehalogenation rates in this case were about
20ꢀ25% of the rates in the presence of methyl viologen (Table 1).
Interestingly, upon removal of the MBP tag via proteolytic cleavage, the purified and cofactorꢀ
reconstituted VcrA also showed dehalogenation activity regardless of whether methyl viologen
was absent or present. The methyl viologenꢀindependent rates were 45ꢀ55% of the rates observed
in the presence of methyl viologen (Table 1). The fact that methyl viologen accelerates the rates
of dechlorination both in VcrAꢀMBP and VcrA suggests that it could create a chemical short cut
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for transferring electrons directly of Co(II) to form the catalytically active Co(I) species, but the
actual rates further depend on steric factors based on the conformation of the enzyme.
EPR studies: Xꢀband CW EPR spectra of VcrAꢀMBP and VcrA were recorded under different
reducing conditions. In the presence of 2 mM 2ꢀmercaptoethanol, the observed EPR spectrum
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(
top trace) of VcrAꢀMBP was nearly axial with gꢀvalues = [2.29, 2.23, 2.004] (blue trace, Fig
3
A). The octet centered about g = 2.004 results from the splitting of the resonance due to the
||
5
9
hyperfine interaction of the unpaired electron with the I = 7/2 Co nucleus (100% natural
5
9
abundance); A ( Co) = 305 MHz. Each of these lines was further split into a triplet due to an
||
14
14
additonal hyperfine interaction with a single I = 1 N nucleus (A ( N) = 52 MHz). These EPR
||
spectral features are consistent with their assignment to a Cob(II)alamin species for which either
the pendant dimethylbenzimidazole (DMB) is bound axially to the cobalt centre, or this DMB
base has been replaced by some other nitrogenous ligand such as a histidine side chain from the
polypeptide.
The CW EPR spectra of VcrAꢀMBP reconstituted with either aquahydroxocobinamide or
dicyanocobinamide are essentially identical to one another, but markedly different from the
spectrum of the cobalaminꢀreconstituted enzyme (Fig S4). The gꢀvalues move slightly upfield
14
and the N ligand hyperfine is lost the when the enzyme is reconstituted with cobinamide
instead of cobalamin. This indicates that the dimethylbenzimidazole base of the cobalamin is
likely serving as a ligand to the VcrAꢀbound cofactor, not a proteinaceous histidine ligand as in
methionine synthase. Recent studies showed that dimethylbenzimidazole was essential for D.
3
9
mccartyi dehalogenation , unlike in Sulfurospirillumꢀtype microorganisms where the lower
ligand is an adenine derivative. Therefore, our use of a dimethylbenzimidazoleꢀcontaining
corrinoid is relevant.
Further treatment of VcrAꢀMBP with the strong reductant Ti(III)ꢀcitrate left the Cob(II)alamin
signal unchanged; however, a new intense signal appeared at g = [1.969, 1.928, 1.852] in the
lowꢀtemperature EPR spectrum (Fig 3A, green trace). This new signal relaxed too quickly to be
observed at temperatures above 60 K (Fig S5), which is consistent with the behaviour of reduced
[4Feꢀ4S] clusters. Integration of the individual EPR signals revealed that approximately 69% of
the total signal corresponded to reduced [4Feꢀ4S] centers and the 31% of the EPR spectrum
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corresponded to Cob(II)alamin, in line with the elemental analysis showing 1ꢀ1.4 Co mol and 8ꢀ
12 mol nonꢀheme iron per mol of enzyme (Table S1).
An additional signal appeared at g = [1.989, 1.928, 1.900] that varied in intensity with each
preparation (Fig 3B). This new signal was greatly diminished following an additional rapid
desalting step (via a 10 kDa cutꢀoff Centricon membrane filter) in the sample preparation (Fig
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S6) and looked similar to signals measured recently for Ti(III)ꢀcontaining compounds . Thus,
we concluded that the features at [1.989, 1.928, 1.900] arise from S = 1/2 Ti(III) species
generated during the reduction.
Interestingly, though Ti(III)citrate should be a sufficiently strong reductant to reduce
1
+
Cob(II)alamin to Co , it seems insufficient to do so for cobalamin bound to VcrAꢀMBP. Only
upon further addition of methyl viologen did the Cob(II)alamin EPR spectrum disappear. The
+
intensity of the [4Feꢀ4S] signal was unchanged (Fig 3B). This spectral change coincided with
the appearance of new features at ≈385 nm and ≈555 nm in the electronic absorption spectrum
that is indicative of Cob(I)alamin. Samples of Ti(III)ꢀcitrate and methyl viologen reduced VcrA
with 1,1ꢀDCE as a substrate or with 50 ꢁM iodoacetamide as an inhibitor did not change the
spectra substantially (data not shown). The EPR spectra of the Ti(III)ꢀreduced, cobalaminꢀ
reconstituted VcrA are similar to those of VcrAꢀMBP. Specifically, we observe signals from
Co(II)Cbl (not shown) as well as signals from two reduced [4Fe4S] clusters and adventitious
Ti(III) species (Fig S7). Both FeS cluster signals observed in samples of VcrA lacking the MBP
tag possess slightly different gꢀtensors than that determined for the FeS cluster in the samples of
VcrAꢀMBP. The origin of these differences is unclear at this time. However, subtle changes in gꢀ
values are expected if the MBP tag influences the structure of VcrA. Secondꢀcoordination sphere
interactions are wellꢀknown to modulate EPR properties of FeS clusters. Additionally, it is
possible that in these samples both clusters in VcrA (two [4Feꢀ4S] clusters being suggested by
iron content analysis) are reduced and in sufficiently close proximity to one another so as to
cause a shifting of some of the resonant field positions due to throughꢀspace spinꢀdipole
interactions. This hypothesis will be evaluated in the future using multifrequency EPR
techniques.
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The intensity of the Cob(II)alamin signal was reduced by approximately 70% upon oxidation by
the presence of 10 mM ferricyanide (Fig 4). Upon addition of up to 50ꢀ75 mM ferricyanide, the
Co(II) signal was completely abolished (data not shown) and no new signals appeared except for
a broad derivative feature, that was also present in spectra of samples containing only 1 mM
ferricyanide. Chemical oxidation in the absence of molecular oxygen yielded oxidized [4Feꢀ4S]
cluster(s) and oxidized cobalamin that are both EPR silent. No EPR evidence for oxidized
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[3Fe4S] clusters was found in the ferricyanideꢀtreated samples .
UV-visible spectrophotometry: Titration of VcrAꢀMBP with Ti(III)ꢀcitrate up to concentrations
of 1.6 mM reduced the intensities of the ironꢀsulfur signal (420 nm) and produced peaks at 385
and 475 nm (Fig S8, panel A). Estimation of the Cob(I)alamin vs. Cob(II)alamin using the
reported extinction coefficients (see methods section) showed only about 10% of the cobalamin
in the Co(I) state. However, if 0.4 mM methyl viologen was added to VcrAꢀMBP, then the
sequential titration with Ti(III)ꢀcitrate produced increasing absorption of three sharp peaks at 366
nm, 385 nm, characteristic of the Co(I) state (Fig S8, Panel B), and 395 nm. A broad peak also
appeared for reduced methyl viologen at 600 nm, and the intensity of the peak for Co(I) around
555 nm increased (Fig S8, panel B). The spectrum of free methyl viologen and Ti(III)ꢀcitrate
overlaps considerably with that of VcrAꢀMBP reduced with methyl viologen and Ti(III)ꢀcitrate
from 350ꢀ 450 nm, and is quite different from that of the unreduced enzyme (Fig S8, panel C).
Further, Cob(I)alamin also spontaneously reoxidizes to the Co(II) state. This makes the accurate
quantification of Cob(I)ꢀ vs Cob(II)alamins in the methyl viologenꢀreduced enzyme difficult.
However, VcrAꢀMBP undergoes a shift of a peak from 530 nm for Co(II) to 555 nm typical for
Co(I) upon reduction; we estimated about 70ꢀ90% of the cobalamin must be in the Co(I) state
ꢀ1
ꢀ1
based on the absorption at 555 nm (calculated extinction 9.0 mM cm ).
In contrast, VcrA (lacking MBP) exhibited quite different UVꢀvis spectroscopic features upon
reduction by Ti(III)ꢀcitrate (Fig S9, Panel A). An intense peak at 388 nm characteristic of
Cob(I)alamin and a smaller peak at 550 nm appeared 1 mM Ti(III) in the absence of methyl
viologen. Concomitant with the appearance of the Cob(I)alamin signal, the 475 nm
Cob(II)alamin maximum was replaced by another at 465 nm (which shows that cobalamin is still
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bound to VcrA). The spectra measured in this experiment are strikingly similar to those of the
4
2
corrinoid iron sulfur protein where Co(I)corrinoid species also play a key catalytic role.
Based on the extinction coefficients estimated in our study, we calculated that about 99% of the
enzymeꢀbound cobalamin was reduced to the Co(I) state at approximately 1.5 mM of Ti(III). The
methyl viologen mediated spectra of VcrA were similar to those of VcrAꢀMBP with methyl
viologen (Fig S9, Panel B). The disappearance of the Co(II)Cbl signal as assessed by EPR
spectroscopy is correlated to the appearance Co(I)Cbl features in the UVꢀvisible spectrum,
demonstrating the presence of Co(I)Cbl in the catalytically competent form of VcrA.
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CONCLUSIONS
We have established a heterologous platform to produce the VcrA enzyme, which can be
reconstituted in the fully active form. This is also the first biochemical report to unambiguously
demonstrate the existence of a bifunctional chloroethene and chloroethane reductive
dehalogenase. This observation has strong implications for the remediation of sites coꢀ
contaminated with chloroethenes and chloroethanes.
Interestingly, although the exact chemical identity of the corrinoid cofactor of native VcrA is
unknown, heterologously expressed VcrAꢀMBP reconstituted with hydroxocobalamin or
adenosylcobalamin has kinetic properties that are indistinguishable from that of native VcrA.
Since we did not use dark/red light conditions, the active species was likely to be
hydroxocobalamin. The use of either cobalamin derivative did not lead to any difference in the
specific activity (data not shown). Based on the findings reported here we propose that native
14
VcrA coordinates Cob(II)alamin with an N axial ligand, which arises most likely from the
8
dimethylbenzimidazole moiety of the cofactor. Earlier corrinoid crossꢀfeeding studies suggested
that dimethylbenzimidazole (DMB), the lower ligand of cobalamins was essential for the
reductive dechlorination of chloroethenes by defined microbial consortia containing D. mccartyi,
which is consistent with our findings. Baseꢀon cobalamins were reported to have Co(II)/Co(I)
reduction potentials of about ꢀ600 mV (SHE) while baseꢀoff cobalamins potentials are around ꢀ
43
500 mV . The exact potential of the Co(II)/Co(I) couple depends on the interaction of the
cobalamin with the polypeptide. The [4Feꢀ4S] clusters which are reducible by Ti(III) in vitro
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may be reduced in vivo possibly by ferrdoxin/flavodoxin. We can distinguish only one class of
EPR signals associated with the reduced [4Feꢀ4S] clusters. Thus, the two putative clusters are
either spectroscopically equivalent or we are only able to observe one of two classes under our
experimental conditions. Furthermore, these clusters do not necessarily have different reduction
potentials. A chemical model study of the dehalogenation of chloroethenes reported a reduction
potential in free solution of ꢀ450 mV for the vinyl radical with much more positive potentials for
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the chloroethyl radicals . Thus, a mechanism where a Cob(I)alamin species donates an electron
to the chlorinated compound, leading to radical dechlorination (Fig 5) may be envisaged. The
vinylꢀtype radical could be finally reduced by either a [4Feꢀ4S] cluster (as in Fig 5B) or a second
Co(I) (as in Fig 5A) and protonated to yield the product.
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18
The recently published crystal structures of PCE dehalogenase and of NpRdhA seem to lend
support to our mechanism. The mechanism is similar to the one proposed by Ragsdale and others
4
6
for the dehalogenation of 3ꢀchloroꢀ4ꢀhydroxy benzoic acid . While the proposed mechanism
serves as a basis for future experiments with recombinant VcrA, our studies reported here
provided novel insights into an environmentally important Dehalococcoidesꢀtype reductive
dehalogenase.
ACKNOWLEDGEMENTS: Funding for this work was provided by ENI and NSF to AMS and
by the National Institutes of Health to (GMꢀ104543) RDB. We thank Dick Winant (PMF at the
PAN Center, Stanford), Guangchao Li (ICPꢀMS at the School of Earth Sciences, Stanford), and
Koshlan MeyerꢀBlackwell and Maeva Fincker for helpful discussions. We thank Maeva Fincker
also for critically reading the manuscript and useful comments.
SUPPORTING INFORMATION AVAILABLE: The figures S1ꢀS9 and table S1 are available
free of charge via the Internet at http://pubs.acs.org.
FIGURES
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Fig 1. SDSꢀPAGE of VcrAꢀMBP purified by NiꢀTED and NiꢀIDA Hisꢀtag affinity
chromatography columns under denaturing conditions. The arrow indicates the band
corresponding to purified VcrAꢀMBP. M is the molecular mass marker. FT = flow through from
the column and E = elution with 250 mM imidazole. Sample 1 was processed with a NiꢀTED
column and sample 2 with a NiꢀIDA column.
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Fig 2. A). Reduction of 1,1ꢀDCE to vinyl chloride (VC, empty circles) and ethene (Et, filled
circles) by VcrAꢀMBP. The curve fitting suggests that the formation of both products follows
secondꢀorder kinetics. B) Kinetics of 1,2 DCA dihaloelimination to ethene by VcrAꢀMBP. The
ꢀ
MichaelisꢀMenten parameters were estimated as K = 1300 μM and Vmax = 450 nmol*min
M
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*
[mg protein] . The filled circles are the measured data points, while the dotted line represents
the simulation based on the MichaelisꢀMenten equation. The crossꢀmarks represent error bars.
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Fig 3 A. X band (9.367 GHz) EPR spectra of VcrAꢀMBP at 15 K under different reducing
conditions. (Top) 2ꢀmercaptoethanolꢀreduced VcrAꢀMBP (solid blue trace) with corresponding
simulation (dashed blue trace) along with 10ꢀfold magnification of features centered at g = 2.00
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to better illustrate the N hyperfine splitting. Ti(III)ꢀreduced VcrAꢀMBP (middle, green trace)
and Ti(III)ꢀreduced VcrAꢀMBP following addition of methylviologen (bottom, red trace) are
also shown. The gꢀvalues are indicated in the figure. Simulation parameters for the
Cob(II)alamin species are as follows: g = [2.29, 2.23, 2.004]; gꢀStrain = [0.05, 0.04, 0.002];
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A( Co) = [50, 40, 305] MHz; A( N) = [5, 5, 52] MHz). Spin quantification of the difference
spectrum from the green and blue traces showed that the FeS signals: cob(II)alamin ratio is
approximately 2:1 (77:34) (See Table S1).
B. Expansion of EPR spectrum of Ti(III)ꢀreduced VcrAꢀMBP (top, solid green trace) and
+
corresponding simulation of the [4Feꢀ4S] contribution (dotted green trace) compared to the
spectrum of Ti(III)ꢀreduced VcrAꢀMBP following addition of methylviologen (red trace).
+
Simulation parameters for the [4Feꢀ4S] species are as follows: g = [1.969, 1.928 1.851]; gꢀ
Strain = [0.017, 0.018, 0.028].
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Fig. 4 Chemical oxidation in the absence of oxygen of 2ꢀmercaptoethanol reduced VcrAꢀMBP:
The top trace shows the Xꢀband EPR spectrum VcrAꢀMBP reduced with 2ꢀmercaptoethanol,
whereas the bottom trace shows the same sample treated with 10 mM ferricyanide. The intensity
of the Cob(II)alamin signal is reduced by about 70%; upon the further addition of ferricyanide
(50ꢀ75 mM) the signal is completely abolished (data not shown). No [3Feꢀ4S] signals were
observed.
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Fig 5. Proposed reaction mechanism(s) for reductive dehalogenation by VcrA; Co(II) is reduced
to Co(I) by a low potential ferredoxin/flavodoxin physiologically or Ti(III) in vitro. Co(I)
ꢀ
donates an electron to the substrate eliminating Cl and generating a substrate radical; this radical
is further reduced by an electron originating either from a second Co(I) as in Scheme A or
directly by a [4Feꢀ4S] cluster as in Scheme B; protonation possibly simultaneous to the second
reduction, leads to the formation of the product. Vinyl chloride is shown as the substrate here,
but the same mechanism could dechlorinate 1,1ꢀDCE and other substrates.
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Table 1 Substrates tested in this study for transformation by VcrA and VcrAꢀMBP, and their
MichaelisꢀMenten parameters. VcrA = enzyme without MBP and His tags; VcrAꢀMBP =
enzyme with His and MBP tags intact. MV = methyl viologen.
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Substrate
VcrAꢀMBP (molar mass 100 kDa)
VcrA, no MBP (molar mass 57 kDa)
ꢀ
ꢀ1
ꢀ1
ꢀ1
Vmax
Kcat [s
Vmax
Kcat [s ]
KM
Vmax
Kcat [s ]
Vmax
Kcat [s ]
K [ꢁM]
M
ꢀ
1
ꢀ
ꢀ
[
nmol*min
]
[nmol*minꢀ
1*(mg
[ꢁM]
[nmol*min
[nmol*min
without
MV
with MV
without
MV
1
1
1
*(mg
*(mg
*(mg
with
MV
ꢀ1
ꢀ1
ꢀ1
protein) ]
with MV
protein)ꢀ1]
without MV
protein) ]
protein) ]
with MV
without
MV
1,1ꢀDCE
VC
295
1050
450
177
630
270
73
265
80
43.8
159
96
50
10
675
2250
722
578
1928
618
300
1006
395
257
862
338
45
13
1
,2ꢀDCA
1300
1300
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ACS Paragon Plus Environment