Anaerobic Microbial Transformation of Halogenated Aromatics and

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Anaerobic Microbial Transformation of Halogenated Aromatics and Fate Prediction Using Electron Density Modelling Myriel Cooper, Anke Wagner, Dominik Wondrousch, Frank Sonntag, Andrei Sonnabend, Martin Brehm, Gerrit Schüürmann, and Lorenz Adrian Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00303 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Environmental Science & Technology

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Anaerobic Microbial Transformation of Halogenated Aromatics

2

and Fate Prediction Using Electron Density Modelling

3

Myriel Cooper,† Anke Wagner,‡,ǂ Dominik Wondrousch,§,ǁ Frank Sonntag,‡

4

Andrei Sonnabend,‡ Martin Brehm,§ Gerrit Schüürmann,§,ǁ and Lorenz Adrian†,‡,*

5

†

Helmholtz-Zentrum für Umweltforschung – UFZ, Isotope Biogeochemistry, Permoserstr. 15,

6

04318 Leipzig, ‡Technische Universität Berlin, Applied Biochemistry, Gustav-Meyer-Allee 25,

7

13355 Berlin, §Helmholtz-Zentrum für Umweltforschung – UFZ, Ecological Chemistry,

8

Permoserstr. 15, 04318 Leipzig and ǁTechnische Universität Bergakademie Freiberg, Institute for

9

Organic Chemistry, Leipziger Strasse 29, 09596 Freiberg, Germany

10 11

Keywords: microbiology, biodegradation, computational modeling, enzyme mechanism,

12

chemical biology, Dehalococcoides, halogenated aromatics, reductive dehalogenation,

13

organohalide respiration, B12

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*Corresponding author: Helmholtz Centre for Environmental Research – UFZ, Isotope

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Biogeochemistry, Permoserstraße 15, 04318 Leipzig, Germany, Tel.: +49 - 341 235 1435,

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e-Mail: [email protected]

17

Running title: Fate of halogenated aromatics in anaerobic environments

1

ACS Paragon Plus Environment


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ABSTRACT

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Halogenated homo- and heterocyclic aromatics including disinfectants, pesticides and

20

pharmaceuticals raise concern as persistent and toxic contaminants with often unknown fate.

21

Remediation strategies and natural attenuation in anaerobic environments often build on

22

microbial reductive dehalogenation. Here we describe the transformation of halogenated anilines,

23

benzonitriles, phenols, methoxylated or hydroxylated benzoic acids, pyridines, thiophenes, furoic

24

acids and benzenes by Dehalococcoides mccartyi strain CBDB1 and environmental fate

25

modelling of the dehalogenation pathways. The compounds were chosen based on structural

26

considerations to investigate the influence of functional groups present in a multitude of

27

commercially used halogenated aromatics. Experimentally obtained growth yields were 0.1-5 x

28

1014 cells mol-1 of halogen released (corresponding to 0.3 to 15.3 g protein mol-1 halogen), and

29

specific enzyme activities ranged from 4.5 to 87.4 nkat mg-1 protein. Chlorinated electron-poor

30

pyridines were not dechlorinated in contrast to electron-rich thiophenes. Three different partial

31

charge models demonstrated that the regioselective removal of halogens is governed by the least

32

negative partial charge of the halogen. Microbial reaction pathways combined with

33

computational chemistry and pertinent literature findings on CoI chemistry suggest that halide

34

expulsion during reductive dehalogenation is initiated through single electron transfer from

35

B12CoI to the apical halogen site.

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2


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INTRODUCTION

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Halogenated aromatic hydrocarbons are widely used e.g. as solvents, biocides, flame retardants

39

or additives to polymers. Commercialized halogenated aromatics often contain non-halogen

40

substituents or heteroatoms in the cycle determining the physicochemical and biological

41

properties in aerobic and anaerobic environments. Halogenated heterocycles can be extremely

42

persistent and toxic as seen with the polyhalogenated dibenzodioxins and dibenzofuranes.

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A number of strictly anaerobic bacteria have been isolated using organohalogen compounds as

44

electron acceptor in an anaerobic respiration including members of the genus Dehalococcoides.1-

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3

46

Dehalococcoides mccartyi strain CBDB1 transforms a wide variety of halogenated aromatic

47

compounds including benzenes, phenols, biphenyls and dioxins1,4-6 and uses the electron transfer

48

for energy conservation.1

Whereas some Dehalococcoides strains are specialized on chlorinated ethenes,2,3

49

Reductive dehalogenation of halogenated aromatic compounds by strain CBDB1 is not random

50

but follows distinct rules, which allow to some extent the prediction of dehalogenation pathways.

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Halogen substituents on aromatic compounds have previously been classified as doubly flanked,

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singly flanked or isolated when they have two, one or no neighboring halogen substituent,

53

respectively.7 Strain CBDB1 reductively dehalogenated doubly flanked substituents of

54

chlorinated benzenes and polychlorinated biphenyls (PCBs) and with a lower rate also singly

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flanked substituents, whereas isolated chlorine substituents were not removed.4,8 It was also

56

shown that strain CBDB1 removes doubly, singly and unflanked bromine substituents from

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bromobenzenes.9

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The hydroxyl group in chlorophenols enhanced dechlorination of a neighboring chlorine

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substituent similar to a chlorine substituent.5 Also halogen substituents singly flanked by one 3



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hydroxyl group were dechlorinated as shown for 2,4,5-trichlorophenol which was dechlorinated

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to 4,5-dichlorophenol. However, the effect of a hydroxyl-group was weaker than the effect of a

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chlorine substituent when it was in meta- or para-position.

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An enhancing effect on dechlorination was observed for the oxygen atom in the heteroaromatic

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ring of dioxins. 1,2,3-tri- and 1,2,3,4-tetrachlorodibenzo-p-dioxin were both dehalogenated to

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2,3-dichlorodibenzo-p-dioxin.6,10 Similarly 1,2,3,7,8-pentachlorodibenzo-p-dioxin was converted

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preferentially to the most toxic congener 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reaction that will

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also occur in nature from highly chlorinated dioxins. The peri-substituent in position 1 was

68

preferred over the doubly flanked substituent in position 2.

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In a previous study, chemical properties of chlorobenzenes, dioxins and chlorophenols were

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modelled using density functional theory calculations at the B3LYP/6-31G(d) level.11 Mulliken

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partial charges were identified as a tool to describe the dehalogenation pathways of these three

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classes of chlorinated persistent organic pollutants catalyzed by strain CBDB1. Lu and

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colleagues correlated more positive partial charges of chlorine substituents with dehalogenation

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pathways of strain CBDB1 previously described in the literature. However, the Mulliken model

75

does not account for differences in electronegativity of atoms within one molecule.12,13 In

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addition, due to the restricted number of available experimental data this model excludes most of

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the commercially used halogenated aromatics and molecules with two or more different halogen

78

substituents.

79

Reductive dehalogenases are the key catalysts for reductive dehalogenation.

In

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Dehalococcoides strains reductive dehalogenases are not specific for only one substrate or even

81

one mechanism as has been shown e.g. for the TceA enzyme of strain 195,14 the BvcA enzyme

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of strain BAV115 and the VcrA enzyme of strain VS16. In strain CBDB1 very broad enzyme 4


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specificity has been shown towards many different halogenated aromatics by the CbrA

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enzyme.9,17,18 Reductive dehalogenases are mostly encoded by operons with two genes: the

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catalytic subunit (rdhA) and a small membrane protein proposed to function as a membrane

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anchor (rdhB).19 Generally, the catalytic subunits contain two binding motifs for iron-sulfur

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clusters and in some cases binding motifs for corrinoid cofactors.20 Although corrinoid binding

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motifs are rare in rdhA genes of Dehalococcoides strains light-reversible inhibition by alkyl-

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iodids indicated the involvement of a corrinoid cofactor in most dehalogenases studied up to

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now.18,21 All yet-purified reductive dehalogenases contained a corrinoid cofactor, with the

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exception of the 3-chlorobenzoate reductive dehalogenase of Desulfomonile tiedjei.22 On the

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basis of the fact that superreduced corrinoids are strong nucleophiles, several reaction

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mechanisms have been proposed including nucleophilic substitution19 and a mechanism with

94

radical intermediates.23-25 Both mechanisms focus on the addition of electrons to the partially

95

positively charged carbon atom from which the halogen substituent is removed. Very recently,

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the 3D-structures of a tetrachloroethene reductive dehalogenase from Sulfurospirillum

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multivorans26 and a soluble, oxygen-tolerant reductive dehalogenase from Nitratireductor

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pacificus pht-3B27 specialized on ortho–halogenated phenolic substrates were resolved providing

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a structural basis to understand organohalide respiration. For the oxygen-tolerant reductive

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dehalogenase a cobalt-halogen interaction during catalysis was suggested.27

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In this study, D. mccartyi strain CBDB1 was cultivated with a broad range of electron

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acceptors containing different heteroatoms (-O; -S; -N), halogen substituents (-Br; -Cl; -F) or

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non-halogen substituents (-NH2; -CN; -OH; -OCH3) to study their influence on reductive

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dehalogenation. Whole-cell activity assays were conducted to determine specific activities. Three

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partial charge models were analysed for their ability to predict dehalogenation pathways. Strain 5



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CBDB1 dehalogenates halogenated anilines, benzonitriles, pyridines, thiophenes, benzoic and

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furoic acids to a different extent. Functional groups enhance or inhibit reductive dehalogenation.

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Comparative analysis of the results together with corresponding data from previous

109

investigations on reductive dehalogenation1,4-6,8 show that halogen net atomic charges, calculated

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by quantum chemistry, are good indicators for the regioselectivity of the observed

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dehalogenation pathways. Associated mechanistic reasoning strongly suggests a primary attack

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of B12CoI at the rear (apical) electron-poor site of the aromatically bound halogen followed by an

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inner-sphere electron-transfer reaction, which contrasts with previous mechanistic reaction

114

models and suggests that the recently proposed halogen-cobalt interaction in an oxygen-tolerant

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cytoplasmic dehalogenase27 is also formed in membrane-bound respiratory reductive

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dehalogenases of strictly anaerobic bacteria. The results promote our ability to predict the

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regioselective dehalogenation of a wide range of halogenated aromatics which is important for

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the evaluation of environmental persistence and toxicity.

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EXPERIMENTAL SECTION

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Chemicals. Chlorinated and brominated aromatic compounds were purchased from Sigma-

121

Aldrich (Seelze, Germany) at a purity of 99%. Titanium(III) citrate was prepared as previously

122

described.5

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Cultivation. Strain CBDB1 was grown in titanium(III) citrate-reduced, carbonate-buffered,

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vitamin amended synthetic medium with hydrogen as electron donor and 5 mM acetate as carbon

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source, as previously described.1 After inoculation, the headspace was pressurized with 20% CO2

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/ 80% N2 (1.5 bar), and hydrogen was added to a pressure of 1.8 bar. Cultivation was performed

127

in the dark at 30 °C without shaking. The electron acceptors were added from either 1 M stock 6


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solutions in acetone at a final concentration of 50 or 100 µM or as crystals (Supporting

129

Information, Table S1). All cultures were initially set up from cultures grown on 30 µM 1,2,3-

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trichlorobenzene. A starting cell density of about 1 x 106 cell ml-1 was chosen. Cell numbers

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were quantified after staining with SYBR-green by direct cell counting on agarose-coated slides

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as previously described.5,28 All cultivations were done in duplicates or triplicates. Additionally,

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chemical controls without inoculum, negative growth controls without electron acceptor and

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positive controls with 30 µM 1,2,3-trichlorobenzene were set-up. Results presented are from

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cultures of the first or second transfer with the respective electron acceptor. First transfers are

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shown when one of the electron acceptors in a compound class did not support growth so that no

137

second transfer was available.

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Analytical Techniques. 1,2,3-trichlorobenzene, chlorinated anilines, benzonitriles, thiophenes

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and pyridines were extracted from 0.5 ml samples with 1 ml of hexane and analyzed using a

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Shimadzu gas chromatograph (GC) 14A equipped with flame ionization detector on a

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Permabond-FFAP capillary column (25 m, 0.25 mm inner diameter, 0.25 µm film thickness,

142

Macherey & Nagel, Düren, Germany). Nitrogen (99.999% purity) served as carrier and make-up

143

gas. The following temperature program was used: initial hold at 55 °C for 1 min; increase at a

144

rate of 6 °C min-1 to 225 °C, with a final hold for 2 min. Injector and detector temperatures were

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250 and 300 °C, respectively.

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Bromophenols from 1 ml culture volume were derivatized with 120 mM sodium bicarbonate

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and 50 mM acetic anhydride for 10 min on a rotary shaker set to 350 rpm at room temperature

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before extracting the sample with 1 ml hexane. Samples were analyzed using a Hewlett Packard

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GC 6890 equipped with flame ionization detector on a HP-5 capillary column (30 m, 0.25 mm

150

inner diameter, 0.25 µm film thickness, Agilent Technologies, Böblingen, Germany). The 7



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temperature program started with an initial hold for 1 min at 55 °C and continued by an increase

152

of 10 °C min-1 to 150 °C, then 10 °C min-1 to 230 °C, and 30 °C min-1 to 260 °C with a final hold

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of 2 min. Injector and detector temperatures were 250 °C.

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Thiophene was analyzed from the headspace of the culture. Two hundred µl of the headspace

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were removed at each time point of analysis and transferred directly into the injector of a

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HP5890II GC equipped with a DB-5 column (30 m, 0.32 mm inner diameter, 0.25 µm film

157

thickness, J&W Scientific, Köln, Germany). Nitrogen (99.999% purity) served as carrier and

158

make-up gas. Injection was done at a column temperature of 35 °C. After 5 min, the column

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temperature was increased to 60 °C at a rate of 5 °C min-1, then with 30 °C min-1 to 300 °C and a

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final hold of 10 min was included. Injector and detector temperatures were 240 and 320 °C,

161

respectively.

162 163

The chlorinated compounds were identified and quantified by injecting single compounds as standards with concentration between 5 and 100 µM.

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Determination of Bromide Concentrations. Bromide was quantified from 1 ml culture

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samples using a DIONEX – ISC2000 Ion Chromatography System equipped with an

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IonPacAS18/AG18 column (4 mm inner diameter, Thermo Fisher Scientific, Bremen, Germany).

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Resting Cell Activity Assay for Reductive Dehalogenases. Dehalogenase activity was

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analyzed based on a microtiter plate-format assay described previously,9 using whole cells as

169

catalyst and reduced methyl viologen as an artificial electron donor. Used halogenated

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compounds are listed in Table S1 (Supporting Information). Final concentrations of 625 µM

171

were added for each tested electron acceptor. Two hundred µl of assay solution were filled in

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each well and the reaction was started by adding 20 or 40 µl of cell suspension. Cells were 2-fold

173

concentrated using a rotary evaporator before being added to the activity test. The microtiter 8


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plate was monitored with a microtiter plate reader within an anaerobic tent at 578 nm every 7.5

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minutes for 15 h at 30 °C. Each plate contained the samples with electron acceptor, negative

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controls without electron acceptor and a positive control with cells and 1,2,3-trichlorobenzene as

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electron acceptor, each in five parallels in addition to three parallel chemical controls with

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growth medium instead of the cells. The growth medium contained 50 µg L-1 vitamin B12. The

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specific activity was calculated from the slope of the linear part of the measured curves after

180

correction using the negative controls. Protein amounts were calculated from cell numbers8,9

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using a value of 30 fg protein per cell.

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Calculation of Partial Charges. Different approaches have been described to compute atomic

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partial charges based on the electronic structure of a molecule. As atomic charges are no

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quantum chemical observables, there is no "canonical" way of obtaining them and all approaches

185

have advantages and disadvantages. In this work, we used atomic charges based on the NPA

186

(natural population analysis) method, which itself is based on the NBO (natural bond orbital)

187

algorithm.29 In short, NBO constructs a unitary transformation of the canonical molecular

188

orbitals (which are the result of the calculation of the electronic structure) into a set of spatially

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confined orbitals resembling the concept of chemical bonding. The NBOs are assigned to atoms

190

and their occupation per atom is integrated, similar to the computation of Mulliken charges.13

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But in contrast to the latter, the NPA method is able to correctly handle a larger variety of bond

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types including those with a high degree of ionicity and features a higher numerical stability as

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well as a much lower dependence on basis set size.30 Mulliken and Hirshfeld partial charges were

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calculated additionally to test whether different charge models reproduce similar trends. The

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Mulliken charge of an atom is obtained by summing up the populations for all basis functions

196

centered on the atom and subtracting them from the nuclear charge. Overlapping populations 9



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between two atoms are divided half in half therefore not taking into account the differences of

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electronegativity between two atoms. While the NBO and the Mulliken model are based on the

199

analysis of wave functions the Hirshfeld model is a density-based population model. It makes

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use of a promolecule which is the sum of the ground-state atomic densities of the atoms of the

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real molecule. The electron density of the real molecule is partitioned onto the atoms of the

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promolecule corresponding to the ratio of the electron densities of the atoms of the promolecule.

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By subtracting the integrated density of an atom of the real molecule from the nuclear charge of

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the free atom of the promolecule the Hirshfeld atomic charge is obtained.

205

The geometries of 58 molecules were optimized using Density Functional Theory level

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B3LYP/6-31G(d,p) in order to obtain a minimum energy structure for each molecule. Correct

207

ground-state geometries for all compounds were confirmed by frequency analysis. All tasks

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including Mulliken,13 Hirshfeld31 (keyword IOP(6/79=1)) and NBO32 population analysis were

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performed using Gaussian 09 revision C.01.33

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To obtain NPA charges with solvation effect included, the CPCM solvation model34,35 was

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applied. Gaussian 09 CPCM calculations of the molecules were carried out with the two solvents

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chlorobenzene and water. Chlorobenzene was used to model the solvation within an enzyme

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pocket, where the effective permittivity is much lower than in water.36 The atomic radii required

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for CPCM were obtained by the UAKS method, as suggested in the literature.37 Additional non-

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electrostatic terms of the model were explicitly switched on ("dis", "rep", "cav"). Geometry

216

optimizations with CPCM were carried out for all molecules to include the solvent effect on

217

geometry.

10


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RESULTS

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Dehalogenation of homo- and heterocyclic aromatic compounds. Strain CBDB1 was

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cultivated with halogenated homo- and heterocyclic aromatic compounds as electron acceptor

221

and hydrogen as electron donor. Reductive dehalogenation was observed for 2,3-dichloroaniline,

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2,3-, 2,5- and 2,6-dichlorobenzonitrile, 2-chloro-6-fluorobenzonitrile, 2,4,6-, 2,4- and 2,6-

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bromophenol, 4-bromo-3,5-dimethoxy- and 4-bromo-3,5-dihydroxybenzoic acid, 2,3- and 2,5-

224

dichlorothiophene, 4,5- and 5-bromo-2-furoic acid, and 3-bromo-2-chloropyridine (Table 1,

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Figure 1 and 2). 2,4- and 2,6-dichloroaniline, and 2,3- and 2,6-dichloropyridine were not

226

dehalogenated within a cultivation period of 140 days.

227

First doses (10 µM to 100 µM or added as crystals, Supporting Information, Table S1) of 2,3-

228

dichloroaniline, benzonitriles, halogenated phenols, thiophenes and 3-bromo-2-chloropyridine

229

were dehalogenated within 70 days of cultivation. Additional doses of 50 µM 2,3-

230

dichloroaniline, 40 - 60 µM 2,4-di-, 2,6-di- or 2,4,6-tribromophenol, 2,3- or 2,5-

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dichlorothiophene and 25 - 30 µM 3-bromo-2-chloropyridine were also dehalogenated (Figure 1

232

and 2). Dehalogenation was detected after 7 days with bromophenols, after 18 days with 3-

233

bromo-2-chloropyridine and after a lag phase of 10 to 30 days with 2,6-dichlorobenzonitrile or

234

2,3- and 2,5-dichlorobenzonitrile (data not shown). In cultures fed with 4,5-dibromo-2-furoic

235

acid 400 µM bromide was released within 126 days (Figure 2). Dehalogenation of 800 µM 5-

236

bromo-2-furoic

237

dihydroxybenzoic acid was observed within 63 days of cultivation (Figure 2).

acid,

340

µM

4-bromo-3,5-dimethoxy-

and

410

µM

4-bromo-3,5-

238

All tested halogenated compounds were stable in chemical controls without strain CBDB1

239

except for 2,4,6-tribromophenol, 4,5-dibromo-2-furoic acid and 3-bromo-2-chloropyridine.

240

These chemical controls contained growth medium instead of culture and therefore also 11



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contained vitamin B12 to account for corrinoid-mediated abiotic catalysis. In chemical controls

242

with 45 µM 2,4,6-tribromophenol, 21.6 µM reacted abiotically within 70 days of incubation to

243

2,4-dibromophenol whereas in parallel cultures with CBDB1 in the same time approximately 50

244

µM of 2,4,6-tribromophenol were transformed to less brominated phenols and phenol. After 126

245

days of incubation, the chemical control with 4,5-dibromo-2-furoic acid contained about 50% of

246

the bromide concentration found in cultures with strain CBDB1. 15 µM of 2-chloropyridine were

247

released in chemical controls of 3-bromo-2-chloropyridine within 49 days, compared to 25.5 µM

248

in cultures inoculated with strain CBDB1 (Figure 2).

249

Dehalogenation Pathways Catalyzed by Strain CBDB1. Dehalogenation pathways were

250

determined to identify which halogen substituent was removed preferentially from a given

251

compound (Table 1). The chlorine substituent in position 2 of 2,3-dichloroaniline was

252

reductively dechlorinated by strain CBDB1 and 3-chloroaniline was formed. The cyano-group of

253

benzonitriles allowed the dechlorination of all ortho-chlorines. Substituents at other positions

254

and also the fluorine substituent in the ortho-position of 2-chloro-6-fluorobenzonitrile were not

255

removed. Strain CBDB1 fully dehalogenated all tested bromophenol congeners to phenol.

256

Intermediates from 2,4,6-tribromophenol were 2,4-dibromo- and 4-bromophenol demonstrating

257

that the ortho-position was the preferred dehalogenation site. 2,4- and 2,6-dibromophenol were

258

debrominated to phenol via 4- and 2-bromophenol, respectively. 4-Bromo-3,5-dimethoxybenzoic

259

acid and 4-bromo-3,5-dihydroxybenzoic acid were both debrominated to the non-halogenated

260

3,5-dimethoxy- and 3,5-dihydroxybenzoic acid.

261

In thiophenes, the position next to the sulfur heteroatom was the preferred dehalogenation site

262

(Figure 1c). 2,3-dichlorothiophene was only dehalogenated to 3-chlorothiophene. 2,5-

263

Dichlorothiophene, having both chlorine substituents flanked by the sulfur heteroatom, was fully 12


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dehalogenated to thiophene. 4,5-Dibromo- and 5-bromo-2-furoic acid were both biotically

265

debrominated by strain CBDB1. The nitrogen heteroatom in pyridines did not support

266

dechlorination in ortho- or meta-position. In contrast, removal of bromine substituents in

267

pyridines was possible as shown with the transformation of 3-bromo-2-chloropyridine to 2-

268

chloropyridine.

269

Growth of Strain CBDB1. To investigate if the different halogenated compounds were used

270

as an electron acceptor in organohalide respiration, strain CBDB1 was cultivated for at least two

271

transfers on compounds that were dehalogenated and cultures were monitored for growth and

272

dehalogenation. Transformation of 2,3-dichloroaniline, chlorinated benzonitriles, brominated

273

phenols, brominated dimethoxy- and dihydroxybenzoic acids, chlorinated thiophenes,

274

brominated furoic acids, and 3-bromo-2-chloropyridine was accompanied by an increase in cell

275

number of strain CBDB1. Initial cell numbers of 5x105 to 6x106 ml-1 and final cell numbers of

276

4x106 to 8x107 ml-1 were observed. An increase in cell numbers was not observed if no electron

277

acceptor was added. From the measured dehalogenation and growth data, molar growth yields of

278

0.1x1014 to 5x1014 cells per mol of halogen ion released were calculated (Table 1).

279

Resting Cell Activity Assay for Reductive Dehalogenases. In activity assays cell

280

suspensions of

strain CBDB1 cultivated with 1,2,3-trichlorobenzene dehalogenated 2,3-

281

dichlorobenzonitrile, all tested brominated phenols, 4-bromo-3,5-dimethoxybenzoic acid, 2,3-

282

dichlorothiophene and 2,5-dichlorothiophene (Table 1). The highest specific activities were

283

found for 2,4,6-tribromophenol (87.4 nkat mg-1), 2,3-dichlorobenzonitrile (50.6 nkat mg-1) and

284

2,3-dichlorothiophene (42.1 nkat mg-1). The lowest activity was determined for 2,5-

285

dichlorothiophene (4.5 nkat mg-1).

13



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Although dehalogenation of 2,3-dichloroaniline, 2,5-, 2,6-dichlorobenzonitrile, 2-chloro-6-

287

fluorobenzonitrile, 4-bromo-3,5-dihydroxybenzoic acid and 5-bromo-2-furoic acid was observed

288

during cultivation of strain CBDB1, no activity was measured in the enzyme activity assays.

289

These results were obtained with cultures grown with 1,2,3-trichlorobenzene as the electron

290

acceptor but also with cultures grown on the same electron acceptor that was later tested in the

291

activity test. All compounds except for 4,5-dibromo-2-furoic acid were stable in activity assays

292

as confirmed by chemical controls without cells.

293

Net Atomic Halogen Charges Predict Regioselectivity of Reductive Dehalogenation.

294

Partial charges were calculated for each atom in the tested halogenated anilines, benzonitriles,

295

benzoic acids, thiophenes, furoic acids and pyridines on the basis of the NBO (ܳ ே஻ை ), Hirshfeld

296

(ܳ ௛௜௥௦௛ ) and Mulliken (ܳ ௠௨௟௟ ) models, separately (Table 2, Supporting Information, Tables S2

297

and S3). Additionally, electron densities were calculated for chlorobenzenes, chlorophenols and

298

selected bromobenzenes (Supporting Information, Tables S4-S7) and compared with previously

299

determined experimental data5,9,18 to analyze correlations between the occurrence of a

300

dehalogenation reaction and electron densities from a broader data basis. A total of 58 molecules

301

were analyzed.

302

All three partial charge models revealed for most of the analyzed compounds that

303

dehalogenation preferentially took place in the position having the most positive charge on the

304

halogen atom (ܳ௑ ) and a more negative partial charge on the carbon atom (ܳ஼ ). On the basis of

305

the most positive ܳ௑ the Hirshfeld system predicted 96% of dehalogenation pathways correctly,

306

the Mulliken and NBO models 88%. Analyzing the most negative ܳ஼ the Hirshfeld, Mulliken

307

and NBO models allowed for a correct prediction of dehalogenation pathways for 80%, 68% and

14


Page 15 of 37


308

76%, respectively. When calculating with phenolates, prediction rates on the basis of the most

309

positive ܳ௑ using the Mulliken and NBO models were improved to 96% (data not shown).

310

While all models calculate the most positive partial charge at the halogen that was

311

preferentially dehalogenated, the NBO model reflected higher electronegativity of chlorine

312

compared to bromine and therefore predicted correctly the preferential removal of bromine from

313

2-chloro-3-bromopyridine.

314

Obtained charges on the halogen atom using the NBO model (ܳ௑ே஻ை ) ranged from -0.312 for

315

the fluorine atom of 2-chloro-6-fluorobenzonitrile to +0.161 for the bromine atom in the ortho-

316

position of 4,5-dibromofuroic acid. Halogen substituents that were dehalogenated showed ܳ௑ே஻ை

317

values between -0.094 (Cl2 of 2,5-dichlorophenolate) and +0.139 (5-bromofuroic acid).

318

The most negative ܳ஼ே஻ை value was calculated for the C2 of 2,3-dichlorothiophene (-0.304) and

319

the most positive values for the C6 atom of 2-chloro-6-fluorobenzonitrile (+0.484) and the C2/6

320

atoms of 2,6-dichloropyridine (+0.198). ܳ஼ே஻ை values of carbon atoms associated with

321

substituents that were dehalogenated were between -0.304 for the C2 carbon atom of 2,3-

322

dichlorothiophene and -0.009 for the chlorine substituted carbons of 2,6-dichlorobenzonitrile.

323

Calculated specific enzyme activities (Table 1) were compared to ܳ௑ே஻ை values of the

324

respective compound. When comparing within one compound class, higher (i.e. more positive)

325

ܳ௑ே஻ை values were paralleled with higher enzyme activity.

326

The NBO model permitted the calculation of σ and π electron distributions. For halogenated

327

anilines, benzonitriles, benzoic acids, thiophenes, furoic acids and pyridines experimentally

328

tested in this study and chlorobenzenes, chlorophenols and selected bromobenzenes from

329

previous studies σ and π electron distributions were modelled based on NBO partial charges

330

reflecting inductive and mesomeric effects, respectively (Supporting Information, Table S8-S10). 15



Page 16 of 37

331

88% of the regioselective dehalogenation reactions catalyzed by strain CBDB1 were predicted

332

correctly when using the halogen atom with the most positive σ partial charge as predictor. In

333

contrast, only 64% of the reactions were correctly predicted when using the halogen with the

334

most positive π electron partial charge.

335

To estimate is the influence of solvation on electron densities within the halogenated electron

336

acceptors NBO charges for halogen and carbon atoms (ܳ௑ே஻ை and ܳ஼ே஻ை ) were calculated for the

337

molecules listed in Table 1 with the solvents water and chlorobenzenes (Supporting Information,

338

Table S11 and S12, respectively). The results demonstrate that solvation did not change the

339

relative values of partial changes within a given molecule to each other.

340

DISCUSSION

341

Dehalococcoides mccartyi strain CBDB1 transforms a much wider range of halogenated

342

aromatics than previously anticipated including aromatics substituted with cyanide, amino,

343

hydroxyl or methoxyl groups and heterocycles with oxygen, sulfur or nitrogen heteroatoms.

344

These functional groups are present in many commercially used halogenated pesticides,

345

pharmaceuticals and flame retardants. We here demonstrate that non-halogen substituents or

346

heteroatoms can promote or inhibit dehalogenation reactions by their influence on the electron

347

density distribution within the molecule. Most of the investigated molecules were used by strain

348

CBDB1 as an electron acceptor for energy conservation via organohalide respiration allowing

349

effective transformation in bioremediation. Growth yields between 0.9 x 1013 and 5.1 x 1014 cells

350

per mol of halogen released were similar to previous findings with other electron acceptors.5,9

351

Growth yields were generally lower for electron acceptors which reacted also abiotically or

352

which contained phenolic groups. For abiotically reacting molecules, dehalogenation might be 16


Page 17 of 37


353

partially uncoupled from growth. Compounds containing phenolic groups inhibit growth by

354

interfering with the proton membrane gradient.5,38

355

The systematic investigation of functional group influence onto reductive dehalogenation may

356

allow for an improved reactivity prediction of newly introduced halogenated aromatics. So far,

357

predictions of dehalogenation pathways were built exclusively on known dehalogenation

358

pathways which did not allow extrapolation onto unknown aryl halides. Therefore, we here

359

studied why and how halogen positions and additional substituents are relevant to reductive

360

dehalogenation.

361

Quantum chemical analysis of aromatic halides ArX across several compound classes using

362

three different partial charge models unravels that the positive partial charge required for

363

reduction of ArX is located on a halogen and not on a carbon atom. In addition the analysis

364

shows that the net halogen charge (ܳ௑ ) can serve as a good predictor of the regioselectivity

365

observed for the reductive dehalogenation by D. mccartyi strain CBDB1. For compounds of

366

which two or more reaction products were formed concurrently the ܳ௑ of simultaneously

367

removed halogens were relatively close to each other (Supporting Information, Table S4 and S6).

368

This is consistent with a previous study in which a correlation between Mulliken charges and

369

reductive dehalogenation was found for chlorinated benzenes, phenols and dioxins.11,39

370

When a molecule was substituted with only one type of halogen, all models correctly predicted

371

the dehalogenation pathway when using the most positive halogen net atomic charge. The

372

Mulliken and the Hirshfeld partial charge model however, were not suitable for the prediction of

373

dehalogenation pathways when a molecules contained both, bromine and chlorine substituents.

374

Also they did not give correct predictions when comparing compounds that only differed by the

375

type of the halogen substituents (e.g. brominated vs. chlorinated benzenes). Then, both charge 17



Page 18 of 37

376

models assigned more negative partial charges to bromine substituents compared to chlorine

377

substituents (Supporting Information, Table S2), thus not reflecting the higher electronegativity

378

of chlorine compared to bromine.

379

Although all partial charge models showed good prediction rates for dehalogenation pathways

380

when using the halogen atomic net charge, each model showed few inconsistencies with

381

experimentally identified dehalogenation pathways. This emphasizes the necessity for the

382

applications of several charge models in parallel for a verification of obtained values.

383

A threshold for reductive dehalogenation according to a specific partial charge value was not

384

identified when analyzing all compound classes together. Possibly, a threshold for reductive

385

dehalogenation can be identified when analyzing more congeners of only one compound class or

386

by including further parameters into the charge calculation. Similarly, partial charges showed no

387

correlation with specific activities when analyzing all compounds together. However, when

388

considering only one compound class less negative halogen atomic net charges were paralleled

389

with higher specific activity.

390

In general, ܳ௑ values were more positive for aromatics with a higher number of halogen

391

substituents, which reflects the observed preferential transformation of highly halogenated

392

compounds (Supporting Information, Tables S2, S4 and S6, for experimentally tested substrates,

393

halogenated benzenes and halogenated phenols, respectively). The values of the net atomic

394

carbon charge performs significantly inferior as a predictor for regioselectivity, but a trend

395

towards more negative ܳ஼ values indicating enhanced reductive dehalogenation was observed.

396

However, a rough negative correlation between carbon and halogen charges was expected due to

397

a polarization of the carbon halogen bond caused by their difference in electronegativity.

18


Page 19 of 37


398

According to our modelled ܳ௑ே஻ை values the cyano-group of benzonitrile induced a more

399

positive charge on the ortho halogen atom than the hydroxyl- and amino-group in 2,6-

400

dichlorophenol and 2,6-dichloroaniline (Table 2). This is in line with the experimental data

401

showing that dehalogenation was most extensive for 2,6-dichlorobenzonitrile and least extensive

402

for chlorinated anilines, when comparing chlorinated benzonitriles, benzenes, phenols and

403

anilines5,8,18. This roughly corresponds with Hammett constants placing the electron withdrawing

404

effect in the order -CN > -X > -OH> -NH2.40 Hence, substituents withdrawing electron density

405

from the halogen enhanced reductive dehalogenation. When density functional theory modelling

406

is not available, Hammett constants might serve as a rule of thumb to estimate the enhancing

407

effect of substituents onto reductive dehalogenation.

408

When investigating reductive dehalogenation of halogenated heteroaromatic compounds with

409

strain CBDB1, electron-rich heteroaromatic compounds such as chlorinated thiophenes were

410

dehalogenated, whereas electron-poor chlorinated pyridines were not dehalogenated. Brominated

411

furoic acids were dehalogenated to non-halogenated reaction products. Carbon atoms flanking

412

the sulfur or oxygen heteroatoms had more negative net carbon charges than carbon atoms

413

flanking the nitrogen heteroatom of chlorinated pyridines. Nitrogen-heteroatom flanking carbon

414

atoms showed one of the most positive partial charges of all analyzed compounds (Table 2).

415

For different halogen substituents an enhanced removal in the order -Br > -Cl > -F was

416

observed, i.e. fluorine was not removed at all, and bromine substituents were removed to a

417

further extent than chlorine substituents, when comparing for instance 2,3-dichloropyridine with

418

2-chloro-3-bromopyridine, 2,6-dichlorobenzonitrile with 2-chloro-6-fluorobenzonitrile, or other

419

brominated with chlorinated molecules tested in the current or in previous studies.9,21 These

420

findings are reflected in the NBO atomic net charges of halogens, which have a more positive 19



Page 20 of 37

421

partial charge for bromine compared to chlorine and for chlorine compared to fluorine in

422

comparable molecules. Also they are in line with reduction potentials reported for

423

monohalogenated benzenes yielding the order Br > Cl > F with Br having the least negative

424

reduction potential.41

425

Previous models suggested a SNAr reaction as mechanistic model for reductive

426

dehalogenation. An enhanced removal of halogens in the order -Br > -Cl > -F, is however in

427

contrast with the decrease of CAr-X bond dissociation energy in the order F > Cl > Br > I,42

428

reflecting the trend of the halogen facility as a leaving group as opposed to the halogen polarity

429

(inductive) effect that governs the readiness for SNAr reactions (Figure 3a). The increase in SNAr

430

reactivity with increasing electron deficiency at the aromatic ipso carbon as site of nucleophilic

431

attack is also not compatible with our present finding that the halogen and not the carbon with

432

the most positive net atomic charge predicts the regioselective dehalogenation pathway.

433

Similarly, observations made for heteroaromatic compounds contradict previous reaction models

434

based on a SNAr mechanism which would predict enhanced removal for halogens substituted to

435

carbons with a more positive partial charge as it was observed for in pyridines.

436

A more realistic option for the underlying reaction mechanisms is a dissociative single electron

437

transfer process (SET)43-45 (Figure 3b) in which initial electron transfer from B12CoI to the halide

438

could proceed in a concerted manner (SET, path 1) or in a consecutive manner involving a

439

radical anion ArX −• (SET, path 2). The generated radical Ar • yields after recombination with

440

B12CoII and protonation the reduced product ArH. If the initial electron transfer step is rate-

441

determining, the reaction rate is expected to correlate with the reduction potential E0

442

(ArX/ArX− •) within a given compound class. Heavy metals M including cobalt with their d8

443

configuration are known for their readiness to form hydrogen bonds as Lewis bases providing 20


Page 21 of 37


444

support for an initial single electron transfer from the corrinoid cobalt to the halogenated

445

substrate. Experimental findings suggest that the hydrogen bond to a metal atom as Lewis base

446

may take place intermediately on the way of a proton transfer to the metal, and thus can also be

447

understood as an arrested protonation. The d8 electron configuration facilitates a stabilizing 3-

448

center-4-electron interaction M···H–Y (Y = H-bond donor atom) with a partial d σHY*

449

electron transfer into the respective antibonding orbital of the Lewis acid HY. The readiness of

450

d8 metals to accept H bonds reflects their electron donor capability.

451

The phenomenon of halogen bonding X···B between a halogen atom X acting as Lewis acid

452

(electron acceptor) through its apical σ-hole and a Lewis base B, suggests that the latter may also

453

be formed with d8 heavy metals such as CoI as electron donor. The ability of B12CoI as H bond

454

acceptor has been demonstrated through computational chemistry, confirming the affinity of its

455

doubly occupied dz2 orbital for attractive interactions with Lewis acids. For Ni0 with a d8 electron

456

configuration as present in CoI, an electron transfer step through a metal attack at the halogen

457

site of X–C has been considered as one of several mechanistic options. An involvement of the

458

apical halogen σ−hole of X-CAr was indirectly supported by the improved predictions on the

459

basis of positive partial σ charges observed in this study. Electron transfer from the dz2 orbital of

460

B12CoI to the apical σ-hole X site of X–CAr driven by the attractive interaction of an emerging

461

Co-halogen bond B12CoI···X–CAr would initially populate the σCX* antibonding orbital (Figure

462

4), leading to a transient σ* radical anion [B12CoII···X–···Ar]• with a substantially weakened bond

463

between CAr and X. Now, the complex is prepared for a concerted dissociation into its

464

components B12CoII, X– and Ar• (Figure 3b, path 1). The neutral radical Ar• is relatively stable

465

due to delocalization of the unpaired electron through its aromatic π system, and together with

466

the thermodynamically stable halide X– pushes the dissociative electron transfer step to the 21



Page 22 of 37

467

product side disfavoring the formation of a radical anion intermediate ArX− •. Yet, it is not clear

468

why electron-poor pyridines did not invoke organohalide respiration, which would be expected

469

to be more reactive in both SNAr (because of the more positively polarized ipso CAr) and SET

470

mechanism (because of a generally larger electron affinity of the molecule). However, our

471

findings highlight the important role of the halogen-carbon bond polarization for the molecule

472

reactivity with Co(I) of reductive dehalogenases.

473

Local reactivity parameters such as the site-specific electrophilicity46 may provide additional

474

insight into the halogen affinity for its role as SET electron acceptor. Also, hydrogen bonds

475

between substrate and the protein environment in the active site of the enzyme will influence

476

substrate reactivity as suggested recently for the binding of ortho-halogenated phenols in the

477

soluble and oxygen-tolerant enzyme of Nitratireductor pacificus (NprdhA).27 Studying the

478

crystal structure of respiratory membrane-bound dehalogenases and their non-phenolic substrates

479

could give a more general picture of the B12-organohalogen interaction. In addition, reaction

480

barriers associated with different mechanisms and for different ligands could be characterized

481

through quantum chemical analyses of the B12CoI catalysis process, following approaches

482

successfully undertaken in the area of P450 catalysis.47,48 The very small active site in the

483

recently described three-dimensional structure of the RdhA protein of Sulfurospirillum

484

multivorans corroborates the proposed initial attack on the halogen atom.26 Similarly, modeling

485

of a NprdhA interaction with its substrate 3,5-dibromo-4-hydroxy benzoic acid suggested a

486

localization of the halogen directly above the cobalt.27 The presently introduced data obtained

487

with growing, organohalide-respiring anaerobic bacteria and electron density modelling together

488

with the characterization of the two reductive dehalogenase structures and EPR spectra26,27

489

strongly suggest the halogen as the primary site of attack for Co(I). These findings can be 22


Page 23 of 37


490

explained by the electronic structure characteristics of the d8 Co(I) supernucleophile, the affinity

491

of d8 heavy metals for hydrogen bonding49 and an apical σ-hole of halogens.

492

D. mccartyi strain CBDB1 is specialized on reductive dehalogenation of halogenated aromatic

493

compounds. The combination of microbiology, computational modeling and enzymatic studies

494

revealed the influence of the electron acceptor’s chemical properties on reductive

495

dehalogenation. Functional groups withdrawing electron density from the halogen enhance

496

reductive dehalogenation. Although other D. mccartyi strains such as strains DCMB510 and 19550

497

catalyze variations of the dehalogenation pathways catalyzed by strain CBDB1 functional groups

498

of the electron acceptors withdrawing electron density from the halogen still appear to have

499

major impact on the pathways. It is undisputed that the three-dimensional conformation of the

500

active site in the enzyme has pivotal influence on the regioselectivity of the catalyzed reaction.

501

Further systematic investigation using model organisms from different dehalogenating bacterial

502

groups will have to explore the influence attributable to the electron distribution within the

503

electron acceptor in comparison with the influence attributable to the structure of the catalyzing

504

enzyme. Together our findings give evidence that B12-halogen interactions take place in

505

anaerobic organohalide respiration and allow for an improved fate prediction of halogenated

506

aromatics in microbially habituated anaerobic environments.

507

AUTHOR INFORMATION

508

Corresponding Author

509 510 511

*Phone: 49-341-235-1435; e-mail: [email protected] Present Addresses ǂ

Technische Universität Berlin, Bioprocess Engineering, Ackerstr. 76, 13355 Berlin, Germany 23



512 513

514

Page 24 of 37

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

515

The authors thank Ulrich Wagner for critical discussion and helpful comments, Bernd Krostitz

516

for technical support. The work was supported by the ERC (project Microflex), DFG (FOR1530)

517

to L.A. and UFZ IP CCF to L.A. and G.S.

518 519

ASSOCIATE CONTENT

520

Supporting Information Available

521

Table S1: Used electron acceptors; Tables S2 to S10: Data of electron density calculation

522

according to the NBO, Hirshfeld and NBO models for different halogenated compounds on

523

halogen and carbon atoms. This information is available free of charge via the Internet at

524

http://pubs.acs.org.

525

24


Page 25 of 37

526


FIGURE LEGENDS

527 528

Figure 1. Transformation of halogenated aromatics in cultures of strain CBDB1. (a) Anilines (○

529

- 2,3-dichloroaniline, ● - 2,4-dichloroaniline; - 2,6-dichloroaniline), (b) benzonitriles (○ - 2,3-

530

dichlorobenzonitrile, ● - 2,5-dichlorobenzonitrile, - 2,6-dichlorobenzonitrile, □ - 2-chloro-6-

531

fluorobenzonitrile), (c) thiophenes (○ - 2,3-dichlorothiophene, ● - 2,5-dichlorohiophene) and (d)

532

pyridines (○ - 2,3-dichloropyridine; ● - 2,6- dichloropyridine). Shown are mean values and

533

standard deviations of two to three parallel cultures. Negative controls without inoculum or with

534

killed inoculum showed no decrease of electron acceptor concentrations. Arrows indicate a re-

535

feeding with electron acceptors 2,3-dichloroaniline (a), 2,3- and 2,5-dichlorothiophene (c).

536 537

Figure 2. Dehalogenation of brominated compounds in cultures of strain CBDB1. (a) Bromide

538

release from 4,5-dibromo-2-furoic acid (4,5-BDrFa) after 126 days of incubation and from 5-

539

bromo-2-furoic acid (5-BrFa), 4-bromo-3,5-dimethoxybenzoic acid (BrDMeOBa) or 4-bromo-

540

3,5-dihydroxybenzoic acid (BrDOHBa) after 63 days of incubation. (b) Formation of phenol

541

from 2,4-dibromophenol (○), 2,6-dibromophenol (▼) or 2,4,6-tribromophenol (●). (c)

542

Transformation of 3-bromo-2-chloropyridine in CBDB1 cultures (■) and in abiotic controls (□,

543

dashed lines). The product 2-chloropyridine accumulated much more in CBDB1 cultures (●)

544

than in chemical controls (○, dashed lines). Arrows indicate re-feeding of CBDB1 cultures with

545

the electron acceptor 3-bromo-2-chloropyridine. Shown are mean values and standard deviations

546

of two parallel cultures of a second transfer.

547

25



Page 26 of 37

548

Figure 3. Possible reaction mechanisms for the reactive dechlorination of substituted aromatics

549

by strain CBDB1. (a) SNAr, nucleophilic aromatic substitution; (b) SET, single electron transfer.

550 551

Figure 4. Single electron transfer model. In the initial step one electron is transferred from the

552

doubly occupied dz² orbital of CoI (d8) to the empty antibonding C-X σ* orbital at the backside of

553

the halogen atom attached to aromatic carbon.

554

26


Page 27 of 37


555

TABLES

556 557 558

Table 1. Dehalogenation reactions catalyzed by strain CBDB1 and molar growth yields when grown on different halogenated compounds and specific dehalogenating activity of resting cells previously grown on 1,2,3-trichlorobenzene. Molar growth yield Electron acceptor

Dehalogenation product(s)

(cells/mol halogen released)

Sp. Act.

(g protein/mol halogen released)

(nkat/mg of protein)

1.7-2.6

n.d.

Aromatic compounds with non-halogenated substituents 2,3-dichloroaniline

→ 3-chloroaniline

5.7 - 8.7 x 1013

2,4-dichloroaniline

not dehalogenated

no growth

n.d.

2,6-dichloroaniline

not dehalogenated

no growth

n.d.

2,3-dichlorobenzonitrile

→ 3-chlorobenzonitrile

2.3 x 1014

6.9

50.6 ± 4.5


→ 3-chlorobenzonitrile

5.1 x 10

14

15.3

n.d.


→ 2-chlorobenzonitrile → benzonitrile

3.5 x 1014

10.5

n.d.

2-chloro-6-fluorobenzonitrile

→ 2-fluorobenzonitrile1

2.3 x 1014

6.9

n.d.

2,4,6-tribromophenol

→ 2,4-dibromophenol → 4-bromophenol → phenol

3.7 x 1013

1.1

87.4 ± 3.7

2,4-dibromophenol

→ 4-bromophenol → phenol

3.0 x 1013

0.9

41.7 ± 3.9

2,6-dibromophenol

→ 2-bromophenol → phenol

3.7 x 10

13

1.1

14.3 ± 1.9

4-bromo-3,5dimethoxybenzoic acid

→ 3,5-dimethoxybenzoic acid

1.8 x 1014

5.4

34.0 ± 4.7

4-bromo-3,5dihydroxybenzoic acid

→ 3,5-dihydroxybenzoic acid

4.4 x 1013

1.3

n.d.

Heterocyclic compounds

559 560

2,3-dichlorothiophene

→ 3-chlorothiophene

9.9 x 1013

3.0

42.1 ± 1.3


→ 2-chlorothiophene → thiophene

9.3 x 1013

2.8

4.5 ± 1.1

4,5-dibromo-2-furoic acid

bromide released, product not determined

0.9 x 1013

0.3

a.r.

5-bromo-2-furoic acid

→ furoic acid

9.5 x 1013

2.9

n.d.

2,3-dichloropyridine

not dehalogenated

no growth

n.d.


not dehalogenated

no growth

n.d.

3-bromo-2-chloropyridine

→ 2-chloropyridine

9.4 x 1013

2.8

30.2 ± 1.7

-

n.d. – no activity detected; a.r. – abiotic reaction, but Br release was higher in biotic reactions 1 A product was detected that was not 2-chlorobenzonitrile, presumably 2-fluorobenzonitrile. 27



Page 28 of 37

561

Table 2 ܳ௑ே஻ை and ܳ஼ே஻ை values for homo- and heteroaromatic compounds tested experimentally

562

in this study. Indicated in bold are positions from which halogen substituents were removed by

563

strain CBDB1. Grey background highlights halogen substituents with most positive ܳ௑ or

564

ܳ஼ values. C2 ܳ௑ே஻ை

C3 ܳ஼ே஻ை

ܳ௑ே஻ை

C4 ܳ஼ே஻ை

ܳ௑ே஻ை

C5 ܳ஼ே஻ை

ܳ௑ே஻ை

C6 ܳ஼ே஻ை

ܳ௑ே஻ை

ܳ஼ே஻ை

Aromatic compounds with non-halogenated substituents 2,3-dichloroaniline

0.008

-0.110 0.019

-0.049

2,4-dichloroaniline

-0.013 -0.074

2,6-dichloroaniline

-0.015 -0.075


0.070

-0.035 0.043


0.047

-0.017


0.050

-0.001

2-chlorobenzonitrile

0.038

-0.010

2-chloro-6-fluorobenzonitrile

0.048

-0.002

2,4,6-tribromophenol

0.072

-0.162

0.077

-0.123

2,4-dibromophenol

0.089

-0.156

0.063

-0.130

2,6-dibromophenol

0.057

-0.169

2-bromophenol

0.073

-0.165

-0.007 -0.069 -0.015 -0.075 -0.067 0.023

-0.052 0.050

-0.001

-0.312 0.484

4-bromophenol

0.049

-0.138

4-bromo-3,5-dimethoxybenzoic acid

0.106

-0.182

4-bromo-3,5-dihydroxybenzoic acid

0.083

-0.221

0.102

-0.147

0.088

-0.156

0.013

0.198

Heterocyclic compounds 2,3-dichlorothiophene

0.071

-0.305 0.037


0.055

-0.285

2-chlorothiophene

0.046

-0.287

-0.124 0.055

4,5-dibromo-2-furoic acid

0.127

5-bromo-2-furoic acid 2,3-dichloropyridine

0.026

0.174


0.013

0.198

3-bromo-2-chloropyridine

0.025

0.175

0.039

0.107

-0.285

-0.249 0.161

0.182

0.139

0.187

-0.106

-0.177

28


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565


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TOC graphic 56x39mm (300 x 300 DPI)



Figure 1 203x198mm (300 x 300 DPI)


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Figure 2 116x64mm (300 x 300 DPI)



Figure 3 66x63mm (600 x 600 DPI)


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Figure 4 495x239mm (96 x 96 DPI)


Anaerobic Microbial Transformation of Halogenated Aromatics and

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