Organic Bromine and Iodine Compounds docx

Both strains IMB-1 and Leisingeria rans strain MB2 degrade methyl bromide by respiration that is mediated by a methyltransferase whereas in Methylomonas rubra that is also able to take u

Environmental Chemistry is a relatively young science Interest in this subject,however, is growing very rapidly and, although no agreement has been reached

as yet about the exact content and limits of this interdisciplinary discipline, thereappears to be increasing interest in seeing environmental topics which are based

on chemistry embodied in this subject One of the first objectives of mental Chemistry must be the study of the environment and of natural chemicalprocesses which occur in the environment A major purpose of this series onEnvironmental Chemistry, therefore, is to present a reasonably uniform view ofvarious aspects of the chemistry of the environment and chemical reactionsoccurring in the environment

The industrial activities of man have given a new dimension to mental Chemistry We have now synthesized and described over five millionchemical compounds and chemical industry produces about hundred and fiftymillion tons of synthetic chemicals annually We ship billions of tons of oil peryear and through mining operations and other geophysical modifications, largequantities of inorganic and organic materials are released from their naturaldeposits Cities and metropolitan areas of up to 15 million inhabitants producelarge quantities of waste in relatively small and confined areas Much of thechemical products and waste products of modern society are released into theenvironment either during production, storage, transport, use or ultimatedisposal These released materials participate in natural cycles and reactionsand frequently lead to interference and disturbance of natural systems

Environ-Environmental Chemistry is concerned with reactions in the environment It

is about distribution and equilibria between environmental compartments

It is about reactions, pathways, thermodynamics and kinetics An importantpurpose of this Handbook, is to aid understanding of the basic distribution andchemical reaction processes which occur in the environment

Laws regulating toxic substances in various countries are designed to assessand control risk of chemicals to man and his environment Science can con-tribute in two areas to this assessment; firstly in the area of toxicology andsecondly in the area of chemical exposure The available concentration(“environmental exposure concentration”) depends on the fate of chemicalcompounds in the environment and thus their distribution and reaction be-haviour in the environment One very important contribution of EnvironmentalChemistry to the above mentioned toxic substances laws is to develop laboratorytest methods, or mathematical correlations and models that predict the environ-

mental fate of new chemical compounds The third purpose of this Handbook is

to help in the basic understanding and development of such test methods andmodels

The last explicit purpose of the Handbook is to present, in concise form, themost important properties relating to environmental chemistry and hazardassessment for the most important series of chemical compounds

At the moment three volumes of the Handbook are planned Volume 1 dealswith the natural environment and the biogeochemical cycles therein, includingsome background information such as energetics and ecology Volume 2 is con-cerned with reactions and processes in the environment and deals with physicalfactors such as transport and adsorption, and chemical, photochemical andbiochemical reactions in the environment, as well as some aspects of pharma-cokinetics and metabolism within organisms.Volume 3 deals with anthropogeniccompounds, their chemical backgrounds, production methods and informationabout their use, their environmental behaviour, analytical methodology andsome important aspects of their toxic effects The material for volume 1, 2 and 3was each more than could easily be fitted into a single volume, and for thisreason, as well as for the purpose of rapid publication of available manuscripts,all three volumes were divided in the parts A and B Part A of all three volumes isnow being published and the second part of each of these volumes should appearabout six months thereafter Publisher and editor hope to keep materials of thevolumes one to three up to date and to extend coverage in the subject areas bypublishing further parts in the future Plans also exist for volumes dealing withdifferent subject matter such as analysis, chemical technology and toxicology,and readers are encouraged to offer suggestions and advice as to future editions

of “The Handbook of Environmental Chemistry”

Most chapters in the Handbook are written to a fairly advanced level andshould be of interest to the graduate student and practising scientist I also hopethat the subject matter treated will be of interest to people outside chemistry and

to scientists in industry as well as government and regulatory bodies It would

be very satisfying for me to see the books used as a basis for developing graduatecourses in Environmental Chemistry

Due to the breadth of the subject matter, it was not easy to edit this book Specialists had to be found in quite different areas of science who werewilling to contribute a chapter within the prescribed schedule It is with greatsatisfaction that I thank all 52 authors from 8 countries for their understandingand for devoting their time to this effort Special thanks are due to Dr F Boschke

Hand-of Springer for his advice and discussions throughout all stages Hand-of preparation

of the Handbook Mrs A Heinrich of Springer has significantly contributed tothe technical development of the book through her conscientious and efficientwork Finally I like to thank my family, students and colleagues for being sopatient with me during several critical phases of preparation for the Handbook,and to some colleagues and the secretaries for technical help

I consider it a privilege to see my chosen subject grow My interest in mental Chemistry dates back to my early college days in Vienna I receivedsignificant impulses during my postdoctoral period at the University of Californiaand my interest slowly developed during my time with the National Research

Environ-Council of Canada, before I could devote my full time of EnvironmentalChemistry, here in Amsterdam I hope this Handbook may help deepen theinterest of other scientists in this subject.

Twentyone years have now passed since the appearance of the first volumes ofthe Handbook Although the basic concept has remained the same changes andadjustments were necessary

Some years ago publishers and editors agreed to expand the Handbook bytwo new open-end volume series: Air Pollution and Water Pollution Thesebroad topics could not be fitted easily into the headings of the first three vol-umes All five volume series are integrated through the choice of topics and by asystem of cross referencing

The outline of the Handbook is thus as follows:

1 The Natural Environment and the Biochemical Cycles,

2 Reaction and Processes,

A recent development is the accessibility of all new volumes of the Handbookfrom 1990 onwards, available via the Springer Homepage http://www.springer de

or http://Link.springer.de/series/hec/ or http://Link.springerny.com/ series/hec/.During the last 5 to 10 years there was a growing tendency to include subjectmatters of societal relevance into a broad view of Environmental Chemistry.Topics include LCA (Life Cycle Analysis), Environmental Management, Sustain-able Development and others.Whilst these topics are of great importance for thedevelopment and acceptance of Environmental Chemistry Publishers and Edi-tors have decided to keep the Handbook essentially a source of information on

“hard sciences”

With books in press and in preparation we have now well over 40 volumesavailable.Authors, volume-editors and editor-in-chief are rewarded by the broadacceptance of the “Handbook” in the scientific community

Except for astatine whose chemistry is largely unknown, fluorine and iodine arethe first and last of the halogens This is shown in a number of ways includingthe successive decrease in the redox potential Hal–/Hal2and the electronegativi-

ty, and increase in the covalent and van der Waals radii The substitution ofhydrogen by fluorine does not greatly alter the structure of organofluorines incontrast to the effect of introducing bulky bromine or iodine substituents.Although fluorides are found abundantly in a range of minerals, the taming ofboth elemental fluorine and the hydrogen fluorides presented serious experi-mental difficulties that were solved only after many years of dangerous work andwere a prelude to the synthesis of organofluorines Bromide is present in seawa-ter at a concentration of 65 ppm and iodide at 0.05 ppm although these concen-trations are greatly exceeded in hypersaline lakes that are the current source ofbromide and iodide

The preparation of both elemental bromine and iodine was accomplishedmore than 60 years before that of elemental fluorine, bromine in 1826 and iodi-

ne in 1811’ and the synthesis of organobromine and organoiodine compoundspresented fewer problems A wide range of organofluorines has achieved in-dustrial importance as refrigerants, surfactants, pharmaceuticals, dyestuffs,whereas the range of organobromine and organoiodine compounds in generaluse is much more limited

Organofluorine compounds exist only in the monovalent state whereas all theother halogens may exist at oxidation levels up to 7 Organoiodine compoundsmay exist in the trivalent and pentavalent states that have seen numerous appli-cations: they have been used extensively in organic synthesis as oxidizing agents[Zhdankin and Stang 2002], benziodoxoles have attracted attention as syntheticreagents for the destruction of chemical weapons [Morales-Rojas and Moss2002] and iodonium salts have been used to develop a silver-free, single-sheetimaging medium [Marshall et al 2002]

The number of naturally occurring organofluorines is structurally limitedand essentially confined to higher plants in contrast to the plethora of organob-romine – and to a lesser extent organoiodine – metabolites produced mostly bymarine biota Iodide is essential for many biota including humans, and organiccompounds of iodine have long attracted interest as a result of the physiologicalimportance of iodinated tyrosines in thyroid function and the antiseptic pro-perty of diiodine released from triiodomethane More recently they have achiev-

ed importance as X-ray contrast agents

Organic compounds of bromine have a greater diversity of application.Dibromoethane was once used extensively in automobile fuel containing tetrae-thyl lead to diminish engine corrosion, while methyl bromide has a long history

of use as fumigant and has attracted attention as a result of concern with globalwarming and ozone depletion In addition, oxidants produced in the brominecycle in the troposphere have been shown to be important in mobilizing ele-mentary Hg to species that are both accessible to biota and accumulate in Arcticsnow [Lindberg et al 2002] Polybrominated aromatic compounds, and especi-ally diphenyl ethers, have been used as flame-retardants and are now widely dis-tributed in the environment A relatively small number of agrochemicals inclu-ding bromoxynil, bromacil and bromethuron have been used

This volume addresses a broad spectrum of the environmental issues unding organic bromine and iodine compounds In assessing their environ-mental significance it is important to assess their partition among the environ-mental compartments and the potential for their long-range dissemination:these issues are discussed by Cousins and Palm Orlando discusses atmosphericchemistry in the context of ozone depletion and global warming, and the signi-ficant difference between the reactions of methyl bromide and methyl iodide areunderscored

surro-Mammalian toxicity is discussed by DePierre and the mechanisms of theirdegradation and transformation by Allard and Neilson There has been consid-erable interest in naturally occurring metabolites in the current debate on thefate and partition of methyl bromide that is – or possibly by the time this ispublished was – important nematocide and is produced in substantial quantities

as a metabolite of marine algae There has also been speculation on the ral occurrence of diphenyl ethers and Neilson discusses plausible mechanismsfor the biosynthesis of representative organic bromine and organic iodine me-tabolites

natu-Once again, it is a particular pleasure to thank the authors who were

prepar-ed to sacrifice their valuable time and take on the additional burden of makingtheir contributions This is particularly appreciated since, in these days of con-tinual stress, potential contributors feel themselves already overburdened withthe demands of seeking financial support and producing publications to justifytheir existence Any success with this volume is entirely due to the contributors,and I feel sure that their effort has been well rewarded in producing an excitingvolume

Lindberg SE, S Brooks, C-J Lin, KJ Scott, MS Landis, RK Stevens, M Goodsite and A Richter (2002) Dynamic oxidation of gaseous mercury in the Arctic troposphere at polar sunrise Environ Sci Technol 36:1245–1256

Marshall JL, SJ Telfer, MA Young, EP Lindholm, RA Minns, L Takiff (2002) A silver-free, sheet imaging medium based on acid amplification Science 297:1516-1521

single-Morales-Royas H and RA Moss (2002) Phosphorolytic reactivity of o-iodosylcarboxylates and

related nucleophiles Chem Rev 102:2497–2521

Zdankin VV and PJ Stang (2002) Recent developments in the chemistry of polyvalent iodine compounds Chem Rev 102:2523–2584

Degradation and Transformation of Organic Bromine and Iodine Compounds: Comparison with their

Chlorinated Analogues

Ann-Sofie Allard1· Alasdair H Neilson2

Swedish Environmental Research Institute Limited IVL, Sweden

An overview is given of the pathways for the degradation and transformation of selected brominated and iodinated aliphatic and aromatic compounds Although greater emphasis is placed on reactions mediated by microorganisms, examples of important abiotic reactions are also given A mechanistic outline of the enzymology is provided when possible and compar-isons are made with the chlorinated analogues which have been more extensively studied.

Keywords. Biodegradation and biotransformation, Abiotic transformation, Aliphatic com-pounds, Aromatic compounds

1 Introduction . 2

2 Aliphatic Compounds . 4

2.1 Halogenated Methanes 6

2.1.1 Methyl Halides 6

2.1.1.1 Methane Monooxygenase Pathway 6

2.1.1.2 Methyl Transfer and Corrinoid Pathways 8

2.1.1.3 Corrinoid Transmethylations in Aerobic and Anaerobic Metabolism of Methyl Halides 9

2.1.2 Di- and Trihalomethanes 12

2.1.2.1 Aerobic Organisms 12

2.1.2.2 Anaerobic Organisms 13

2.2 Halogenated Alkanes and Related Compounds with Two or More Carbon Atoms 15

2.3 Halogenated Ethenes 20

2.4 Haloalkanols 22

2.5 Haloaldehydes 23

2.6 Haloalkanoates 23

2.7 Halogenated Ethers 26

2.8 Reductive Loss of Halogen 27

2.9 Brominated and Iodinated Alkanes and Related Compounds as Metabolic Inhibitors 30

3 Abiotic Reactions . 31

3.1 Photohydrolytic Reactions 31

3.2 Reductive Reactions 32

© Springer-Verlag Berlin Heidelberg 2003

4 Aromatic Compounds : Aerobic Reactions . 34

4.1 Hydrocarbons 34

4.1.1 Degradation and Growth 34

4.1.2 Metabolism Without Growth 35

4.1.3 Biotransformation to Dihydrodiols 39

4.2 Benzoates 40

4.2.1 Dioxygenation 41

4.2.1.1 Dehalogenation of 2-Halogenated Benzoates 41

4.2.1.2 Loss of Halogen in 4-Halogenated Phenylacetates 43

4.2.1.3 Halohydrolases 44

4.2.1.4 Reductive Dehalogenation 45

4.2.1.5 Denitrification 45

4.2.1.6 Fungi 45

4.3 Phenols 46

4.3.1 O-Methylation of Halogenated Phenols 48

4.3.2 Fungal Metabolism 49

4.4 Amines 49

5 Alternative Mechanisms of Dehalogenation . 51

5.1 Peroxidase 51

5.2 Dehalogenation by a Polychaete 51

5.3 Dehalogenation by Thymidylate Synthetase 51

6 Anaerobic Reactions 52

6.1 Introduction 52

6.2 Halogenated Hydrocarbons 54

6.2.1 Polyhalogenated Benzenes 54

6.2.2 PCBs 55

6.2.3 PBBs and Diphenylmethanes 56

6.3 Anaerobic Degradation of Benzoates 59

6.3.1 Dehalogenation 59

6.3.2 Oxidation and Reduction of Aromatic Carboxylates and Aldehydes 60

6.4 Phenols 60

7 Concluding Comments . 61

8 References . 62

1

Introduction

In the course of preparing this chapter it became evident that relatively few stud-ies were directed primarily to brominated compounds Many were concerned with chlorinated compounds, and some brominated analogues were fortuitously included It was therefore necessary to glean the literature on chlorinated

com-pounds and extract details on their brominated and iodinated analogues thatwere sometimes included It was then decided to include results for selected chlo-rinated compounds for several reasons: (i) for comparison with their brominatedanalogues; (ii) when studies of the brominated compounds were lacking and had

been carried out only with the chlorinated analogues; (iii) when studies only with

chlorinated analogues illustrated important principles of metabolism

Attention is drawn to a selection of reviews that cover various aspects of halogenation [23, 53, 55, 84, 94, 162, 188, 226]

de-Three different metabolic situations have been encountered: (i) growth at theexpense solely of the brominated compound; (ii) loss of bromide during incu-bation with cell suspensions or enzymes; (iii) inclusion of a brominated substrate

in the course of enzymological studies It is worth noting that for some genated substrates, evidence for diminution of its concentration has beendemonstrated in spite of the absence of dehalogenation This may plausibly be at-tributed to simple biotransformations such as oxidation or dehydrogenation un-der aerobic conditions

halo-It is important to note the different experimental procedures that have beenused Experiments under anaerobic conditions have been carried out under a va-riety of conditions using: (i) pure cultures; (ii) metabolically stable mixtures oforganisms; (iii) unselected suspensions of soil or sediment The last can lead toproblems in interpretation since the sample used for assay will generally containsome of the putative degradation products

A cardinal issue that not been addressed here is the accessibility of brominatedcompounds – especially hydrocarbons and phenolic compounds – to the appro-priate organisms in suspended matter or in the sediment phase containing or-ganic carbon This is only noted parenthetically with references to some repre-sentative illustrations from the relevant literature from chlorinated analogues.Attention is drawn in the text to some taxonomic changes For simplicity, thesesynonymies are duplicated in the table below

Hyphomicrobium sp strain CM2 Hyphomicrobium chloromethanicum Methylobacterium sp strain CM4 Methylobacterium chloromethanicum

Pseudomonas paucimobilis Sphingomonaspaucimobilis

Rhodococcus chlorophenolicum Mycobacterium chlorophenolicum Clostridium thermoautotrophica Moorella thermoautotrophica

For chlorinated compounds, the greatest attention has been given to groups ofsubstances that are considered environmentally unacceptable, for example, low

molecular mass chlorinated aliphatic compounds used as solvents, chiral propionates incorporated into agrochemicals, hexachlorocyclohexane, PCBs, andpentachlorophenol used as a wood preservative Although brominated organiccompounds are important as agrochemicals, pharmaceuticals and flame-retar-dants, and iodinated compounds as X-ray contrast agents, these have been stud-ied less exhaustively than their chlorinated analogues In addition, the appro-priate compounds may not be commercially available as substrates and it mayseem unusually academic to synthesize them.

chloro-Reliance has of necessity been placed on the metabolism of chlorinated pounds, particularly for the structures of those enzymes that have been deter-mined by X-ray analysis Greatest weight has been placed on studies in which thebiochemistry of degradation and transformation has been elucidated, and inwhich comparison among the halogens is possible

com-There have been major methodological developments during recent years.These include the success in obtaining crystals of enzymes that enable the ap-plication of X-ray analysis to the study of enzyme mechanisms: these provide im-portant details and where available the results of such studies have been included.Although 13C NMR has been used generally with cell suspensions, the availabil-ity of on-line LC-NMR opens this to wider application in establishing the struc-ture of transient metabolites There have been substantial advances in establish-ing the genetics of degradation, and procedures for comparing amino acid andnucleotide sequences among groups of enzymes This has made it possible to es-tablish relationships between enzymes from different organisms and encouragedspeculation on their evolution This aspect has not, however, been treated here inthe depth that it deserves

It is worth noting that – with the exception of simple bromophenols – ally no investigations have been directed to the biodegradation of the plethora

virtu-of naturally occurring brominated organic compounds that are discussed byNeilson

in the chapter by Cairns and Palm and, on the basis of the “replacement ciple”, attention has been redirected to the use of propargyl bromide [248] Poly-chlorinated ethanes and ethenes have been extensively used as solvent and de-greasing agents in the metallurgical industry and concern has arisen over theiradverse health effects This has stimulated efforts to study their degradability and

prin-to find replacements: indeed this applies equally prin-to all polyhalogenated aliphaticcompounds.

An outline of the primary C-Br fission reactions encountered during thedegradation of brominated alkanes, alkenes, brominated alkanols and bro-moalkanoates is given in Fig 1 Further reactions are, of course, possible, whenreactive intermediates such as epoxides or ethenes are formed

Fig 1. Outline of primary reactions involved in degradation or transformation of haloalkanes and related compounds

Degradation in the Natural Environment It has been established that the

biodegradation of methyl bromide occurs in a number of natural environments.For example, a methylotrophic bacterium IMB-1 isolated from agricultural soilwas able to degrade methyl bromide to CO2[75] The degradation of methyl bro-mide in a mixed soil bacterial flora occurred at concentrations several powers often lower than have been used in previous experiments (ten parts per billion byvolume) [82] At these low concentrations, neither chemical degradation noranaerobic degradation occurred This potential has been examined in freshwa-ter, estuarine, and seawater samples in which non-bacterial degradation waseliminated from the results by lack of inhibition of degradation by methyl fluo-ride: both methyl bromide and methylene dibromide were examined but only inthe freshwater sample was degradation observed [68] Two strains were able todegrade methyl bromide at concentrations of the mixing ratio of troposphericmethyl bromide with surface water and displayed no evidence of a threshold con-

centration for uptake [70] Both strains IMB-1 and Leisingeria rans strain MB2 degrade methyl bromide by respiration that is mediated by a methyltransferase whereas in Methylomonas rubra that is also able to take up

methylohalidovo-methyl bromide at comparably low concentrations degradation of the substrates

is mediated by a monooxygenase

Strains Involved in Degradation There are two distinct mechanisms for the

degradation of halomethanes involving (1) methane monooxygenase and (2) rin-dependent, and the biodegradation of methyl bromide has been demon-strated in both groups

cor-2.1.1.1

Methane Monooxygenase Pathway

A number of strains with monooxygenase activity have been examined and

it is convenient to add some comment on the enzyme since various types of hane monooxygenase have played important roles in the degradation of halo-

met-genated aliphatic compounds The enzyme exists in both a soluble and a ticulate form of which the former has been more extensively studied The enzyme consist of three components: a hydroxylase, a regulatory protein that

par-is not directly involved in electron transfer between the hydroxylase, and a thirdprotein that is a reductase containing FAD and an [2Fe-2S] cluster Details ofthe structure of the hydroxylase and the mechanism of its action involvingthe FeIII-O-FeIIIat the active site are given in a review [112] The particulateenzyme contains copper or both copper and iron, and the concentration ofcopper determines the catalytic activity of the enzyme [194] For example,

trichloroethene is degraded by Methylosinus trichosporium strain OB3b under

copper limitation when the soluble monooxygenase is formed, but not duringgrown with copper sufficiency when the particulate form is synthesized [152].Examples of methyl bromide-degrading strains include the following:

1 The soluble methane monooxygenase from Methylococcus capsulatus (Bath)

is able to oxidize chloro- and bromomethane, but not iodomethane with thepresumptive formation of formaldehyde [36]

2 The methane degrading Methylosinus trichosporium OB3b has been shown to

degrade both methyl bromide and dibromomethane [9, 196] and the

propane-degrading Mycobacterium vaccae JOB5 methyl bromide (Table 1) [196] The degradation pathway for Methylosinus trichosporium OB3b was ex-

amined in an elegant study using 13C NMR with [13C]CH3Br and [13C]CH2Br2

as substrates Although the expected formaldehyde from the former could not

be demonstrated possibly on account of its rapid further transformation, theformation of CO was shown for CH2Br2by 13C NMR, and the involvement ofmethane monooxygenase was supported by inhibition of activity by acety-lene [9] Although it was postulated that the initial reactions were monoxy-genation followed by loss of hydrogen bromide, more recent studies on themetabolism of both methyl chloride and methyl bromide show that the reactions are corrinoid-dependent (see below) It is also worth noting thecontrasting mechanisms for the degradation of methyl chloride anddichloromethane One of the fascinating results of the studies with methylchloride is the similarity of pathways proposed for aerobic and strictly anaer-obic bacteria

3 Suspensions of Nitrosomonas europaea were able to oxidize a number of

halo-genated alkanes more effectively in the presence of NH4+that was oxidized tonitrite (Table 2) [221]

Table 1. Concentrations of bromide (µmol) in cultures in which the substrates hadbeen graded The theoretical values are 4.5 for methyl bromide and 11 for dibromoethane [196]

NA = not available.

Methyl Transfer and Corrinoid Pathways

This pathway is used by a number of organisms, and a general outline is given:

1 Strain IMB-1 is able to grow at the expense of methyl bromide [239] and belongs to a group of organisms that are also able to degrade methyl io-dide but unable to use formaldehyde or methanol [176] A single gene clus-

ter contained six open reading frames: cmuC, cmuA, orf 146, paaE, hutI, and part of metF Although CmuA from this strain had a high homology with the methyl transfer of Methylobacterium chloromethanicum and Hyphomi- crobium chloromethanicum, CmuB that has been identified in these strains was not detected A study with Hyphomicrobium chloromethanicum strain CM2 revealed a cmu gene cluster containing ten open reading frames: folD (partial), pduX, orf153, orf 207, orf 225, cmuB, cmuC, cmuA, fmdB, and paaE (partial) CmuA, CmuB, and CmuC from this strain showed a high similarity to those from Methylobacterium chloromethanicum (Table 3)

[239] and it was postulated that the pathway for chloromethane degradation

in this strain was similar to that in Methylobacterium chloromethanicum

[126]

2 Methylobacterium sp CM4 [222, 223] is able to degrade methyl chloride, and details of the metabolism by this strain (now classified as Methylobacterium chloromethanicum) have been resolved and are discussed below.

Table 2. Loss of haloalkanes by Nitrosomonaseuropaea during oxidation of NH4+ Substrate remaining after 24 h as % of the initial amount [222]

Table 3. Summary of Hyphomicrobium chloromethanicum methyltransferase genes and

iden-tity (%) with representative proteins [239]

FolD Methylene tetrahydrofolate

Cyclohydrolase/dehydrogenase

and orf414 (34) CmuA Methyltransferase and corrinoid M chloromethanicum CM4 cmuA (80)

protein

Corrinoid Transmethylations in Aerobic and Anaerobic Metabolism of Methyl Halides

The existence of corrinoids in anaerobic bacteria in substantial concentrations

is well established and their metabolic role in acetogenesis and in esis understood Their involvement in degradation pathways of aerobic organ-isms is more recent, and it has emerged that their roles under these different con-ditions are similar These issues are explored in the following paragraphs with aview to illustrating the similar metabolic pathways used by both aerobes andanaerobes

methanogen-Corrinoids are involved in aerobic degradation as noted below for the

degra-dation of methyl chloride by the aerobic Methylobacterium sp strain CM4, and

also C1degradation by Methylobacterium extorquens [34] Methyl corrins are key

components in transmethylation and examples illustrating the similarity of ways in aerobic and anaerobic metabolism will be summarized In the followingdiscussion, tetrahydrofolate or tetrahydromethanopterin (Fig 2) are implicated

path-in the form of their methyl (CH3), methylene (CH2), methine (CH), and formyl(CHO) derivatives (Fig 3) The formation of a CH3-Co bond is integral and gen-erally the 5,6-dimethylbenziminazole is displaced by histidine

Aerobic Degradation of Methyl Chloride – Methylotrophic bacteria have been

isolated that are able to use methyl chloride aerobically as the sole source ofenergy and carbon The substrate is metabolized to formaldehyde and under-goes subsequent oxidation either to formate and CO2 or incorporation via theserine pathway A study using a strain CC495 that is similar to the strain IMB-1noted above revealed the complexity of this reaction [38] while details hademerged from a somewhat earlier of methyl chloride degradation by the aero-

bic Methylobacterium sp strain CM4 (Methylobacterium chloromethanicum).

Cobalamin was necessary for growth with methyl chloride, though not forgrowth with methylamine, and use of mutants containing a miniTn5 insertion

Fig 2. Partial structures of tetrahydrofolate (H F) and tetrahydromethanopterin (H MPT)

and enzyme assays revealed the mechanism of the degradation involving initialmethyl transfer to a Co(I) corrinoid followed by oxidation via tetrahydrofolates

to formyltetrahydrofolate and thence to formate with production of ATP (Fig 4)[222]

Anaerobic Degradation of Methyl Chloride – The anaerobic methylotrophic

ho-moacetogen Acetobacterium dehalogenans is able to grow with methyl chloride

and CO2and uses a comparable pathway for dehydrogenation of the methylgroup involving tetrahydrofolate, a corrinoid coenzyme while the activity of COdehydrogenase and the methyl tetrahydrofolate produce acetate (Fig 5) [130].Some of the gene products are shared with those involved in metabolism ofmethyl chloride [222] (Table 4) The methyl transfer reactions and those involved

in the subsequent formation of acetate have been explored for the demethylase

of this strain [101] and also resemble closely those for the aerobic metabolism ofmethyl chloride by aerobic methylotrophs

Fig 3. Dehydrogenation of CH 3 -tetrahydrofolate to CHO-tetrahydrofolate

Fig 4. Degradation of methyl chloride by Methylobacterium chloromethanicum (Redrawn from

[222])

Fig 5. Degradation of methyl chloride by Acetobacterium halogenans (Redrawn from [101,

130])

halogenases closely resemble each other The mechanism for dechlorination

in-volves a glutathione S-transferase that produced dideuteroformaldehyde from dideuterodichloromethane from cell extracts of Hyphomicrobium sp strain DM2

[72], so that neither elimination-addition nor oxidation-reduction mechanismsare possible Cell extracts of this strain were able to dehalogenate a number of di-halomethanes (Table 5) [197] There are two different dichloromethane-dehalo-

genating glutathione S-transferases, neither of which contain metals, the enzyme from Hyphomicrobium sp strain DM 4 with an a6 and that from Methylobac- terium sp strain DM 11 with an a2 structure [105] Glutathione S-transferase is

also involved in the degradation of isoprene (2-methyl-buta-1,3-diene) by

Rhodococcus sp strain AD45 [215] and is able to transform cis- and

trans-1.2-dichlorethene to the epoxides with formation of glyoxal The enzyme has beenpurified [216] and has a wide range of substrate specificity (Table 6) Glutathione

is also involved in the dechlorination of hexachlorocyclohexane catalyzed by

LinD in Sphingomonas paucimobilis strain UT26 [141] and the dehalogenation of tetrachlorohydroquinone by Flavobacterium sp [246].

The degradation of dibromo- and tribromomethanes has been examined der different conditions: (a) an enrichment culture from seawater was able to de-

un-Table 4. Genes, inferred function and identity (%) to representatives [222]

folD 5,10-Methylene-H 4folate dehydrogenase/ FolD (E coli) 49 5,10-methenyl-H 4 folate cyclohydrolase

purU 10-Formyl-H 4 folate hydrolase PurU (Corynebacterium sp. 47

metF 5,10-Methylene-H 4 folate reductase Orf (Saccharomyces cerevisiae) 24

grade 14CH2Br2to 14CO2; (b) degradation was studied in a marine strain of lobacter marinus strain A45 and new type I methanotrophic strains designated

Methy-KML E-1 and Methy-KML E-2: the first was able to degrade methyl bromide and momethane whereas the last was able to degrade tribromomethane but not di-bromomethane [69] It has been established that strains bearing plasmids formonooxygenation of toluene are able to dechlorinate chloroform: for example,

dibro-the toluene 4-monooxygenase from Pseudomonas mendocina strain KR1 [127], and the generalized toluene 2-, 3-, and 4-monooxygenase from Pseudomonas stutzeri strain OX1[32] (halogenated alkenes are discussed elsewhere in this

chapter)

2.1.2.2

Anaerobic Organisms

Acetobacterium dehalogenans is able to degrade dichloromethane and the

path-ways resemble formally that for the anaerobic degradation of methyl chloride

(Fig 5) A strain of Dehalobacterium formicoaceticum is able to use only

dichloromethane as a source of carbon and energy forming formate and acetate[138] The pathway involves initial synthesis of methylene tetrahydrofolate ofwhich two-thirds is degraded to formate with generation of ATP while the otherthird is dehydrogenated, transmethylated, and after incorporation of CO formsacetate with production of ATP (Fig 6) The formation of [13C]formate,[13C]methanol, and [13CH3]CO2H was elegantly confirmed using a cell suspensionand [13C]CH2Cl2 It was suggested that a sodium-independent F0F1-type ATP syn-thase exists in this organism in addition to generation of ATP from formylte-trahydrofolate

A strain of Acetobacterium woodii strain DSM 1930 dehalogenated

tetra-chloromethane to ditetra-chloromethane as the final chlorinated product, while thecarbon atom of [14C]tetrachloromethane was recovered as acetate (39%), CO2(13%), and pyruvate (10%) [49] Since the transformation of tetrachloromethane

to chloroform and CO2 is a non-enzymatic corrinoid-dependent reaction [50, 77]

it seems safe to assume operation of the acetyl-CoA synthase reaction and thesynthesis of acetate that also takes place during the degradation of

dichloromethane by Dehalobacterium formicoaceticum and in which the CO2originates from the medium [139]

Synthesis of Corrinoid-Dependent Reactions – It is appropriate to bring gether a number of related reactions These resemble those noted above even

to-Table 6. Substrate specificity of S-glutathione transferase from Rhodococcus sp strain AD45

though they do not involve halogenated substrates The metabolism of acetate

and lactate by Desulfomaculatum acetoxidans and Archaeoglobus fulgidus

re-spectively and the pathways are given (Fig 7), and are clearly essentially tical to those used for anaerobic degradation of methyl chloride and

iden-dichloromethane In a wider context methanogenesis by Methanosarcina eri is worth noting: methane can be synthesized from CO2and H2, or acetate, ormethanol or methylamine There are, however, important differences from the re-actions noted above:

bark-1 Use of tetrahydromethanopterin in place of tetrahydrofolate

2 The involvement of an aminofuran as acceptor of the formate produced from

Halogenated Alkanes and Related Compounds with Two or More Carbon Atoms

Alkanes with a Single Halogen Atom – The monooxygenase in

ammonia-oxidiz-ing Nitrosomonas europaea has been examined for oxidation of halogenated

ethanes and resulted in production of acetaldehyde presumably by initial

termi-nal hydroxylation (Table 7) [158] while a strain of Nitrosomonas europaea was shown to oxidize a number of substrates including dibromoethane, and cis- and trans-dibromoethene (Table 2) [221].

The rates of dehalogenation of a range of 1-substituted haloalkanes was

ex-amined in an Arthrobacter sp strain HA1 (Table 8) and the enzyme was a

halo-hydrolase that produced the corresponding alkanol and dehalogenated a muchwider range of substrates than could be used for growth [179] Further investi-gations with the same strain confirmed that the reaction was hydrolytic, showedthat there were three dehalogenases, examined the pattern of induction, and ex-tended the range of growth substrates to 1-brominated alkanes C10, C12, C14, and

C [180] Pseudomonas sp strain ES-2 was able to grow with a range of

bromi-Fig 7a,b. Comparison of degradation of: a acetate by Desulfomaculatum acetoxidans; b lactate

by Archaeoglobus fulgidus (Redrawn from [202a])

nated alkanes that greatly exceeded the range of chlorinated or unsubstitutedalkanes: bromoalkanes with chain lengths of C6to C16, and C18could all be uti-lized [187] A range of chlorinated, brominated, and iodinated alkanes C4to C16

was incubated with resting cells of Rhodococcus rhodochrous NCIMB 13064 [40],

and dehalogenation assessed from the concentration of halide produced(Table 9) The range of substrates is impressive and the yields were approximatelyequal for chloride and bromide and greater than for iodide

Possibly more remarkable is the metabolic capacity of species of

mycobacte-ria including the human pathogen Mycobacterium tuberculosis strain H37Rv [96] The specific activities in extracts of M avium and M smegmatis to a range

of halogenated alkanes is given in Table 10 On the basis of aminoacid and DNAsequences, the strain that was used contained three halohydrolases and the de-bromination capability of a selected number of other species of mycobacteria is

given in Table 11 The haloalkane dehalogenase gene from M avium has been

cloned and partly characterized [97]

Table 7. Rate of formation of acetaldehyde [nmol/min/mg protein] by whole cells of

Table 9. Dehalogenation of long-chain alkyl halides by Rhodococcus rhodochrous strain

NCIMB 13064 measured as halide release (1-chlorobutane=100) The symbols in parentheses designate growth at the expense of the substrate [40]

Table 10. Relative specific activity (µmol alkanol produced/mg protein/min) of extracts of

Mycobacterium avium MU1 and M smegmatis CCM 4622 to halogenated alkanes [96]

Table 11. Specific activity (µmol bromide produced/mg protein/min) of dehalogenase from

selected species of Mycobacterium towards 1,2-dibromoethane [96]

Table 12. Relative substrate activities (1-chlorobutane (=100) of crude halohydrolase from

Rhodococcus erythropolis Y2 towards a,w-dihaloalkanes [172]

Alkanes with More than a Single Halogen Atom – Some strains are able to use

a,w-dichlorinated alkanes for growth, and the activity of the hydrolase from Rhodococcus erythropolis strain Y2 was high for 1,2-dibromoethane, 1,2-di- brompropane, and the a,w-dichloroalkanes (Table 12) [172] whereas the range

of a,w-dichlorinated alkanes that was used for growth of Pseudomonas sp strain

273 was limited to the C9 and C10substrates [238]

Dehalogenase activity was demonstrated in a strain of Acinetobacter GJ70 that could degrade some a,w-dichloroalkanes, and 1-bromo- and 1-iodopro-

pane Although 1,2-dibromoethane could be converted to 2-bromoethanol, thiscould not be used for growth possibly due to the toxicity of bromoacetaldehydeand the unsuitability of dihydroxyethane as growth substrate [92] In a laterstudy the enzyme from this strain showed dehalogenase activity towards a widerange of substrates including halogenated alkanes, alkanols and ethers [95](Table 13)

It seems valuable to provide a brief summary of the degradation of

1,2-dichloroethane by Xanthobacter autotrophicus strain GJ10 which has been used

to delineate all stages of the metabolism (Fig 8) The activity of a haloalkane halogenase initiates degradation and is discussed in this section, while the alka-nol dehydrogenase, the aldehyde dehydrogenase, and the haloacetate dehalo-genase are discussed in subsequent sections The enzyme responsible for

de-dehalogenase activity has been purified from Xanthobacter autotrophicus strain

GJ10, consists of a single polypeptide chain with a molecular mass of 36 kDa, andwas able to dehalogenate chlorinated, and both brominated and iodinated alka-nes [103] Details of the mechanism have been explored using an ingeniousmethod of producing crystal at different stages of the reaction [224] The over-all reaction involves a catalytic triad at the active site: Asp124binds to one of thecarbon atoms, and hydrolysis with inversion is accomplished by cooperation ofAsp260and His289 with a molecule of water bound to Glu56(Fig 9) In a Mycobac- terium sp strain GP1 that belongs to the group of fast-growing mycobacteria, 1,2-

dibromoethane could be used as a source of carbon and energy Although it wasconverted into the epoxide by a haloalkane dehalogenase that could be used forgrowth and thereby circumvent the production of toxic bromoacetaldehyde,degradation of the epoxide was unresolved [156] It is worth noting that, in con-

trast, 1,2-dichloroethane can be used for growth by Xanthobacter autrotrophicus

strain GJ10: the pathway is shown in Fig 8, and the appropriate enzymes have

been demonstrated [93] The second step in the degradation of clohexane by Sphingomonas paucimobilis UT26 is carried out by the hydrolytic

g-hexachlorocy-dehalogenation of 1,3,4,6-tetrachlorocyclohexa-1,4-diene to hexa-2,5-diene-1,4-diol [141] and the enzyme is also able to carry out debromi-nation (Table 14) [142]

2,5-dichlorocyclo-Pseudomonas putida strain G786) that harbors the CAM plasmid

debromi-nated some polybromidebromi-nated ethanes under anaerobic conditions [89], for

exam-ple 1,1,2,2-tetrabromoethane, was reduced to a mixture of cis- and

trans-1,2-dibromoethene that were also formed from 1,2-dichloro-1,2-dibromoethane(Fig 10)

Table 13. Rates of dehalogenation with purified dehalogenase from Acinetobacter sp strain

GJ70 relative to 1-bromopropane (=100) [95]

Fig 8. Degradation of 1,2-dichloroethane by Xanthobacter autotrophicus strain GJ10 (Redrawn

from [94])

Fig 9. Active site of haloalkane dehalogenase with 1,2-dichloroethane as substrate (Redrawn from [224])

Table 14. Selected substrates dehalogenated by the dehalogenase (LinB) from Sphingomonas paucimobilis strain UT26 (chlorobutyrate=100) [142]

Fig 10a, b Metabolism of: a 1,2-dibromo-1,2-dichloroethane; b tetrabromoethane by

Pseudomonas putida G786 (Redrawn from [89])

(a)

(b)

Haloepoxides – Xanthobacter sp strain Py2 is able to grow with propene or

propene oxide and the pathway is dependent on the presence or absence of CO2[189] This strain is also able to degrade 1-fluoro-, 1-chloro-, and 1-bromo-2,3-epoxypropane with decreasing ease (Table 15) [190] The toxicity of the epi-halohydrins occurs in the opposite order, and for 1-chloro-2,3-epoxypropane thefirst detectable metabolite was chloroacetone that is formed by an isomerizationand not by a hydrolytic reaction: details of the further metabolism of chloroace-tone remained unresolved

2.3

Halogenated Ethenes

The degradation of chlorinated ethenes has attracted considerable attention ences in [53]) under aerobic conditions by ammonia monooxygenase, by solublemethane monooxygenase, and by toluene 2-, 3-, and 4-monooxygenases (Table 16)

(refer-The soluble methane monooxygenase from Methylosinus trichosporium OB3b

produced a number of products from trihaloethenes [58]: formate fromtrichloroethene and tribromoethene, and glyoxylate from trifluoroethene, to-gether with the volatile fluoral, chloral, and bromal respectively (Fig 11) The

controlled oxidation of trichloroethene by Methylosinus trichosporium OB3b

pro-duced both trichloroethene epoxide and trichloroacetaldehyde, and the last wastransformed biologically to trichloroethanol and trichloroacetate by a Can-nizarro-like dismutation: the formation of chloroform and formate were abioticreactions [149]

A strain of a methane-oxidizing bacterium was able to degrade ethene when a growth substrate such as methane or methanol was present [113].The reactions proceeded through an intermediate epoxide from which formate,

trichloro-Table 15. Relative time (min) required for degradation of haloepoxides by Xanthobacter sp.

strain Py2 and toxicity (% activity remaining after 2 h incubation with propene) [190]

degradation (min) after 2 h)

Table 16. Toluene monooxygenases and the products from hydroxylation of toluene

CO, and glyoxylic acid were formed, while its spontaneous chemical degradationled to dichloroacetate by chloride migration (Fig 12) [113].

There are several toluene monooxygenases that can oxidize halogenatedethenes Comparably with the methane monooxygenases, the purified toluene 2-

monooxygenase from Burkholderia cepacia strain G4 produced CO, formate, and

glycolate from trichloroethene in an approximate ratio of 4:2:1, but neitherchloroacetic acids nor chloral were formed [150] Trichloroethene and 1,1-dichloroethene were degraded stoichiometrically to chloride by the toluene

monooxygenase in Pseudomonas stutzeri strain OX1 that is able to carry out 2-,

3-, and 4-monooxidation of toluene [32] The degradation of a selection of genated ethenes by the toluene monooxygenases is given in Table 17 [127].Considerable effort has been devoted to the anaerobic dechlorination ofchloroethenes and is discussed elsewhere in this chapter

halo-Fig 11 Products from the activity of methane monooxygenase on: a trifluoroethene; b

tri-bromoethene

Fig 12. Generalized degradation of trihaloethenes by methanotrophs (Redrawn from [113])

Table 17. Degradation of halogenated substrates by toluene-oxidizing strains given as trations remaining µmol/l after 3 h incubation with20 µmol/l substrate The dibromoethenes were used as a 1:1 mixture ofthe isomers with a total concentration of 20 µmol/l [127]

concen-Strain 1,2-Dichloro- Trichloro- Chloroform cis-1,2-Dibro-

Monooxygenases were from the following strains:

T4O: Pseudomonas mendocina strain KR1

T3O: Pseudomonas pickettii strain PKO1

T2O: Burkholderia cepacia strain G4.

Table 18. Substrate specificity of haloalkanol dehalogenase from Arthrobacter sp strain AD2,

and rates relative to that for 1,3-dichloro-propan-2-ol [214]

Table 19. Relative substrate activities (1,3-dichloropropan-2-ol=100) of halohydrin halide lyase

B from Corynebacterium sp strain N-1074 to various substrates [143]

Fig 13. Transformation of 1,3-dibromopropan-2-ol of halohydrin hydrogen-halide lyase in

Corynebacterium strain N-1074 (Redrawn from [145])

2.4

Haloalkanols

The conversion of vicinal haloalkanols to epoxides is of considerable interest and

was apparently first recognized in a strain of Flavobacterium [24] Their tion is stereospecific to produce the trans-epoxide from erythro 3-bromobutan- 2-ol and the cis- from threo 3-bromobutan-2-ol, and the epihalohydrins are able

forma-to react with halide forma-to produce 2-hydroxy-1,3-dihalobutanes [8] The enzyme has

been purified from Arthrobacter strain AD2 that is able to utilize

3-chloro-1,2-propandiol for growth, has a molecular mass of 29 Da, and consists of two equalsubunits [214] It is able to form epoxides from vicinal chlorinated and bromi-nated alkanols (Table 18) and carry out transhalogenation between epihalohy-drins and halide ions By contrast, the halohydrin hydrogen-halide lyase B from

Corynebacterium sp strain N-1074 is able to hydrolyze some brominated

2-halo-gen-substituted alkanols (Table 19) [143] as well as carrying out the

transfor-mation of 1,3-dichloro-propan-2-ol to the epichlorohydrin enriched in the

R-iso-mer (Fig 13) Haloalkanol dehalogenase activity has also been found in a number

of different bacteria (Table 20) [188]

The complete degradation of 1,2-dichloroethane by Xanthobacter icus strain GJ10 requires the synthesis of 2-chloroethanol dehydrogenase This

autotroph-has been purified and is a quinoprotein showing the typical absorption at 345 nmand a shoulder at 410 nm, has a maximal activity at pH 9–9.4 and a temperature

of 40 °C [93]

Haloaldehydes

In the degradation of haloalkanes, the initially formed aldehyde is genated to the carboxylic acid by an NAD-dependent dehydrogenase that was iso-lated from a 1,2-dichloroethane-degrading bacterium strain DE2 [198]

dehydro-2.6

Haloalkanoates

Halogenated alkanoic acids are produced from the aldehydes during the dation of 1,2-dihaloethanes The degradation of chlorinated alkanoic acids hasbeen extensively investigated and the dehalogenases have been grouped accord-ing to their reactions with 2-chloropropionate to carry out hydrolysis with orwithout inversion and the effect of sulfhydryl-inhibitors [55] The enzymology

degra-of 2-haloalkanoate dehalogenases is discussed in detail in [188]

By contrast, the brominated analogues have not been so systematically

exam-ined and only a summary account can be given Organisms able to utilize

n-un-decane were able to debrominate 6-bromohexanoate but not 2-bromohexanoate[154] Halohydrolases have been isolated from a number of bacteria and dis-played activity against 2-haloacetates and, for some, 2-chloropropionate:

1 The halohydrolase H-2 from Delftia acidovorans (Moraxella sp strain B)

pro-duced glycollate from 2-chloro-, 2-bromo-, and 2-iodoacetate [102]

2 Xanthobacter autotrophicus GJ 10 is able to grow at the expense of short-chain

halogenated hydrocarbons and carboxylic acids The strain produces two stitutive dehalogenases that differ in their heat stability, in their pH optima,and in their substrate specificity (Table 21) [91] It was proposed that the me-tabolism of 1,2-dichloroethane is proceeded by hydrolytic dehalogenation, de-hydrogenation, and dehalogenation of the resulting chloroacetate to glycolate.The specific activities of these enzymes in this strain are given in Table 22 [93]

con-3 The haloacid dehalogenase from a strain of Xanthobacter autotrophicus GJ

that was able to utilize 1,2-dichloroethane using a haloalkane hydrolase, ahaloalkanol dehydrogenase, an aldehyde dehydrogenase, and lastly a haloaciddehydrogenase has been purified and characterized It was able to dehalo-genate a restricted range of halogenated substrates (Table 23), and produced

Table 20. Activity of haloalcohol dehalogenases relative to 1,3-dichloropropanol for selected strains [188]

NA: not available. C = Corynebacterium sp strain N-1074: dehalogenase Ia

A = Pseudomonas sp strain AD1 D = Corynebacterium sp strain N-1074: dehalogenase Ib

B = Arthrobacter sp strain AD2 E = Arthrobacter erithii strain H10a.

lactate from L-2-chloropropionate but not from the D-isomer [217].A strain of

Pseudomonas sp strain YL is also able to catalyze hydrolysis not only of

2-haloalkanoic acids but also of long-chain halogenated substrates (Table 24)[114] In both cases, inversion takes place at the halogenated carbon atom Two2-haloacid dehalogenases have been isolated from this strain [114] differing

in their thermostability and their stereospecificities One of them (L-DEX) incells grown at the expense of 2-chloropropionate is thermostable, is inducible

by 2-chloropropionate, and catalyzed the dehalogenation of both D- and Lchloropropionate with inversion and production ofD- and L-lactates The other(L-DEX) is produced in cells grown with 2-chloroacrylic acid, is not thermallystable, and acts on L-2-haloalkanoic acids to produce the D-hydroxyalkanoate.The substrate specificities of the two enzymes are given (Table 25) These re-

-2-sults may be compared with the constitutive dehalogenases from ter autotrophicus GJ 10, one of which is thermally stable and dehalogenates

Xanthobac-Table 23. Relative rates of dehalogenation (chloroacetate=100) for Pseudomonas strain CBS 3 [140] and Xanthobacter autotrophicus strain GJ10 M50 [217]

Table 21. Specificity of dehalogenation in extracts of Xanthobacter autotrophicus strain GJ10,

effect of pH and % activity after heat treatment [91]

Table 22. Specific activities of enzymes involved in the degradation of 1,2-dichloroethane by

Xanthobacter autotrophicus strain GJ10 [93]

1,2-Dichloroethane 2-Chloroethanol Chloroacetaldehyde Chloroacetate

Table 24. Activities of dehalogenase L-DEX from Pseudomonas sp strain YL for long-chain

halogenated alkanoates ( L -2-chloropropionate=100)[114]

broadly similar except for access to the active site [166] The structure of the

haloacid dehalogenase from Xanthobacter autotrophicus GJ is quite different

from that of the haloalkane hydrolase from the same strain and the catalytictriad is missing: Asp124is on another strand and the pair His289and Asp260have

no counterpart [166] The following catalytic mechanism was proposed: thesubstrate is bound to Ser114via its carboxylate group, Asp8is available to form

an ester intermediate with fission of the C-Cl bond, and there is an unresolvedsite for an activated water molecule analogous to the His289Asp260residues in

the haloalkane dehalogenase In the Pseudomonas sp strain YL structure,Asp10

forms the ester with the substrate and Arg41is the acceptor of the chloride anion (Fig 14) [111]

4 A dehalogenase (II) found when a cosmid gene bank of Pseudomonas strain CB3 constructed in Escherichia coli Hb 101 was screened, was purified and as-

sayed for activity to a number of substrates (Table 23) and removed chloride

from (R)-2-chloropropionic acid with inversion of configuration [140].

5 An apparently unique enzyme has been found in Pseudomonas sp strain 113

that is able to hydrolyze both D- and L-2-chloropropionate to the yalkanoic acids with inversion of configuration of both substrates (references

hydrox-in [144]), and it has been proposed [144] that the enzyme has a shydrox-ingle commoncatalytic site for both enantiomers

6 The purple non-sulfur anaerobic phototrophic bacteria Rhodospirillum rubrum, Rh photometricum, and Rhodopseudomonas palustris DSM 123 were

able to grow at the expense of chloroacetate, 2-chloropropionate, pionate, and 2-bromopropionate by dehalogenation to acetate or propionate:low concentrations of bromoacetate were used only by the last strain [129].

3-chloropro-It emerged that one limitation to utilization of haloalkanoates was the toxicity ofthe substrates This was shown for bromoacetate that inhibited growth of

Pseudomonas putida strain PP3 [233] although a mutant of Xanthobacter totrophicus GJ10 overexpressed the bromoacid dehalogenase and was able to

au-grow with substrate concentrations between 10 mmol/l and 25 mmol/l [218] A

strain of Pseudomonas cepacia MBA4 was able to utilize bromoacetate as sole

source of carbon and energy, and the inducible dehalogenase (designated IVa)was isolated and purified [206] Its activity is given in Table 26 It was subse-quently shown that although this dehalogenase (DehIVa) had a 68% identity to

the dehalogenase (DehCI) from Pseudomonas sp strain CB3 the former is

dimeric and the latter monomeric [205]

2.7

Halogenated Ethers

Two strains of an Ancylobacter aquaticus AD25 and AD27 were isolated by

en-richment using 2-chloroethylvinyl ether and the dehalogenase displayed activitytowards a range of halogenated alkanes (Table 27) [212] and the degradation of

Fig 14. Reaction intermediates of L -2-haloacid dehalogenation (Redrawn from [112])

some halogenated ethers has been described (Table 28) [213] The C-O-C bond

is not fissioned enzymatically in the substrate and chemical hydrolysis is involved

in two possible pathways (Fig 15) For the sake of completeness attention isdrawn to investigations on the biodegradation of vinyl chloride: in both, elegantuse was made of13C NMR of whole cell suspensions One used Methylosinus trichosporium OB3b (Fig 16) [29] and the other a strain of Pseudomonas

sp.(Fig 16) [30]: whereas the first involved chloroethene epoxide, the second wascarried out by direct hydrolytic fission of the C-Cl bond

2.8

Reductive Loss of Halogen

In contrast to all of the above reactions that are not dependent on the presence

of oxygen, reductive loss of bromine has been demonstrated in anaerobic

bacte-ria that depend on its absence The reduction of tetrachloroethene to

cis-1,2-Table 26. Dehalogenase specific activities (µmol substrate converted/mg protein/min) of

Pseudomonas cepacia strain MBA4 under different growth conditions [206]

Growth substrate Assay substrate

Table 27. Relative activities (%) of haloalkane dehalogenase from Ancylobacter aquaticus [212]

Table 28. Specific activity (n mol halide/min/mg protein) of dehalogenase inextracts of strains

AD25 and AD27 of Ancylobacter aquaticus towards halogenated ethers [213]

2-Chloroethylvinyl ether 2-Hydroxyethylmethyl ether 323 46

ND = not determined.

dichloroethene by the enteric organism Enterobacter (Pantoea) agglomerans may

be noted [185] as one of the few examples of the metabolic role of riaceae.

Enterobacte-Dehalorespiration in which dehalogenation is coupled to the synthesis of ATP

has been demonstrated for halogenated ethenes in Dehalospirillum multivorans, Dehalobacter restrictus, Desulfuromonas chloroethenica, and strains of Desulfi- tobacterium sp (references in [84]), and in Dehalococcoides ethenogenes that is

capable of reductively dehalogenating tetrachloroethene to ethene [121, 125] The

Fig 15. Degradation of 2-chloroethylvinyl ether by Ancylobacter aquaticus (Redrawn from

[213])

Fig 16a,b. Degradation of vinyl chloride by: a Pseudomonas sp (Redrawn from [30]); b

Me-thylosinus trichosporium OB-3b (Redrawn from [29])

electron donors were generally H2or pyruvate, or for Dehalococcoides genes methanol The chloroethene reductases contain cobalamine and Fe-S clus-

ethene-ters, and the range of brominated substrates for trichloroethene reductase from

Dehalococcoides ethenogenes is given in Table 29 [121] In addition, the TCE

re-ductive dehalogenase from this organism is able to debrominate substrates taining C2, C3, and C4or C5carbon atoms, albeit in decreasing ease: an illustra-tive reaction is the debromination of tribromoethene to dibromoethenes, vinylbromide, and ethene Further examples include the debromination methanogenicbacteria of 1,2-dibromoethane to ethene and 1,2-dibromoethene to ethyne by(Fig 17) [15] Details of these dehalogenations have emerged from studies withmethanogens The formation of ethene from 1,2-dichloroethane with hydrogen

con-as electron donor hcon-as been demonstrated in cell extracts of Methanobacterium thermoautotrophicum DH, and in Methanosarcina barkeri this reaction has been

shown to involve cobalamin and F430using Ti(III) as reductant [85] Further ments are given in the section on abiotic reactions

com-It is also worth pointing out that the aerobic Pseudomonas putida G786

car-rying the cytochrome P450CAMplasmid is also able to carry out selective tive debromination [110] The reductive debromination of BrCCl3with formation

reduc-of HCCl3by cytochrome P450CAMhas been shown [28] and examined with agreater range of substrates (Table 30) [110]

Reductive dehalogenation is one of the series of reactions involved in the

degradation of g-hexachlorocyclohexane (aaaeee) [141]: (i) initial elimination

Table 29. Substrate range and products of trichloroethene reductase for selected brominated substrates [121]

Fig 17. Reductive debromination by methanogenic bacteria (Redrawn from [15])

Table 30. Products from reductive debromination of halogenated alkanes with cytochrome P450 CAM [110]

catalyzed by LinA, (ii) hydrolysis by LinB, and (iii) reductive loss of chloride from2,5-dichlorohydroquinone catalyzed by glutathione (Fig 18) The last reactionalso occurs during the degradation of pentachlorophenol [246].

Both the active enzyme, the heat-inactivated enzyme from Dehalospirillum multivorans, and cyanocobalamin are capable of dehalogenating haloacetates

(Table 31) [147] and the rate of abiotic dehalogenation depends on the catalystthat is used (Table 32)

2.9

Brominated and Iodinated Alkanes and Related Compounds as Metabolic Inhibitors

Most of the reactions of inhibitors depend on the chemical reactivity of nated and iodinated alkanes and related compounds, and are consistent bothwith the chemical reactivity of these compounds and their biological reactivitythat has been exemplified in hydrolytic or reductive reactions:

bromi-1 Methyl iodide and propyl iodide bind to corrinoid coenzymes and thereby hibit their activity The reaction is reversible in the presence of tungsten-fila-ment white light, and these reagents have been used to establish the require-ment for corrins in biological reactions, for example, (i) the synthesis ofacetate from CO2by Clostridium thermoaceticum [64], (ii) the biosynthesis of

in-Fig 18. Dehalogenation of hexachlorocyclohexane by Sphingomonas paucimobilis strain UT 26

and reactions catalyzed by LinA, LinB, LinC and LinD (Redrawn from [141])

Table 31. Rate (sec –1) of dehalogenation by native enzyme from Dehalospirillum multivorans,

heat-inactivated enzyme and cyanocobalamin [147]

Native enzyme Inactivated enzyme

Table 32. Rates (sec –1 ) of dechlorination of trichloroacetate with various catalysts and reduced methylviologen as electron donor [147]

nation of PCBs [249], and alters the community structure of an anaerobic

trichloroethene-degrading consortium [35] It is worth noting that gens may reduce this to ethene [15]

methano-3 Iodoacetate is a well-established inhibitor of the activity of enzymes ing sulfhydryl groups

contain-3

Abiotic Reactions

It must be admitted at the beginning that very few of these reactions involve prehensive examination of brominated compounds The justification for the in-clusion of chlorinated compounds lies in the putative similarity of reactions in-volving their brominated analogues and the expectation that fission of the C-Brbond will be more easily accomplished than that of the C-Cl bond The literature

com-on chlorinated compounds is enormous so that com-only some illustrative examplesrelevant to brominated compounds are given

One recurring issue is best addressed at the beginning Considerable activityhas been given to putative chemical reactions in which, for example, porphyrins

or corrins or related compounds are invoked Enzyme mediated reactions will most always involve these catalysts, so that the distinction between enzymaticand non-enzymatic reactions seems blurred

al-3.1

Photohydrolytic Reactions

The chemical hydrolysis of halogenated alkanes is normally slow but is considerablyaccelerated (≈105) in the presence of light For example, dibromoethane is hy-drolyzed to 2-bromoethanol that is then converted to the epoxide followed by hydrolysis to dihydroxyethane [25] The corresponding reaction with 1,2-dibromo-3-chloropropane is more complex and produces a number of products although thetransformation of 1-chloro-2,3-dihydroxypropane is very much slower (Fig.19) [31]

Reductive Reactions

These have been extensively studied partly with a view to simulating biologicalreactions and their possible role in degrading chlorinated ethanes and ethenesthat are used as degreasing solvents It is, however, worth noting that althoughthis use has diminished and these are sufficiently valuable to justify recovery:

1 In view of concern with the fate of halogenated acetic acids in water ution systems, their reactions been studied in deoxygenated water containingfinely divided Fe0 Successive reductive removal of halogen occurred with for-mation of chloroacetate from trichloroacetate and acetate from tribromoac-etate [86]

distrib-2 Cr(II) has been used to bring about dehalogenation of alkyl halides and results

in the production of alkyl radicals The order of ease of reduction is generallyiodides > bromides > chlorides and tertiary halides are most reactive and pri-mary halides least (Table 33) [26, 27] Rates for selected vicinal halides are given (Table 34) (references in [23]) Vicinal dichlorides such as tetra-

chloroethene are degraded by zero-valent metals by b-elimination to produce

dichloroacetylene and finally acetylene [167]

Fig 19. Photohydrolysis of 1,2-dibromo-3-chloropropane (Redrawn from [31])

Table 33. Products and yields (%) from the reduction of alkyl halides by chromous sulfate in dimethylformamide [26]

3 The stereospecificity of dehalogenation of vicinal dibromides to olefins wasexamined for reducing agents including Cr(II), iodide and Fe0[204].Whereas,

for dibromostilbene the (E)-stilbene represented >70% of the total olefin duced, for threo-dibromopentane reduction by Cr(II) produced ca 70% of (E)-

pro-pent-2-ene, and iodide and Fe0only <5% of this

4 The reactions of alkyl halides with Fe(II) deuteroporphyrin IX have been amined and some examples are given in Table 35 [227] Three classes of reac-tion were observed: (i) hydrogenolysis, (ii) elimination to alkenes, and (iii)coupling of alkyl free radicals, and further discussion is given in [23]

ex-5 In the context of model systems for Factor F-430 that is involved in the minal step in the biosynthesis of methane and that is able to dechlorinate CCl4successively to CHCl3and CH2Cl2[107], nickel (I) isobacteriochlorin anionwas generated electrolytically and used to examine the reactions with alkylhalides in dimethylformamide [79] The three classes of reaction were thesame as those observed with Fe(II) deuteroporphyrin IX.A selection of the re-actants and products is given in Table 36

ter-6 Considerable attention has been directed to dehalogenation mediated by rinoids and porphyrins in the presence of a chemical reductant (references in[62, 67, 240]) The involvement of corrinoids and porphyrins is consistent with

cor-Table 35. Rates (M –1 sec –1 ) of reaction of alkyl halides with Fe(II) deuteroporphyrin IX [227]

Table 36. Reaction of alkyl halides with Ni(I) octaethylisobacteriochlorin anion in dimethyl formamide [79]

butane

butane

butane

the occurrence of analogous mechanisms for biological reactions Illustrationsare provided by the dechlorination and elimination reactions carried out bytitanium(III) citrate and hydroxocobalamin [19, 67] For example, in aqueoussolution cobalamin with titanium(III) as reductant dehalogenated tetra-chloroethene to the trichloroethene radical and thence to the dichlorovinylradical, chloroacetylene and acetylene [67].

7 Although not involving brominated compounds, the dechlorination of chloroethane to tetrachloroethene by hydrogen sulfide in the presence of 5-methylbenzo[1,4]quinone electron acceptor should be noted [155], and hasbeen confirmed in the sulfide-containing anoxic hypolimnion from a lake[132]

hexa-8 The dehalogenation of haloacetates by heat-killed cells of Dehalospirillum multivorans and cyanocobalamin has already been noted above.

in biota in Scandinavia, Japan, the United States and the Eastern North Atlantic[122], and in samples of air from the Great Lakes [195] In addition a number of

agrochemicals contain bromine including 5-bromo-3-sec-butyl-6-methyl uracil

(bromacil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), and some lurea herbicides (e.g., metobromuron)

pheny-4.1

Hydrocarbons

The mechanisms used for the metabolism of halogenated aromatic compoundsdiffer considerably from those for aliphatic substrates, and will be briefly sum-marized

4.1.1

Degradation and Growth

Bacterial degradation of hydrocarbons under aerobic conditions involves twoconsecutive initial reactions before fission of the aromatic ring: dioxygenationwith the introduction of both atoms of O2to produce a cis-dihydrodiol, and de-

hydrogenation to a catechol This is followed by ring fission by either of two ways The mechanisms have been extensively explored with both halogenated