Kohlenstoff-, Chlor- und Wasserstoffisotopeneffekte, um Dehalogenierungsmechanismen in Abbaureaktionen chlorierter Ethene zu identifizieren
Zusammenfassung der Projektergebnisse
Because of their widespread use as industrial solvents, chlorinated alkanes and ethenes are among the most notorious groundwater contaminants. Biodegradation under reducing conditions often leads to the accumulation of even more toxic products such as cis-dichloroethene (cis-DCE) and vinyl chloride (VC) from tetrachloroethene (PCE) or trichloroethene (TCE). Despite its relevance for bioremediation of contaminated sites, knowledge about underlying biochemical degradation mechanisms has been incomplete. Product formation has been little understood, and any insight has been difficult to transfer from lab to field. Here, with 13C/12C and 37Cl/35Cl isotope effect measurements in bacteria and model reactants, the project has broken ground in revealing underlying (bio)chemical mechanisms in bacterial chlorinated ethene degradation. First, the importance of outer-sphere reductive single electron transfer (OS-SET) – hitherto the equivalent of a consensus mechanism for polychlorinated ethene biodegradation – could be toned down. Non-existent chlorine isotope effects of chlorinated ethenes with all aqueous OS-SET model reactants contrasted with pronounced Cl isotope fractionation in all natural reductive dehalogenations reported to date suggesting that OS-SET is an exception rather than the rule in environmental transformations of chlorinated ethenes. Second, reductive dehalogenation of chloroethenes by reduced Vitamin B12 – the structural element that lies at the heart of bacterial reductive dehalogenases – was shown to involve cobalamin chloroethyl carbanions as crossroads of competing pathways. Nucleophilic substitution and nucleophilic addition of Vitamin B12 – previously considered distinct pathways – were found to share addition of cobalamin as same initial step, based on evidence from isotope effects, kinetics, product formation and high-resolution mass spectrometry of intermediate Co-complexes. Third, the same pattern of these contrasting, pathway-dependent isotope effects was discovered also in bacterial chlorinated ethene biodegradation. This observation provides surprising evidence of different mechanistic motifs in reductive dehalogenases despite cobalamin as common cofactor. The putative lack of suitable specific reductive dehalogenases may, therefore, provide a potential explanation to the long-standing question of why bioremediation frequently stalls at cis-DCE. Fourth, we established an analytical method that allows also for chlorine isotope analysis of selected chlorinated alkanes. With the resultant approach of 13C/12C and 37Cl/35Cl isotope effects we discovered that dehalogenases catalysing different underlying biochemical transformation mechanisms are at work also in bacterial reductive dehalogenation of chloroform. Fifth, experiments with reduced Vitamin B12 revealed that one of these mechanisms involved a second-order nucleophilic substitution (SN2) of Cl by cob(I)alamin to form chloroalkyl cobalamin intermediates. Evidence was, again, provided by observed products, radical trap experiments, UV-Vis, and high resolution mass spectra. Together with our contributions from project phase 1 our work has, therefore, played a critical role in (i) advancing the use of isotope effects to transfer insight from model reactants to organisms, (ii) highlighting the role of cob(I)alamin as nucleophile in reductive dehalogenation, (iii) revealing an unexpected mechanistic diversity in bacterial reductive dehalogenases (iv) to fundamentally change our view on underlying mechanisms in bacterial chloroethene degradation.
Projektbezogene Publikationen (Auswahl)
- Compound-Specific Chlorine Isotope Analysis of Tetrachloromethane and Trichloromethane by Gas Chromatography-Isotope Ratio Mass Spectrometry vs Gas Chromatography-Quadrupole Mass Spectrometry: Method Development and Evaluation of Precision and Trueness, Analytical Chemistry, 89 (2017), pp 3411–3420
B. Heckel, D. Rodríguez-Fernández, C. Torrentó, A. Meyer, J. Palau, C. Domènech, M. Rosell, A. Soler, D. Hunkeler, M. Elsner
(Siehe online unter https://doi.org/10.1021/acs.analchem.6b04129) - Reductive Outer-Sphere Single Electron Transfer Is an Exception Rather than the Rule in Natural and Engineered Chlorinated Ethene Dehalogenation, Environ. Sci. Technol. 51 (2017), pp. 9663−9673
B. Heckel, S. Cretnik, S. Kliegman, O. Shouakar-Stash, K. McNeill, M. Elsner
(Siehe online unter https://doi.org/10.1021/acs.est.7b01447) - Chlorinated Ethene Reactivity with Vitamin B12 Is Governed by Cobalamin Chloroethylcarbanions as Crossroads of Competing Pathways, ACS Catalysis 8 (2018) pp. 3054−3066
B. Heckel, K. McNeill, M. Elsner
(Siehe online unter https://doi.org/10.1021/acscatal.7b02945) - Mechanistic dichotomy in bacterial trichloroethene dechlorination revealed by carbon and chlorine isotope effects, Environ. Sci. Technol. 53 (2019) pp. 4245−4254
C. Lihl, L.M. Douglas, S. Franke, A. Pérez-de-Mora, A.H. Meyer, M. Daubmeier, E.A. Edwards, I. Nijenhuis, B. Sherwood Lollar, M. Elsner
(Siehe online unter https://doi.org/10.1021/acs.est.8b06643) - Reductive dehalogenation of trichloromethane by two different Dehalobacter restrictus strains reveal opposing dual element isotope effects, Environ. Sci. Technol. 53 (2019) pp. 2332−2343
B. Heckel, E. Phillips, E. Edwards, B. Sherwood Lollar, M. Elsner, M.M. Manefield, M. Lee
(Siehe online unter https://doi.org/10.1021/acs.est.8b03717)
