In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH 3 Cob(III)] to 2-mercaptoethanesulfonic acid ( Biomethylation and hydride generation of group 15 and 16 metals and metalloids (As, Se, Sb, Te, and Bi) by microorganisms are widespread phenomena in anaerobic habitats including landfills, sewage sludge fermentation, alluvial soils, and, as recently shown, the gut of mice and humans (5-6, 20, 22-23, 29). While these processes have a drastic effect on metal(loid) mobility and toxicity, little is known about the pathways involved in the biological formation of methyl and hydride metal(loid) species. For the methylation of arsenic, a metal(loid)-specific methyltransferase has been identified (2,24,28,33). For instance, genes encoding arsenite methyltransferases such as ArsM, which catalyzes the stepwise methylation of arsenic in S-adenosyl methionine (SAM)-dependent reactions, are found in numerous prokaryotic and eukaryotic genomes. As ArsM confers resistance against increased arsenic concentrations and is expressed in response to elevated arsenic concentrations, arsenic methylation by ArsM is considered a deliberate detoxification mechanism (24). Furthermore, methylcobalamin [CH 3 Cob(III)]-dependent methylation of As, Se, Sb, Te, Hg, and Bi has been reported for numerous anaerobic prokaryotes (4,19,21). In particular, autotrophic sulfate-reducing bacteria as well as methanoarchaea were suggested to be responsible for this process, as CH 3 Cob(III) and CH 3 Cob(III)-dependent enzymes are integral parts of physiological pathways such as carbon fixation via the reductive acetyl-coenzyme A (CoA) pathway and methanogenesis. Hence, these organisms contain high concentrations of corrinoids (17). However, it is unclear whether the different metal(loid)s are methylated by the same pathways and whether metal(loid) methylation is a deliberate enzymatic process, as in the case of ArsM, or whether it arises from side reactions of the basal physiological pathways. Interestingly, nonenzymatic methylation of some metal(loid)s, like As and Hg, by CH 3 Cob(III) under reductive conditions was assumed by some authors (32,34).To identify metal(loid) methylation pathways which could increase understanding of the multielement biomethylation observed in anaerobic habitats, we focused on methanoarchaea. Almost all methanoarchaea studied are capable of methylating a bro...

No abstract

a Metal(loid)s are subject to many transformation processes in the environment, such as oxidation, reduction, methylation and hydride generation, predominantly accomplished by prokaryotes. Since these widespread processes affect the bioavailability and toxicity of metal(loid)s to a large extent, the investigation of their formation is of high relevance. Methanogenic Archaea are capable of methylating and hydrogenating Group 15 and 16 metal(loid)s arsenic, selenium, antimony, tellurium, and bismuth due to side reactions between central methanogenic cofactors, methylcobalamin (CH 3 Cob(III)) and cob(I)alamin (Cob(I)). Here, we present systematic mechanistic studies on methylation and hydride generation of Group 15 and 16 metal(loid)s by CH 3 Cob(III) and Cob(I). Pentavalent arsenical species showed neither methylation nor reduction as determined by using a newly developed oxidation state specific hydride generation technique, which allows direct determination of tri-and pentavalent arsenic species in a single batch. In contrast, efficient methylation of trivalent species without a change in oxidation state indicated that the methyl transfer does not proceed via a Challenger-like oxidative methylation, but via a non-oxidative methylation. Our findings also point towards a similar mechanism for antimony, bismuth, selenium, and tellurium. Overall, we suggest that the transfer of a methyl group does not involve a free reactive species, such as a radical, but instead is transferred either in a concerted nucleophilic substitution or in a caged radical mechanism. For hydride generation, we propose the intermediate formation of hydridocobalamin, transferring a hydride ion to the metal(loid)s.

Methylated metal(loid) compounds are formed in the environment by abiotic as well as enzymatically catalyzed transfer of a methyl group. Due to the increased mobility and toxicity in comparison to the inorganic precursors, the investigation of the formation process is of high relevance. Though the natural abundance carbon isotope ratio can give important insights toward their origin as well as the biochemical methyl transfer process, so far, these species have not been investigated by carbon isotope ratio mass spectrometry (IRMS). This is due to the analytical challenge to precisely determine the natural isotope distribution of trace amounts of metal(loid)-bound carbon in complex organic matrixes. To overcome this problem, we tested the concept of selective derivatization of nonvolatile organometal(loid)s by hydride generation (HG) followed by purge and trap (P-T) enrichment, heart-cut gas chromatography (hcGC), and subsequent analysis by GC/IRMS. Parameter optimization of HG/P-T/hcGC was conducted using online coupling to element-sensitive ICPMS (inductively coupled plasma mass spectrometry) detection. The purity of the HG/P-T/hcGC fraction was verified by GC/MS. For the model substance trimethylarsine oxide (TMAsO), an excellent agreement of the delta(13)C-value analyzed by HG/P-T/hcGC-GC/IRMS was achieved in comparison to the bulk delta(13)C-value, which shows that no significant isotope fractionation occurred during hydride generation and subsequent separation. The optimized method showed good reproducibility and a satisfying absolute detection limit of 4.5 microg TMAsO (1.2 microg(carbon)). This method was applied to the analysis of TMAsO in compost. The low delta(13)C value of this compound (-48.38 +/- 0.41 per thousand) indicates that biomethylation leads to significant carbon fractionation. HG/P-T/hcGC-GC/IRMS is a promising tool for investigation of the biomethylation process in the environment.

In the environment, the methylation of metal(loid)s is a widespread phenomenon, which enhances both biomobility as well as mostly the toxicity of the precursory metal(loid)s. Different reaction mechanisms have been proposed for arsenic, but not really proven yet. Here, carbon isotope analysis can foster our understanding of these processes, as the extent of the isotopic fractionation allows to differentiate between different types of reaction, such as concerted (SN2) or stepwise nucleophilic substitution (SN1) as well as to determine the origin of the methyl group. However, for the determination of the kinetic isotope effect the initial isotopic value of the transferred methyl group has to be determined. To that end, we used hydroiodic acid for abstraction of the methyl group from methylcobalamin (CH3Cob) or S-adenosyl methionine (SAM) and subsequent analysis of the formed methyl iodide by gas chromatography (GC) isotope ratio mass spectrometry (IRMS). In addition, three further independent methods have been investigated to determine the position-specific δ (13)C value of CH3Cob involving photolytic cleavage with different additives or thermolytic cleavage of the methyl-cobalt bonding and subsequent measurement of the formed methane by GC-IRMS. The thermolytic cleavage gave comparable results as the abstraction using HI. In contrast, photolysis led to an isotopic fractionation of about 7 to 9 ‰. Furthermore, we extended a recently developed method for the determination of carbon isotope ratios of organometal(loid)s in complex matrices using hydride generation for volatilization and matrix separation before heart-cut GC and IRMS to the analysis of the low boiling partly methylated arsenicals, which are formed in the course of arsenic methylation. Finally, we demonstrated the applicability of this methodology by investigation of carbon fractionation due to the methyl transfer from CH3Cob to arsenic induced by glutathione.