The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron-and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments. , methylmercury, MeHg) is a potent neurotoxic compound (1). It is biomagnified in the food webs of aquatic systems, reaching high concentrations in carnivorous fish, thus posing a risk to human health (2). Understanding the mechanism of inorganic Hg methylation and the parameters that control the extent of methylation in the environment is thus essential for relating patterns of Hg pollution to human exposure. The production of MeHg has been linked to obligate anaerobic bacteria in the δ-Proteobacteria, including ironand sulfate-reducing bacteria (FeRB and SRB) that live in soil and sediments (3-6). Although mechanisms of Hg(II) methylation by methylating enzymes have been proposed for some time (7,8), the mechanism of Hg(II) uptake by the bacteria has remained obscure. The dominant view is that cellular uptake occurs by passive diffusion of neutral Hg(II) complexes, particularly sulfide complexes, through external membranes, leading to accidental methylation of some of the intracellular Hg(II) (9). However, this view is based on indirect data and modeling, as the precipitation of metal sulfides in the medium and the extensive Hg binding to the surface of the organisms (10-12) have made it difficult to directly measure Hg(II) uptake in methylating bacteria.In previous work (13), we demonstrated that the cysteine complex of Hg(II) was available to the FeRB Geobacter sulfurreducens PCA and that Hg(II) was likely transported into the cell via an unknown facilitated transport mechanism. Here we examine the energy dependence and specificity of Hg(II) uptake and methylation by both G. sulfurreducens and ...

2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum ATCC 39723 is a member of a class of Fe(II)-containing hydroquinone dioxygenases that is involved in the mineralization of the pollutant pentachlorophenol. This enzyme has not been extensively characterized, despite its interesting ring-cleaving activity and use of Fe(II), which are reminiscent of the well-known extradiol catechol dioxygenases. On the basis of limited sequence homology to the extradiol catechol dioxygenases, the residues ligating the Fe(II) center were originally proposed to be H159, H227, and E276 (Xu et al. in Biochemistry 38:7659-7669, 1999). However, PcpA has higher sequence homology to a newly reported, crystallographically characterized zinc metalloenzyme that has a similar predicted fold. We generated a homology model of the structure of PcpA based upon the structure of this zinc metalloenzyme. The homology model predicts that the tertiary structure of PcpA differs significantly from that of the extradiol dioxygenases, and that the residues ligating the Fe(II) are H11, H227, and E276. This structural model was tested by mutating each of H11, H159, H227, and E276 to alanine. An additional residue that is predicted to lie near the active site and is conserved among PcpA, its closest homologues, and the extradiol dioxygenases, Y266, was mutated to phenylalanine. Of these mutants, only H159A retained significant activity, thus confirming the active-site location predicted by the homology-based structural model. The model provides an important basis for understanding the origin of the unique function of PcpA.

The new ligand cis,cis-1,3,5-tris-(E)-(tolylideneimino)cyclohexane (TACH-o-tolyl) forms a 1:1 complex with iron(II). Addition of substituted phenolates forms 1:1:1 ligand:iron:phenolate complexes, which have been characterized both in the solid state and in solution. There is complete binding of the phenolate to the complex only when there are ortho-halogens on the phenolate. The tertiary complexes with ortho-halo-substituted phenolates exhibit short Fe-halogen distances, and the complex containing a non-coordinating but similarly sized ortho-methyl phenolate has a significantly different conformation and coordination geometry. Therefore, it is likely that the metal-halogen interaction stabilizes the complexes. The iron(II)-halogen interaction in these complexes may explain the substrate specificity of PcpA and LinE, enzymes that preferentially bind phenols and hydroquinones containing halogen substituents in ortho positions.

In the undergraduate curriculum, the focus of NMR spectroscopy is predominately on molecules containing 1H and 13C as the sole NMR-active nuclei. Teaching students about the impact of NMR-active heteronuclei such as 19F or 31P can be challenging to fit in the undergraduate curriculum, especially if NMR instrumentation and/or a range of compounds are not available. We present an activity to be conducted in class that exposes undergraduates to how heteronuclear NMR spectroscopy can be used to aid in structure elucidation when NMR-active heteroatoms are present. The purpose of the activity is both to expand student understanding of NMR applications as well as further their understanding of the underlying principles of NMR. The activity utilizes a comparative analysis of 1H and 13C NMR spectra of a compound containing no NMR-active heteroatoms with a structurally analogous compound containing I > 0 heteroatoms. Students are tasked with predicting the impact of the heteroatom on the NMR spectrum, followed by the assignment of signals in the actual NMR spectra.

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