Biochemical Mechanism of Mercury Methylation Our goal is to determine the biochemical mechanism of mercury (Hg) methylation, an important contaminant transformation that occurs in hypoxic subsurface environments. We will characterize HgcA and HgcB, the two proteins shown to be required for Hg methylation by anaerobic microorganisms. In collaboration with the Oak Ridge National Laboratory (ORNL) Hg Science Focus Area (SFA) program, we have produced HgcB containing high levels of its [4Fe- 4S] cofactor and a soluble cobalamin (Cbl) binding domain of HgcA. Recent work has led to the following working hypotheses: (a) the two iron-sulfur clusters in HgcB receive electrons from a low-potential oxidoreductase (pyruvate ferredoxin oxidoreductase, hydrogenase, CO dehydrogenase, etc.); (b) HgcB transfers these reducing equivalents to the Cbl cofactor of HgcA, converting it from Co(III) to the supernucleophilic Co(I) state; (c) Cys73 and the C- terminal vicinal cysteine residues (Cys95 and Cys96 in Desulfovibrio desulfuricans ND132) of HgcB bind Hg; and (d) HgcA catalyzes the methyltetrahydrofolate (CH3-H4folate)-dependent methylation of the Co(I)-Cbl to generate methyl-Co(III) followed by transfer of the methyl group of methyl-Co(III) to Hg(II) producing MeHg. Our two experimental objectives are to: (1) characterize the structure and function of HgcB and examine its roles as a Hg carrier and redox partner to HgcA and PFOR and (2) characterize the interactions and the methylation reactions involving HcgB and HgcA. Our experiments will use a wide variety of biophysical and biochemical techniques allowing us to characterize the multidimensional roles of these proteins in binding heavy metals, performing electron transfer reactions, and catalyzing methyl transfers, ultimately generating a potent and toxic neurotoxin. The experiments include spectroscopy (NMR, EPR, resonance Raman, etc.), kinetics (steady-state and transient), electrochemistry and binding measurements (surface plasmon resonance, isothermal calorimetry, NMR, etc). Our results will uncover the biochemical mechanism of MeHg production, revealing a fundamental understanding of the novel methyl transfer reactions catalyzed by HgcA and HgcB. This work will be generally relevant to the microbiological transformations of heavy metals. The kinetic parameters will provide key input for metabolic and reactive transport models that can be used by the ORNL group and others to predict Hg cycling from single organisms to ecosystems. Thus, our work will help understand the processes that control the fate and transformation of Hg in aquatic environments, which is important for mitigating risk to humans and ecosystems in which these Hg-methylating organisms thrive. .
In the work described in this proposal, we will determine the enzymatic mechanism by which anaerobic microbes generate the highly toxic and pervasive compound, methylmercury. We will characterize the properties of the two involved enzymes (HgcA and HgcB) and determine the enzymatic steps for transfer of the methyl group from the biological methyl donor to the relatively nontoxic mercury substrate to generate the toxic methylmercury product. Our work may promote development of effective strategies to reduce exposure to this neurotoxin, which is significant regionally, nationally, and globally.