Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

Abstract

Methylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pair hgcAB, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized in vivo, the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood. Here, we report the kinetics of Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen sensitive, irreversible, and follows Michaelis-Menten kinetics, with an apparent K_m of 3.2 nM and V_max of 19.7 fmol · min^-1 · mg^-1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Interestingly, increasing thiol/Hg(II) ratios did not impact Hg methylation rates, which suggests that HgcAB-mediated Hg methylation effectively competes with cellular thiols for Hg(II), consistent with the low apparent K_m Supplementation of 5-methyltetrahydrofolate or pyruvate did not enhance MeHg production, while both ATP and a nonhydrolyzable ATP analog decreased Hg methylation rates in cell lysates under the experimental conditions. These studies provide insights into the biomolecular processes associated with Hg methylation in anaerobic bacteria.IMPORTANCE The concentration of Hg in the biosphere has increased dramatically over the last century as a result of industrial activities. The microbial conversion of inorganic Hg to MeHg is a global public health concern due to bioaccumulation and biomagnification of MeHg in food webs. Exposure to neurotoxic MeHg through the consumption of fish represents a significant risk to human health and can result in neuropathies and developmental disorders. Anaerobic microbial communities in sediments and periphyton biofilms have been identified as sources of MeHg in aquatic systems, but the associated biomolecular mechanisms are not fully understood. In the present study, we investigate the biochemical mechanisms and kinetics of MeHg formation by HgcAB in sulfate-reducing bacteria. These findings advance our understanding of microbial MeHg production and may help inform strategies to limit the formation of MeHg in the environment.

Keywords: HgcAB; anaerobic bacteria; environmental microbiology; enzyme kinetics; mercury methylation; methylmercury.

FIG 1

Structural features and proposed mechanism of HgcAB-mediated Hg methylation. (A) Cartoon representation of sequence-based structural models of HgcA and HgcB. Key components and features are labeled as follows: TMD, transmembrane domain; CBD, corrinoid-binding domain; [4Fe-4S], iron-sulfur cluster; C, cysteine). (B) Proposed roles of HgcA and HgcB in Hg methylation (C1 precursor, one-carbon compound; Co(I)/(III), cobalt center of a corrinoid cofactor and its oxidation state; H₄folate, tetrahydrofolate; CH₃-H₄folate, 5-methyltetrahydrofolate; Hg(II), mercuric mercury; CH₃Hg⁺, methylmercury.

FIG 2

Time dependence of Hg methylation and MeHg demethylation in ND132. (A) Hg methylation in cell lysates of ND132 WT (blue) and ΔhgcAB (red) (1.5 mg/ml total protein concentration) under strictly anaerobic conditions at 32°C in the presence of 30 nM Hg(II). The blue line shows a nonlinear fit of the concentration data and the gray dashed line shows a linear fit (R² = 0.98) for time points between 0 and 2 h to determine initial rates. (B) MeHg concentrations in ND132 cell lysates of WT (blue) and ΔhgcAB (red) as a function of time under a similar experimental setup as that in panel A but with 5 nM MeHg as the substrate. Error bars represent standard deviation between duplicate sets of samples (n = 2).

FIG 3

Dependence of Hg methylation rates on pH, temperature, and total protein concentration. Hg methylation rates in cell lysates of ND132 WT measured as a function of (A) pH (4, 5, 6, 7, 8, and 9), (B) temperature (4, 24 [room temperature], 32, 40, and 50°C), and (C and D) total protein concentration. Samples were incubated with 30 nM Hg(II) and harvested for MeHg analysis at 2 h, except in panel C, where samples were harvested at 2 min after Hg(II) addition. pH 7, 32°C, and strictly anaerobic conditions correspond to standard experimental conditions. Error bars represent standard deviation between duplicate sets of samples (n = 2).

FIG 4

Dependence of Hg methylation rates on Hg substrate concentration. (A) Initial rates of Hg methylation in ND132 cell lysates (1.5 mg/ml total protein) in response to increasing concentrations of added Hg(II) from 0.5 nM to 60 nM at 32°C for 2 h under strictly anaerobic conditions for WT (blue circles) and ΔhgcAB strains (red triangles). Hg methylation rates in WT cell lysates were fitted to the Michaelis-Menten equation with a V_max of 1.77 ± 0.03 nM/h and K_m of 3.2 ± 0.26 nM (blue line). (B) Percentage of Hg(II) converted to MeHg within 2 h as a function of initial Hg(II) concentration. Error bars represent standard deviation between duplicate set of samples (n = 2).

FIG 5

Effect of ambient oxygen and key cellular metabolites on Hg methylation. (A) Hg methylation rates in WT ND132 cell lysates and ΔhgcAB under aerobic (exposed to ambient oxygen) and anaerobic conditions (N₂ environment, <0.6 ppm O₂). (B) Hg methylation rates in WT ND132 cell lysates and ΔhgcAB under unamended conditions compared to the Hg methylation rates for samples amended with 16.8 μM CH₃-H₄folate and 10 mM pyruvate (Pyr). (C) Effect of 2 mM ATP and 10 mM ATP on the concentration of MeHg produced at 2 h in the presence of 30 nM Hg(II) compared to that in the unamended sample. ATP was supplemented before the addition of Hg(II) (ATP addition at 0 h) or 1 h after the addition of Hg(II) (ATP addition at 1 h). (D) Effect of 5 mM nonhydrolyzable ATP analog AMP-PNP on MeHg production compared to the unamended sample. Data were analyzed with a two-sample t test. Not significant (ns), P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Error bars represent the standard deviation between duplicates (n = 2).