Mechanism, physiological context, and universality of HgcAB-dependent microbial mercury methylation: the surviving open core of the hgcAB discovery question

The 2013 discovery that a two-gene cluster, hgcA and hgcB, is required for microbial mercury methylation posed a now-classic set of open questions: the chemical mechanism of methyl transfer to Hg(II), the identity of in vivo substrates and accessory factors, the native physiological function of the genes, and whether hgcAB is a universal marker for methylation. The 2024 in vitro reconstitution of the purified HgcAB complex resolved one central part by showing that S-adenosyl-L-methionine (SAM), not methyltetrahydrofolate, is the methyl donor, and confirmed cobalt-thiolate ligation by the conserved HgcA cysteine. The remaining core is open and precise: (i) whether methyl transfer from methylcob(III)alamin-HgcA to Hg(II) proceeds homolytically (by a methyl radical) or heterolytically (by a methyl carbanion) — the two density-functional theory (DFT) studies that have addressed it directly disagree; (ii) the identity and coordination of the in vivo Hg substrate and of the physiological electron donor that regenerates the Co(I) state; (iii) the native physiological function of hgcAB and the selective forces behind its sporadic phylogenetic distribution; and (iv) whether hgcAB is universal, i.e., whether alternative methylation systems exist and whether all hgcA-carrying lineages actually methylate.

Background

Methylmercury ($CH_3Hg^+$, MeHg) is a potent neurotoxin that is produced from inorganic mercury ($Hg^{2+}$) mainly by anaerobic microorganisms in sediments, soils, and wetlands, and bioaccumulates in aquatic food webs. For decades the biomolecular basis of this reaction was unknown. Building on older work (Choi, Chase and Bartha 1994) showing that mercury methylation in Desulfovibrio desulfuricans LS is an enzymatic, corrinoid-dependent process, Parks et al. (2013) identified a two-gene cluster, hgcA and hgcB, whose deletion abolishes methylation in the sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 and the iron-reducing bacterium Geobacter sulfurreducens PCA. hgcA encodes HgcA, a membrane-associated member of the corrinoid protein family: corrinoids (such as cobalamin, vitamin B12) are cobalt-containing ring cofactors whose metal center cycles between the +1, +2, and +3 oxidation states; methyl-corrinoids carry a methyl group on the cobalt and can donate it to acceptors. HgcA carries a strictly conserved cysteine in a 'cap helix' motif (consensus N(V/I)WCA(A/G)GK), proposed to coordinate the cobalt as a lower axial thiolate ligand, and a C-terminal transmembrane domain. hgcB encodes HgcB, a small ferredoxin-like protein with two [4Fe-4S] iron-sulfur clusters (cysteine-bound inorganic cubane clusters that mediate electron transfer), proposed to reduce the corrinoid cobalt from Co(II) to the super-reduced Co(I) state that accepts a methyl group. Parks et al. proposed a catalytic model in which a methyl group derived from 5-methyltetrahydrofolate ($CH_3$-$H_4$folate, a folate cofactor carrying a labile methyl group, as used in the Wood-Ljungdahl acetyl-CoA pathway — an ancient carbon-fixation pathway whose corrinoid iron-sulfur protein, CFeSP, is the closest structural relative of HgcA's corrinoid-binding domain) is transferred to cobalamin on HgcA, forming $CH_3$-cob(III)alamin-HgcA, from which the methyl moves to an $Hg^{2+}$ substrate, plausibly a bis-thiolate ($Hg(SR)_2$-type) complex, by either a methyl-radical ($CH_3^{\cdot}$) or methyl-carbanion ($CH_3^-$) route. They also observed that hgcAB orthologs are present in all then-confirmed methylators and absent from non-methylators, but occur sporadically across Deltaproteobacteria, Firmicutes, and methanogenic archaea — a patchy distribution suggesting lateral gene transfer and/or extensive gene loss — and that deletion of hgcAB does not impair laboratory growth, leaving the genes' native physiological role unexplained. Site-directed mutagenesis (Smith et al. 2015) confirmed that the conserved HgcA cysteine and cap-helix structure are essential for activity. The first cell-free methylation assays (Date et al. 2019) established HgcAB-dependent kinetics but left the methyl donor unidentified, and a 2020 coevolution-based structural model (Cooper et al. 2020) gave the only available structure of the complex, since no experimental HgcA or HgcAB structure exists. In 2024, Zheng et al. achieved the decisive in vitro reconstitution: purified HgcAB is a cobalamin/iron-sulfur enzyme in which S-adenosyl-L-methionine (SAM, the ubiquitous biological methyl-donating cofactor) — not $CH_3$-$H_4$folate and not a separate folate-dependent methyltransferase — donates the methyl group via a two-step reaction involving a methylcob(III)alamin intermediate and Co-thiolate ligation from the conserved cysteine. This corrected the source model's methyl-donor premise while leaving the downstream chemistry and the biological context unresolved.

Problem Statement

Building on the 2013 discovery of hgcAB and on the 2024 finding that S-adenosyl-L-methionine is the methyl donor for HgcAB-dependent methylation, determine the remaining molecular mechanism, physiological context, and universality of microbial mercury methylation, namely: (1) the detailed chemical mechanism of methyl transfer from $CH_3$-cob(III)alamin-HgcA to the $Hg^{2+}$ substrate — whether it proceeds homolytically, by methyl radical ($CH_3^{\cdot}$) transfer (the concerted single-transition-state radical transfer computed by Demissie et al. 2016 being one realization of this branch), or heterolytically, by methyl carbanion ($CH_3^-$) transfer as computed by Zhou et al. 2014 — and how the unresolved conflict between these two theoretical pictures is settled, including the role of the conserved cysteine Co-thiolate ligation; (2) the identity and coordination state of the in vivo $Hg^{2+}$ substrate delivered to HgcA (e.g., which cellular thiol complexes) and the identity of the physiological electron donor(s) that reduce HgcB/corrinoid to regenerate the Co(I) state in the catalytic cycle; (3) the native physiological function(s) of hgcA and hgcB beyond mercury methylation, and the evolutionary history (vertical inheritance with loss versus lateral gene transfer) and selective forces, including any fitness benefit of methylmercury production, that govern the sporadic phylogenetic distribution of the hgcAB cluster; and (4) whether hgcAB is a universal marker for microbial mercury methylation — whether any organisms lacking hgcAB methylate mercury by an alternative system, and conversely whether all organisms carrying hgcA (including hgcA-only lineages and fused hgcAB variants) are functional methylators. These four parts are interrelated, separately addressable open aspects of one overarching problem: a complete molecular and biological account of how anaerobic microorganisms produce methylmercury. A resolution of the overarching problem requires addressing all four parts.

References

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