Introduction
The C-Hg bond in organomercurials is exceptionally stable with respect to protolytic cleavage under mild physiological conditions, and yet organomercurial lyases catalyze the reaction with a rate acceleration of roughly 107 over the reaction rate in solution—raising considerable interest, both from a chemical perspective and for potential bioremediation efforts, in how they do this. Mechanistic studies have provided evidence that the enzyme's active-site cysteines are involved, but varied roles for these thiol groups have been proposed (Fig. 1)1, 2, 3, 4. In an elegant recent study, Melnick and Parkin5 use model compounds to help distinguish between the proposed mechanisms. The most important conclusion from their work is the clear demonstration that the mercury in the R-Hg substrate must be complexed by two thiolate ligands to become sufficiently activated toward protolysis by a separate acid donor.
Figure 1: Mechanistic hypotheses showing varied roles of conserved active-site cysteine residues in protolysis.
(a) Both conserved cysteine thiolates chelate Hg to activate the C-Hg bond, and either a third nonconserved cysteine or an as-yet-unidentified residue (B-H) acts as proton donor in the rate-limiting transition state2. (b) One conserved cysteine thiolate binds Hg, and the second conserved cysteine thiol becomes liganded to Hg2+ and concomitantly donates its proton to C in the rate-limiting transition state3. (c) As in b, one conserved cysteine thiolate binds Hg, and the second conserved thiolate becomes liganded in the rate-limiting transition state; but another (unidentified) residue facilitates S-H deprotonation and concomitant protonation of C ref. 4. (d) Structure of the monoligated complex of R-Hg (R = Me or Et) with the tris(2-mercapto-1-t-butylimidazolyl)hydroborato ligand used in the Melnick and Parkin study, which is proposed to be in equilibrium with the tris-coordinated form based on NMR analysis5.
Full size image (21 KB)Mercury is well known for its toxicity to living organisms. Inorganic mercuric compounds (HgX2) and organomercurials (R-Hg-X), in which the Hg is formally in the +2 oxidation state, are primarily responsible for the toxicity. The ability to exchange their weaker 'X' ligands (for example, chloride ion) for high-affinity anionic sulfur and selenium groups of cysteine and selenocysteine residues can lead to pleiotropic effects, as these residues often serve important catalytic, redox or structural roles in proteins. Elemental mercury itself (Hg0) has little affinity for cellular ligands and is toxic only if it becomes oxidized to the +2 state in the cell. Taking advantage of both the low toxicity of elemental mercury and its high volatility, aerobic bacteria have evolved the ingenious strategy of eliminating mercuric and organomercurial compounds from their environment through reduction of Hg2+ to Hg0 (ref. 6). To accomplish this, they couple the activity of two enzymes: organomercurial lyase (MerB) and mercuric ion reductase. In the overall pathway, MerB is responsible for protolytically cleaving the C-Hg bond of the organomercurial to produce a hydrocarbon product and mercuric ion, which is passed on to the reductase for reduction to elemental mercury. In light of the increased prevalence of the potently toxic organomercurial methyl mercury (CH3-Hg-X), a result of the increased overall mercury burden in the environment due to human activities, there is significant interest in understanding how the lyase protolytically cleaves the C-Hg bonds under such mild physiological conditions, as this may guide efforts to design catalysts for remediation of these contaminants.
As the carbon in the C-Hg bond of organomercurials is formally a carbanion, protolytic cleavage of the bond is viewed as an electrophilic substitution at the carbanion, with H+ acting as the incoming electrophile and [HgX]1+ as the leaving electrophile from a simple R-Hg-X compound. Because Hg2+ essentially never exists without two ligands, this reaction can only occur if [HgX]1+ is activated by the addition of at least one ligand to Hg before (mechanism in Fig. 1a) or concomitant with (mechanism in Fig. 1b or Fig. 1c) protolytic cleavage. Chemical precedent7 suggests that higher-affinity ligands provide the highest degree of activation of R-Hg-X toward protolysis. MerB enzymes have two conserved cysteines at the active site and, in the best-characterized enzyme, a third unconserved cysteine, which could potentially serve as Hg ligands and/or acid donors3, 8. Based on initial mechanistic studies of MerB1 and unpublished studies of single cysteine-to-alanine mutants, Moore et al. previously proposed the mechanism shown in Figure 1a, in which the two conserved cysteines chelate the Hg and the thiol of the third cysteine is a possible candidate for acid donor2. Early model studies by Gopinath and Bruice using dithiols as ligands in aqueous solutions agreed with Figure 1a, but suggested a carboxylic acid could serve as acid donor9. In retrospect, the results of these studies may also be explained by the model in Figure 1c, which has been proposed to be an energetically favorable transition state on the basis of computational modeling4. The mechanism in Figure 1b stemmed from the observation that in the absence of any other thiols, MerB, with the nonconserved cys-teine mutated to serine, can cleave an R-Hg-Cl substrate in a single turnover reaction as well as the wild-type enzyme, whereas mutation of either conserved cysteine to serine eliminates the reaction. Although this is still consistent with the mechanism in Figure 1a, where the acid donor is a residue other than a thiol, Pitts and Summers suggested that one of the two conserved cysteine thiols might concomitantly protonate C and form a second Hg-S bond (Fig. 1b)3.
The new model study by Melnick and Parkin provides strong support for the mechanism in Figure 1a5. Using compounds with either one or three mercaptoimidazole groups, which lack acidic protons on sulfur, the authors synthesized and characterized stable R-Hg-[thiolate]n complexes (Fig. 1d). Subsequent addition of thiophenol to initiate the protolytic reaction showed that the monothiolate (n = 1) complex is unreactive at temperatures where MerB functions. However, addition of a second equivalent of monomercaptoimidazole ligand, which gave an equilibrium mixture of mono- and dithiolate (n = 2) complexes, allowed C-Hg cleavage to proceed at room temperature. The complex synthesized from the tris-mercaptoimidazole chelator reacted even faster but, curiously, showed ligation of Hg by only one thiolate in its crystal structure. However, NMR data demonstrated that the complex interconverts between mono-, bis- and tris-thiolate ligation, providing further support for the idea that higher-order thiolate ligation leads to greater activation toward protolysis of the C-Hg bonds by a separate acid donor under mild conditions.
The model studies of Melnick and Parkin provide convincing evidence for the role of the two conserved cysteine thiolates in MerB in the chelation and activation of the Hg-C bond for protolysis. A key mechanistic question that remains, however, is the identity of the acid donor 'B-H' in the mechanism (Fig. 1a). The model studies show that a third thiol can act as an acid donor, but data from studies of the cysteine-to-serine mutation strongly suggest that a different residue serves as donor. It would be useful to perform additional studies in the model system to test the ability of mimics of other amino acid functional groups to act as donors for protolysis. Likewise, further studies of the alanine mutation of the nonconserved cysteine, as well as studies of other potential donor residues that may be identified from structural data, will help to sort out the identity of the acid donor. Aside from the implications for the MerB mechanism itself, two other points are raised by the model studies. On the one hand, the studies demonstrate the feasibility of developing synthetic ligands for remediation efforts that may be used under fairly mild conditions. On the other hand, a perhaps more intriguing question raised by the observation of the facile cleavage of methyl-Hg by the tris-ligated compound is whether fortuitous ligation and activation of methyl-Hg at multithiol sites in proteins in the brain is responsible for the potent toxicity of methyl-Hg.
