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Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.


H3O+-assisted removal of Hg(SCH3)2 from the MerB active site (extended conformation)(A) Asp99-protonated Int3′ surrounded by water molecules; (B) proton transfer from Asp99 to Cys159 (transition state); (C) regenerated active site with released Hg(SCH3)2. Relevant distances (in Ångstrom) are highlighted.
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fig-7: H3O+-assisted removal of Hg(SCH3)2 from the MerB active site (extended conformation)(A) Asp99-protonated Int3′ surrounded by water molecules; (B) proton transfer from Asp99 to Cys159 (transition state); (C) regenerated active site with released Hg(SCH3)2. Relevant distances (in Ångstrom) are highlighted.

Mentions: In the “extended” conformation of Int3′, the Hg(SCH3)2 moiety lies quite far from Asp99, which modifies the mechanistic analysis due to the impossibility of Asp99-attachment to the metal upon the release of Cys159. In contrast to the previous analysis, in this conformation the solvated H3O+ is unstable even before including bulk solvation effects implicitly through the PCM model. Instead, two separate minima arise: an unproductive intermediate featuring a proton on the Asp99 residue (Fig. 7A), and the Cys159-protonated product featuring a free Hg(SCH3)2 (Fig. 7C). Both minima lie ≈10 kcal mol−1 below the postulated initial (meta-stable) conformation featuring a solvated H3O+ (Fig. 7B).


Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

H3O+-assisted removal of Hg(SCH3)2 from the MerB active site (extended conformation)(A) Asp99-protonated Int3′ surrounded by water molecules; (B) proton transfer from Asp99 to Cys159 (transition state); (C) regenerated active site with released Hg(SCH3)2. Relevant distances (in Ångstrom) are highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525700&req=5

fig-7: H3O+-assisted removal of Hg(SCH3)2 from the MerB active site (extended conformation)(A) Asp99-protonated Int3′ surrounded by water molecules; (B) proton transfer from Asp99 to Cys159 (transition state); (C) regenerated active site with released Hg(SCH3)2. Relevant distances (in Ångstrom) are highlighted.
Mentions: In the “extended” conformation of Int3′, the Hg(SCH3)2 moiety lies quite far from Asp99, which modifies the mechanistic analysis due to the impossibility of Asp99-attachment to the metal upon the release of Cys159. In contrast to the previous analysis, in this conformation the solvated H3O+ is unstable even before including bulk solvation effects implicitly through the PCM model. Instead, two separate minima arise: an unproductive intermediate featuring a proton on the Asp99 residue (Fig. 7A), and the Cys159-protonated product featuring a free Hg(SCH3)2 (Fig. 7C). Both minima lie ≈10 kcal mol−1 below the postulated initial (meta-stable) conformation featuring a solvated H3O+ (Fig. 7B).

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.