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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.


Pathways for Hg removal from MerB, starting from an attacking thiol (“thiol-based” mechanism) or an attacking thiolate (“thiolate-based” mechanism).In both mechanisms, primed-numbered intermediates arise from the attack of a thiol and a thiolate, whereas intermediates numbered with unprimed numbers arise from the attack of two species with the same protonation state (either two thiols or two thiolates).
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fig-1: Pathways for Hg removal from MerB, starting from an attacking thiol (“thiol-based” mechanism) or an attacking thiolate (“thiolate-based” mechanism).In both mechanisms, primed-numbered intermediates arise from the attack of a thiol and a thiolate, whereas intermediates numbered with unprimed numbers arise from the attack of two species with the same protonation state (either two thiols or two thiolates).

Mentions: A large number of mechanistic pathways for Hg2+ removal from the active site of MerB is possible (Fig. 1), depending on the protonation state of each mercury-attacking ligand (thiol vs. thiolate), on whether Cys96 or Cys159 is first ejected from the coordination sphere of the Hg ion, and on whether the protonation state of Asp99 changes throughout the cycle. Our density-functional computations show that extraneous methanethiol is not nucleophilic enough to directly the attack of the enzyme-bound Hg2+. The moderate acidity of the thiol, however, allows it to transfer a proton to one of the Hg2+ ligands (either Cys159 or Asp 99), in a process which both weakens the ligand-to-metal bond and transforms the thiol into a (much more nucleophilic) thiolate (Fig. 2). Proton transfer to Cys159 (Fig. 2B) occurs with a small barrier (12.3–12.8 kcal mol−1 in MP2, 7.8–8.0 kcal mol−1 using DFT) and is moderately exergonic by 7–9 kcal mol−1. This activation barrier is very similar to the barrier found experimentally (Hong et al., 2010) for the initial attack of MerB-bound mercury by free glutathione (2.5 × 104 M−1 s−1, which translates to 11.4 kcal mol−1).


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

Silva PJ, Rodrigues V - PeerJ (2015)

Pathways for Hg removal from MerB, starting from an attacking thiol (“thiol-based” mechanism) or an attacking thiolate (“thiolate-based” mechanism).In both mechanisms, primed-numbered intermediates arise from the attack of a thiol and a thiolate, whereas intermediates numbered with unprimed numbers arise from the attack of two species with the same protonation state (either two thiols or two thiolates).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-1: Pathways for Hg removal from MerB, starting from an attacking thiol (“thiol-based” mechanism) or an attacking thiolate (“thiolate-based” mechanism).In both mechanisms, primed-numbered intermediates arise from the attack of a thiol and a thiolate, whereas intermediates numbered with unprimed numbers arise from the attack of two species with the same protonation state (either two thiols or two thiolates).
Mentions: A large number of mechanistic pathways for Hg2+ removal from the active site of MerB is possible (Fig. 1), depending on the protonation state of each mercury-attacking ligand (thiol vs. thiolate), on whether Cys96 or Cys159 is first ejected from the coordination sphere of the Hg ion, and on whether the protonation state of Asp99 changes throughout the cycle. Our density-functional computations show that extraneous methanethiol is not nucleophilic enough to directly the attack of the enzyme-bound Hg2+. The moderate acidity of the thiol, however, allows it to transfer a proton to one of the Hg2+ ligands (either Cys159 or Asp 99), in a process which both weakens the ligand-to-metal bond and transforms the thiol into a (much more nucleophilic) thiolate (Fig. 2). Proton transfer to Cys159 (Fig. 2B) occurs with a small barrier (12.3–12.8 kcal mol−1 in MP2, 7.8–8.0 kcal mol−1 using DFT) and is moderately exergonic by 7–9 kcal mol−1. This activation barrier is very similar to the barrier found experimentally (Hong et al., 2010) for the initial attack of MerB-bound mercury by free glutathione (2.5 × 104 M−1 s−1, which translates to 11.4 kcal mol−1).

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.