Mercury(II) reductase

 

Mercuric reductase is responsible for detoxifying and volatilising mercury as Hg0. The catalytic centre is activated when in the diol form, rather than the disulfide. This preliminary, activating reduction step is proposed to involve another NADPH molecule. Both forms are present under physiological conditions and have been identified by structural data [PMID:16114877, PMID:2067577].

 

Reference Protein and Structure

Sequence
P00392 UniProt (1.16.1.1) IPR021179 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas aeruginosa (Bacteria) Uniprot
PDB
1zk7 - Crystal Structure of Tn501 MerA (1.6 Å) PDBe PDBsum 1zk7
Catalytic CATH Domains
3.30.390.30 CATHdb 3.50.50.60 CATHdb (see all for 1zk7)
Cofactors
Fadh2(2-) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.16.1.1)

mercury(2+)
CHEBI:16793ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
mercury(0)
CHEBI:16170ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Mer A, Mercurate(II) reductase, Mercuric ion reductase, Mercuric reductase, Mercury reductase, Reduced NADP:mercuric ion oxidoreductase,

Enzyme Mechanism

Introduction

The first steps of this mechanism are responsible for moving the Hg(II) ion from the N-terminus of the protein to where the FAD is bound. The NADPH substrate transfers a hydride to the FAD cofactor. The FAD cofactor now attacks the mercury centre, eliminating Cys136 and forming a carbon-mercury intermediate. Cys136 abstracts a proton from the flavin-mercury adduct, initiating a reductive elimination of the mercury metal and thiolate Cys141. The active site is regenerated when Cys558' is reprotonated.

Catalytic Residues Roles

UniProt PDB* (1zk7)
Tyr62, Met9 Not found, Not found Help stabilise and hold in place the reactive intermediates formed during the course of the reaction. hydrogen bond acceptor, electrostatic stabiliser
Cys11, Cys141, Cys14, Cys136, Cys558, Cys559 Not found, Cys47A, Not found, Cys42A, Cys464A(AA), Cys465A(AA) The cysteine "pairs" act as nucleophiles to reduce the Hg(II) to Hg(0). The two N terminal Cys residues (Cys11/Cys14) form part of the soft-metal binding domain. covalently attached, nucleofuge, nucleophile, metal ligand, proton donor, proton acceptor, activator, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, coordination to a metal ion, decoordination from a metal ion, overall product formed, enzyme-substrate complex cleavage, hydride transfer, cofactor used, elimination (not covered by the Ingold mechanisms), native state of cofactor regenerated, native state of enzyme regenerated, inferred reaction step

References

  1. Ledwidge R et al. (2005), Biochemistry, 44, 11402-11416. NmerA, the Metal Binding Domain of Mercuric Ion Reductase, Removes Hg2+from Proteins, Delivers It to the Catalytic Core, and Protects Cells under Glutathione-Depleted Conditions†,‡. DOI:10.1021/bi050519d. PMID:16114877.
  2. Ledwidge R et al. (2010), Biochemistry, 49, 8988-8998. NmerA of Tn501Mercuric Ion Reductase: Structural Modulation of the pKaValues of the Metal Binding Cysteine Thiols,. DOI:10.1021/bi100537f. PMID:20828160.
  3. Schue M et al. (2008), Biometals, 21, 107-116. Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn501 mer operon. DOI:10.1007/s10534-007-9097-4. PMID:17457514.
  4. Cummings RT et al. (1992), Biochemistry, 31, 1020-1030. Interaction of Tn501 mercuric reductase and dihydroflavin adenine dinucleotide anion with metal ions: implications for the mechanism of mercuric reductase mediated mercury(II) reduction. DOI:10.1021/bi00119a010. PMID:1310417.
  5. Schiering N et al. (1991), Nature, 352, 168-172. Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. DOI:10.1038/352168a0. PMID:2067577.
  6. Moore MJ et al. (1990), Acc Chem Res, 23, 301-308. Organomercurial lyase and mercuric ion reductase: nature's mercury detoxification catalysts. DOI:10.1021/ar00177a006.

Step 1. The catalytic centre is activated towards its function in the presence of NADPH. The group reduces a disulfide bond between Cys141 and Cys136, forming the dithiol species which is necessary for catalysis to occur. A charge transfer complex exists between the FAD cofactor and Cys141 [DOI:10.1021/ar00177a006]. The two N terminal Cys residues (Cys11/Cys14) form part of a soft-metal binding domain. The positive charge of the N terminus, as well as the presence of specific interactions with surrounding residues lowers the pKa of these cysteine thiols relative to unstructured peptide analogues, enhancing their nucleophilic character and therefore their reactivity with Hg over other peptide sequences. Thus, in this step the distal sulfur of Cys11 acts as a nucleophile towards the Hg(II) complex.

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Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys14 electrostatic stabiliser, increase nucleophilicity
Cys11 activator, metal ligand
Cys11 nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 2. The thiolate anion formed on the substrate acts as a base towards the protonated Cys11.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys14 electrostatic stabiliser, hydrogen bond acceptor
Cys11 activator, covalently attached, metal ligand
Cys11 proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. The adjacent sulfur of Cys14 acts as a nucleophile towards the mercury-enzyme adduct. This releases the mercury free thiol-thiolate molecule with concomitant formation of a bridged thio-mercury enzyme complex.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys14 activator
Cys11 activator, metal ligand
Cys14 nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, coordination to a metal ion, decoordination from a metal ion, enzyme-substrate complex formation

Step 4. The charged sulfur of the eliminated group removes a proton from the positively charged mercury-cysteine adduct. The dithiol equivalent of the substrate is released.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) hydrogen bond acceptor
Cys14 metal ligand, covalently attached
Cys11 metal ligand, covalently attached
Cys47A polar interaction
Cys14 proton donor

Chemical Components

proton transfer, overall product formed, intermediate formation

Step 5. The mercury metal is transferred from the N terminal binding site to a thiol pair situated more closely within the active site [PMID:16114877]. Cys559AA acts as a nucleophile towards the Hg(II) metal, displacing it from the metal binding motif.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) metal ligand
Cys14 activator, covalently attached
Cys11 activator, covalently attached, metal ligand
Cys11 nucleofuge
Cys465A(AA) nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, enzyme-substrate complex formation, intermediate formation

Step 6. The Cys11 thiolate now acts as a base towards the positively charged Cys559AA, regenerating one of the soft-metal binding motif thiols.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 electrostatic stabiliser, steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) metal ligand, covalently attached
Cys14 activator, covalently attached, metal ligand, electrostatic stabiliser
Cys11 activator
Cys11 proton acceptor
Cys465A(AA) proton donor

Chemical Components

proton transfer, intermediate formation

Step 7. Cys558AA now attacks the mercury, forming a sulfide-mercury link while eliminating the Cys14 thiolate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) covalently attached, electrostatic stabiliser, metal ligand
Cys14 activator
Cys11 electrostatic stabiliser
Cys42A hydrogen bond acceptor
Cys14 nucleofuge
Cys464A(AA) nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, enzyme-substrate complex formation, intermediate formation

Step 8. The thiolate form of Cys14 abstracts a proton from the positively charged sulfur of Cys558AA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) activator, covalently attached, electrostatic stabiliser
Cys14 activator
Cys11 electrostatic stabiliser
Cys42A hydrogen bond acceptor
Cys14 proton acceptor
Cys464A(AA) proton donor

Chemical Components

proton transfer, intermediate formation

Step 9. Cys136 acts as a nucleophile towards the close proximity mercury-thiol adduct, forming another metal-ligand bridging intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand, electrostatic stabiliser
Met9 steric role, increase nucleophilicity
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) activator
Cys42A activator
Cys465A(AA) nucleofuge
Cys42A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex formation, enzyme-substrate complex cleavage

Step 10. The resulting positively charged sulfur is deprotonated by Cys559AA

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand, electrostatic stabiliser
Met9 steric role, increase nucleophilicity
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) activator
Cys42A activator
Cys465A(AA) proton acceptor
Cys42A proton donor

Chemical Components

proton transfer, intermediate formation

Step 11. Cys141 displaces Cys558AA within the metal coordination sphere.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A activator
Cys42A covalently attached
Cys464A(AA) nucleofuge
Cys47A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, coordination to a metal ion, decoordination from a metal ion, enzyme-substrate complex formation, enzyme-substrate complex cleavage, intermediate formation

Step 12. The NADPH substrate transfers a hydride to the FAD cofactor.

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Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A metal ligand
Cys42A metal ligand

Chemical Components

hydride transfer, intermediate formation, overall reactant used, overall product formed

Step 13. The FAD cofactor now attacks the mercury centre, eliminating Cys136 and forming a carbon-mercury intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A activator, metal ligand
Cys42A activator
Cys42A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, cofactor used, enzyme-substrate complex cleavage

Step 14. Cys136 abstracts a proton from the flavin-mercury adduct, initiating a reductive elimination of the mercury metal and thiolate Cys141.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A activator
Cys42A activator
Cys42A proton acceptor

Chemical Components

proton transfer, elimination (not covered by the Ingold mechanisms), native state of cofactor regenerated, overall product formed, enzyme-substrate complex cleavage

Step 15. The active site is regenerated when Cys558AA is reprotonated. In this inferred return step, the identiy of the proton donor is not clear.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys464A(AA) proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, overall reactant used, inferred reaction step
Download: Image, Marvin File

Step 1. The catalytic centre is activated towards its function in the presence of NADPH. The group reduces a disulfide bond between Cys141 and Cys136, forming the dithiol species which is necessary for catalysis to occur. A charge transfer complex exists between the FAD cofactor and Cys141 [DOI:10.1021/ar00177a006]. The two N terminal Cys residues (Cys11/Cys14) form part of a soft-metal binding domain. The positive charge of the N terminus, as well as the presence of specific interactions with surrounding residues lowers the pKa of these cysteine thiols relative to unstructured peptide analogues, enhancing their nucleophilic character and therefore their reactivity with Hg over other peptide sequences. Thus, in this step the distal sulfur of Cys11 acts as a nucleophile towards the Hg(II) complex.

Step 2. The thiolate anion formed on the substrate acts as a base towards the protonated Cys11.

Step 3. The adjacent sulfur of Cys14 acts as a nucleophile towards the mercury-enzyme adduct. This releases the mercury free thiol-thiolate molecule with concomitant formation of a bridged thio-mercury enzyme complex.

Step 4. The charged sulfur of the eliminated group removes a proton from the positively charged mercury-cysteine adduct. The dithiol equivalent of the substrate is released.

Step 5. The mercury metal is transferred from the N terminal binding site to a thiol pair situated more closely within the active site [PMID:16114877]. Cys559AA acts as a nucleophile towards the Hg(II) metal, displacing it from the metal binding motif.

Step 6. The Cys11 thiolate now acts as a base towards the positively charged Cys559AA, regenerating one of the soft-metal binding motif thiols.

Step 7. Cys558AA now attacks the mercury, forming a sulfide-mercury link while eliminating the Cys14 thiolate.

Step 8. The thiolate form of Cys14 abstracts a proton from the positively charged sulfur of Cys558AA.

Step 9. Cys136 acts as a nucleophile towards the close proximity mercury-thiol adduct, forming another metal-ligand bridging intermediate.

Step 10. The resulting positively charged sulfur is deprotonated by Cys559AA

Step 11. Cys141 displaces Cys558AA within the metal coordination sphere.

Step 12. The NADPH substrate transfers a hydride to the FAD cofactor.

Step 13. The FAD cofactor now attacks the mercury centre, eliminating Cys136 and forming a carbon-mercury intermediate.

Step 14. Cys136 abstracts a proton from the flavin-mercury adduct, initiating a reductive elimination of the mercury metal and thiolate Cys141.

Step 15. The active site is regenerated when Cys558AA is reprotonated. In this inferred return step, the identiy of the proton donor is not clear.

Products.

Introduction

The first steps of this mechanism are responsible for moving the Hg(II) ion from the N-terminus of the protein to where the FAD is bound. The NADPH substrate transfers a hydride to the FAD cofactor. The FAD cofactor now attacks Cys141, forming an enzyme-FAD complex with loss of Hg(0). Reductive elimination initiated by the thiolate of Cys136 regenerates the cofactor. The active site is regenerated when Cys558' is reprotonated.

Catalytic Residues Roles

UniProt PDB* (1zk7)
Tyr62, Met9 Not found, Not found Help stabilise and hold in place the reactive intermediates formed during the course of the reaction. hydrogen bond acceptor, electrostatic stabiliser
Cys11, Cys141, Cys14, Cys136, Cys558, Cys559 Not found, Cys47A, Not found, Cys42A, Cys464A(AA), Cys465A(AA) The cysteine "pairs" act as nucleophiles to reduce the Hg(II) to Hg(0). The two N terminal Cys residues (Cys11/Cys14) form part of the soft-metal binding domain. covalently attached, nucleofuge, nucleophile, metal ligand, proton donor, proton acceptor, activator, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, coordination to a metal ion, decoordination from a metal ion, overall product formed, enzyme-substrate complex cleavage, hydride transfer, cofactor used, elimination (not covered by the Ingold mechanisms), native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Ledwidge R et al. (2005), Biochemistry, 44, 11402-11416. NmerA, the Metal Binding Domain of Mercuric Ion Reductase, Removes Hg2+from Proteins, Delivers It to the Catalytic Core, and Protects Cells under Glutathione-Depleted Conditions†,‡. DOI:10.1021/bi050519d. PMID:16114877.
  2. Schue M et al. (2008), Biometals, 21, 107-116. Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn501 mer operon. DOI:10.1007/s10534-007-9097-4. PMID:17457514.
  3. Cummings RT et al. (1992), Biochemistry, 31, 1020-1030. Interaction of Tn501 mercuric reductase and dihydroflavin adenine dinucleotide anion with metal ions: implications for the mechanism of mercuric reductase mediated mercury(II) reduction. DOI:10.1021/bi00119a010. PMID:1310417.

Step 1. The catalytic centre is activated towards its function in the presence of NADPH. The group reduces a disulfide bond between Cys141 and Cys136, forming the dithiol species which is necessary for catalysis to occur. A charge transfer complex exists between the FAD cofactor and Cys141 [DOI:10.1021/ar00177a006]. The two N terminal Cys residues (Cys11/Cys14) form part of a soft-metal binding domain. The positive charge of the N terminus, as well as the presence of specific interactions with surrounding residues lowers the pKa of these cysteine thiols relative to unstructured peptide analogues, enhancing their nucleophilic character and therefore their reactivity with Hg over other peptide sequences. Thus, in this step the distal sulfur of Cys11 acts as a nucleophile towards the Hg(II) complex.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 electrostatic stabiliser
Cys14 electrostatic stabiliser
Tyr62 electrostatic stabiliser
Met9 steric role
Tyr62 hydrogen bond acceptor
Cys14 increase nucleophilicity
Cys11 activator, metal ligand, nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 2. The thiolate anion formed on the substrate acts as a base towards the protonated Cys11.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys14 electrostatic stabiliser, hydrogen bond donor
Cys11 activator, covalently attached, metal ligand, proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. The adjacent sulfur of Cys14 acts as a nucleophile towards the mercury-enzyme adduct. This releases the mercury free thiol-thiolate molecule with concomitant formation of a bridged thio-mercury enzyme complex.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys14 activator
Cys11 activator, metal ligand
Cys14 nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, coordination to a metal ion, decoordination from a metal ion, enzyme-substrate complex formation

Step 4. The charged sulfur of the eliminated group removes a proton from the positively charged mercury-cysteine adduct. The dithiol equivalent of the substrate is released.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) hydrogen bond acceptor
Cys14 metal ligand, covalently attached
Cys11 metal ligand, covalently attached
Cys47A polar interaction
Cys14 proton donor

Chemical Components

proton transfer, overall product formed, intermediate formation

Step 5. The mercury metal is transferred from the N terminal binding site to a thiol pair situated more closely within the active site [PMID:16114877]. Cys559AA acts as a nucleophile towards the Hg(II) metal, displacing it from the metal binding motif.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) metal ligand
Cys14 activator, covalently attached
Cys11 activator, covalently attached, metal ligand
Cys465A(AA) nucleophile
Cys11 nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, enzyme-substrate complex formation, intermediate formation

Step 6. The Cys11 thiolate now acts as a base towards the positively charged Cys559AA, regenerating one of the soft-metal binding motif thiols.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 electrostatic stabiliser, steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) metal ligand, covalently attached
Cys14 activator, covalently attached, metal ligand, electrostatic stabiliser
Cys11 activator
Cys465A(AA) proton donor
Cys11 proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 7. Cys558AA now attacks the mercury, forming a sulfide-mercury link while eliminating the Cys14 thiolate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) covalently attached, electrostatic stabiliser, metal ligand
Cys14 activator
Cys11 electrostatic stabiliser
Cys42A hydrogen bond acceptor
Cys464A(AA) nucleophile
Cys14 electrofuge, electrophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, enzyme-substrate complex formation, intermediate formation

Step 8. The thiolate form of Cys14 abstracts a proton from the positively charged sulfur of Cys558AA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand
Met9 steric role, electrostatic stabiliser
Tyr62 hydrogen bond acceptor, electrostatic stabiliser
Cys465A(AA) activator, covalently attached, electrostatic stabiliser
Cys14 activator
Cys11 electrostatic stabiliser
Cys42A hydrogen bond acceptor
Cys14 proton acceptor
Cys464A(AA) proton donor

Chemical Components

proton transfer, intermediate formation

Step 9. Cys136 acts as a nucleophile towards the close proximity mercury-thiol adduct, forming another metal-ligand bridging intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand, electrostatic stabiliser
Met9 steric role, increase nucleophilicity
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) activator, nucleofuge
Cys42A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex cleavage, enzyme-substrate complex formation

Step 10. The resulting positively charged sulfur is deprotonated by Cys559AA

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator, metal ligand, electrostatic stabiliser
Met9 steric role, increase nucleophilicity
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys465A(AA) activator
Cys42A activator, proton donor
Cys465A(AA) proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 11. Cys141 displaces Cys558AA within the metal coordination sphere.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) activator
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A activator
Cys42A covalently attached
Cys464A(AA) nucleofuge
Cys47A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, coordination to a metal ion, decoordination from a metal ion, enzyme-substrate complex formation, enzyme-substrate complex cleavage, intermediate formation

Step 12. The NADPH substrate transfers a hydride to the FAD cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A metal ligand
Cys42A metal ligand

Chemical Components

hydride transfer, intermediate formation, overall reactant used, overall product formed

Step 13. The FAD cofactor attacks Cys141, forming an enzyme-FAD complex with loss of Hg(0).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A electrophile
Cys42A nucleofuge
Cys47A electrofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, cofactor used

Step 14. Cys136 abstracts a proton from the enzyme-FAD adduct, initiating a reductive elimination of thiolate Cys141.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met9 steric role
Tyr62 electrostatic stabiliser, hydrogen bond acceptor
Cys47A nucleofuge
Cys42A proton acceptor

Chemical Components

proton transfer, elimination (not covered by the Ingold mechanisms), native state of cofactor regenerated, overall product formed, enzyme-substrate complex cleavage

Step 15. The active site is regenerated when Cys558AA is reprotonated. In this inferred return step, the identiy of the proton donor is not clear.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys464A(AA) proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The catalytic centre is activated towards its function in the presence of NADPH. The group reduces a disulfide bond between Cys141 and Cys136, forming the dithiol species which is necessary for catalysis to occur. A charge transfer complex exists between the FAD cofactor and Cys141 [DOI:10.1021/ar00177a006]. The two N terminal Cys residues (Cys11/Cys14) form part of a soft-metal binding domain. The positive charge of the N terminus, as well as the presence of specific interactions with surrounding residues lowers the pKa of these cysteine thiols relative to unstructured peptide analogues, enhancing their nucleophilic character and therefore their reactivity with Hg over other peptide sequences. Thus, in this step the distal sulfur of Cys11 acts as a nucleophile towards the Hg(II) complex.

Step 2. The thiolate anion formed on the substrate acts as a base towards the protonated Cys11.

Step 3. The adjacent sulfur of Cys14 acts as a nucleophile towards the mercury-enzyme adduct. This releases the mercury free thiol-thiolate molecule with concomitant formation of a bridged thio-mercury enzyme complex.

Step 4. The charged sulfur of the eliminated group removes a proton from the positively charged mercury-cysteine adduct. The dithiol equivalent of the substrate is released.

Step 5. The mercury metal is transferred from the N terminal binding site to a thiol pair situated more closely within the active site [PMID:16114877]. Cys559AA acts as a nucleophile towards the Hg(II) metal, displacing it from the metal binding motif.

Step 6. The Cys11 thiolate now acts as a base towards the positively charged Cys559AA, regenerating one of the soft-metal binding motif thiols.

Step 7. Cys558AA now attacks the mercury, forming a sulfide-mercury link while eliminating the Cys14 thiolate.

Step 8. The thiolate form of Cys14 abstracts a proton from the positively charged sulfur of Cys558AA.

Step 9. Cys136 acts as a nucleophile towards the close proximity mercury-thiol adduct, forming another metal-ligand bridging intermediate.

Step 10. The resulting positively charged sulfur is deprotonated by Cys559AA

Step 11. Cys141 displaces Cys558AA within the metal coordination sphere.

Step 12. The NADPH substrate transfers a hydride to the FAD cofactor.

Step 13. The FAD cofactor attacks Cys141, forming an enzyme-FAD complex with loss of Hg(0).

Step 14. Cys136 abstracts a proton from the enzyme-FAD adduct, initiating a reductive elimination of thiolate Cys141.

Step 15. The active site is regenerated when Cys558AA is reprotonated. In this inferred return step, the identiy of the proton donor is not clear.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Amelia Brasnett