Peroxidase

 

Horseradish peroxidase C (HRPC) is the most studied member of the class III peroxidases. These enzymes occur in higher plants and catalyse the oxidation of phenolic compounds to phenol radicals using hydrogen peroxide which is reduced to water. Since plant peroxidases generally have broad substrate specificity and produce highly reactive radical products which can participate in non-enzymatic reactions, the biological functions of these enzymes have been difficult to verify. They have been proposed to have roles in lignin production and cell wall formation, for example by catalysing the formation of monolignol radicals which subsequently polymerise to lignin, and by catalysing cell wall cross-linking reactions involving ferulic acid.

 

Reference Protein and Structure

Sequence
P00433 UniProt (1.11.1.7) IPR000823 (Sequence Homologues) (PDB Homologues)
Biological species
Armoracia rusticana (Horseradish) Uniprot
PDB
7atj - RECOMBINANT HORSERADISH PEROXIDASE C1A COMPLEX WITH CYANIDE AND FERULIC ACID (1.47 Å) PDBe PDBsum 7atj
Catalytic CATH Domains
1.10.520.10 CATHdb 1.10.420.10 CATHdb (see all for 7atj)
Cofactors
Heme b (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.11.1.7)

hydrogen peroxide
CHEBI:16240ChEBI
+
phenol
CHEBI:15882ChEBI
→
water
CHEBI:15377ChEBI
+
phenolic radical
CHEBI:137811ChEBI
Alternative enzyme names: Japanese radish peroxidase, Extensin peroxidase, Guaiacol peroxidase, Heme peroxidase, Horseradish peroxidase (HRP), Lactoperoxidase, Oxyperoxidase, Protoheme peroxidase, Pyrocatechol peroxidase, Scopoletin peroxidase, Plant peroxidase, Soybean peroxidase (SBP), Coprinus cinereus peroxidase, Arthromyces ramosus peroxidase,

Enzyme Mechanism

Introduction

The reaction occurs in three steps:
The enzyme is first oxidised by hydrogen peroxide and is then reduced in two sequential one-electron transfer steps from reducing substrates (typically small molecule phenol derivatives). The mechanism of the first step is believed to be common to all haem peroxidases. HOOH is deprotonated by His 42 to produce singly ionised Fe(III)-coordinated peroxide. The O-O bond of the coordinated peroxide is now heterolytically cleaved to generate an Fe(IV)=O species and a hydroxide ion, which is protonated by His 42. Arg 38 is though to promote the reaction by stabilising negative charge in the transient Fe(III)-OOH intermediate and associated transition states.

In the second step, a reducing phenol substrate binds to the active site and transfers an electron to haem and a proton to His 42. The proton transfer is thought to occur via an active site water molecule, and is proposed to be assisted by Arg 38 which forms a hydrogen bond to the substrate phenolic oxygen.

The product radical now dissociates from the active site and a second reducing phenol substrate is bound. This second reducing substrate transfers an electron to the haem, and a proton (via an active site water) to the Fe-coordinated O atom. This is accompanied by proton transfer from His 42 to the Fe-coordinated oxygen, so that the overall result is formation of a water molecule and regeneration of the resting state haem Fe(III).

Catalytic Residues Roles

UniProt PDB* (7atj)
Arg68 Arg38A Stabilises negative charge in the transient Fe(III)-OOH intermediate and associated transition states. Assists proton transfer from the phenol substrates to either His 42 or the ferryl oxygen. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
His72 His42A Deprotonates hydrogen peroxide to give singly ionised Fe(III)-coordinated peroxide. Protonates the departing hydroxide ion during heterolytic cleavage of the O-O bond in the coordinated peroxide. Accepts a proton from the phenol substrate (via an active site water molecule). Protonates the Fe-coordinated oxygen in the final step of the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
His200 His170A Acts as the heme axial ligand. metal ligand
Asn100 Asn70A Modifies the pKa of His 42. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
*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

proton transfer, bimolecular nucleophilic substitution, coordination to a metal ion, decoordination from a metal ion, overall reactant used, heterolysis, electron transfer, radical formation, redox reaction, cofactor used, rate-determining step, radical propagation, intermediate formation, overall product formed, proton relay, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Meno K et al. (2002), Acta Crystallogr D Biol Crystallogr, 58, 1803-1812. Structural analysis of the two horseradish peroxidase catalytic residue variants H42E and R38S/H42E: implications for the catalytic cycle. DOI:10.1107/s090744490201329x. PMID:12351824.
  2. Derat E et al. (2006), J Am Chem Soc, 128, 13940-13949. An Efficient Proton-Coupled Electron-Transfer Process during Oxidation of Ferulic Acid by Horseradish Peroxidase:  Coming Full Cycle. DOI:10.1021/ja065058d. PMID:17044722.
  3. Derat E et al. (2006), J Phys Chem B, 110, 10526-10533. The Poulos−Kraut Mechanism of Compound I Formation in Horseradish Peroxidase:  A QM/MM Study. DOI:10.1021/jp055412e. PMID:16722763.
  4. Gajhede M (2001), Biochem Soc Trans, 29, 91-98. Plant peroxidases: substrate complexes with mechanistic implications. DOI:10.1042/0300-5127:0290091. PMID:11356134.
  5. Henriksen A et al. (1999), J Biol Chem, 274, 35005-35011. The Structures of the Horseradish Peroxidase C-Ferulic Acid Complex and the Ternary Complex with Cyanide Suggest How Peroxidases Oxidize Small Phenolic Substrates. DOI:10.1074/jbc.274.49.35005. PMID:10574977.

Step 1. His42 deprotonates hydrogen peroxide. The resulting anion coordinates to the Fe(III) centre of Hem350, displacing water and forming Cpd0.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond acceptor, hydrogen bond donor
Arg38A hydrogen bond donor, electrostatic stabiliser
Asn70A increase basicity, hydrogen bond acceptor, electrostatic stabiliser
His170A metal ligand
His42A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, coordination to a metal ion, decoordination from a metal ion, overall reactant used

Step 2. The O-O bond of Cpd0 undergoes heterolytic cleavage and the resulting hydroxide is protonated by His42. The porphryin ring and Fe(III) donate an electron each to the oxygen bound to the metal.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond donor, electrostatic stabiliser
Arg38A hydrogen bond donor, electrostatic stabiliser
Asn70A hydrogen bond acceptor, increase acidity
His170A metal ligand
His42A proton donor

Chemical Components

heterolysis, proton transfer, electron transfer, radical formation, redox reaction, cofactor used, rate-determining step

Step 3. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring, forming a phenolic radical and Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg38A hydrogen bond donor, electrostatic stabiliser
His42A hydrogen bond acceptor, hydrogen bond donor
Asn70A hydrogen bond acceptor, increase basicity, electrostatic stabiliser
His170A metal ligand
His42A proton acceptor

Chemical Components

proton transfer, electron transfer, radical propagation, redox reaction, intermediate formation, overall product formed, overall reactant used, proton relay

Step 4. The oxygen of Cpd1 is protonated by water, which is in turn protonated by His42.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond donor
Arg38A hydrogen bond donor, electrostatic stabiliser
Asn70A hydrogen bond acceptor, increase acidity
His170A metal ligand
His42A proton donor

Chemical Components

proton transfer, proton relay

Step 5. The porphyrin ring transfers an electron to the Fe(IV) centre to give an isomer of Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond donor, hydrogen bond acceptor
Arg38A hydrogen bond donor
Asn70A hydrogen bond acceptor
His170A metal ligand

Chemical Components

electron transfer, radical propagation, intermediate formation

Step 6. Arg38 transfers a proton to the oxygen of Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond donor, hydrogen bond acceptor
Arg38A hydrogen bond donor
Asn70A hydrogen bond acceptor
His170A metal ligand
Arg38A proton donor

Chemical Components

proton transfer

Step 7. Upon binding of the second phenol derivative, Arg38 deprotonates Cpd2. The doubly-protonated form of Cpd2 is less stable in the presence of the phenol derivative than in the presence of the phenolic radical. Therefore when the phenolic radical product dissociates and the phenolic derivative substrate associates, there is proton transfer to form the more stable isomer of Cpd2.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
His42A hydrogen bond acceptor, hydrogen bond donor
Arg38A hydrogen bond acceptor
Asn70A hydrogen bond acceptor
Arg38A proton acceptor

Chemical Components

proton transfer

Step 8. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring radical, forming a phenolic radical.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
His42A hydrogen bond acceptor, hydrogen bond donor
Arg38A electrostatic stabiliser, hydrogen bond donor
Asn70A hydrogen bond acceptor, electrostatic stabiliser, increase basicity
His42A proton acceptor

Chemical Components

proton transfer, electron transfer, radical propagation, native state of cofactor regenerated, overall product formed, overall reactant used, proton relay

Step 9. The metal-bound hydroxide is protonated by water, which is in turn protonated by His42.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His42A hydrogen bond donor
Arg38A electrostatic stabiliser, hydrogen bond donor
Asn70A increase acidity, hydrogen bond acceptor
His170A metal ligand
His42A proton donor

Chemical Components

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

Step 1. His42 deprotonates hydrogen peroxide. The resulting anion coordinates to the Fe(III) centre of Hem350, displacing water and forming Cpd0.

Step 2. The O-O bond of Cpd0 undergoes heterolytic cleavage and the resulting hydroxide is protonated by His42. The porphryin ring and Fe(III) donate an electron each to the oxygen bound to the metal.

Step 3. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring, forming a phenolic radical and Cpd1.

Step 4. The oxygen of Cpd1 is protonated by water, which is in turn protonated by His42.

Step 5. The porphyrin ring transfers an electron to the Fe(IV) centre to give an isomer of Cpd1.

Step 6. Arg38 transfers a proton to the oxygen of Cpd1.

Step 7. Upon binding of the second phenol derivative, Arg38 deprotonates Cpd2. The doubly-protonated form of Cpd2 is less stable in the presence of the phenol derivative than in the presence of the phenolic radical. Therefore when the phenolic radical product dissociates and the phenolic derivative substrate associates, there is proton transfer to form the more stable isomer of Cpd2.

Step 8. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring radical, forming a phenolic radical.

Step 9. The metal-bound hydroxide is protonated by water, which is in turn protonated by His42.

Products.

Introduction

The reaction occurs in three steps:
The enzyme is first oxidised by hydrogen peroxide and is then reduced in two sequential one-electron transfer steps from reducing substrates (typically small molecule phenol derivatives). The mechanism of the first step is believed to be common to all haem peroxidases. HOOH is deprotonated by a water molecule which is deprotonated by His 42 to produce singly ionised Fe(III)-coordinated peroxide. The O-O bond of the coordinated peroxide is now heterolytically cleaved to generate an Fe(IV)=O species and a hydroxide ion, which is protonated by His 42. Arg 38 is though to promote the reaction by stabilising negative charge in the transient Fe(III)-OOH intermediate and associated transition states.

In the second step, a reducing phenol substrate binds to the active site and transfers an electron to haem and a proton to His 42. The proton transfer is thought to occur via an active site water molecule, and is proposed to be assisted by Arg 38 which forms a hydrogen bond to the substrate phenolic oxygen.

The product radical now dissociates from the active site and a second reducing phenol substrate is bound. This second reducing substrate transfers an electron to the haem, and a proton (via an active site water) to the Fe-coordinated O atom. This is accompanied by proton transfer from His 42 to the Fe-coordinated oxygen, so that the overall result is formation of a water molecule and regeneration of the resting state haem Fe(III).

Catalytic Residues Roles

UniProt PDB* (7atj)
Arg68 Arg38A Stabilises negative charge in the transient Fe(III)-OOH intermediate and associated transition states. Assists proton transfer from the phenol substrates to either His 42 or the ferryl oxygen. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
His72 His42A Deprotonates a water molecule which deprotonates hydrogen peroxide to give singly ionised Fe(III)-coordinated peroxide. Protonates the departing hydroxide ion during heterolytic cleavage of the O-O bond in the coordinated peroxide. Accepts a proton from the phenol substrate (via an active site water molecule). Protonates the Fe-coordinated oxygen in the final step of the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
His200 His170A Acts as the heme axial ligand. metal ligand
Asn100 Asn70A Modifies the pKa of His 42. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
*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

proton transfer, coordination to a metal ion, decoordination from a metal ion, overall reactant used, rate-determining step, heterolysis, electron transfer, radical formation, redox reaction, cofactor used, radical propagation, intermediate formation, overall product formed, proton relay, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Meno K et al. (2002), Acta Crystallogr D Biol Crystallogr, 58, 1803-1812. Structural analysis of the two horseradish peroxidase catalytic residue variants H42E and R38S/H42E: implications for the catalytic cycle. DOI:10.1107/s090744490201329x. PMID:12351824.
  2. Vidossich P et al. (2010), J Phys Chem B, 114, 5161-5169. On the role of water in peroxidase catalysis: a theoretical investigation of HRP compound I formation. DOI:10.1021/jp911170b. PMID:20345187.
  3. Gajhede M (2001), Biochem Soc Trans, 29, 91-98. Plant peroxidases: substrate complexes with mechanistic implications. DOI:10.1042/0300-5127:0290091. PMID:11356134.
  4. Henriksen A et al. (1999), J Biol Chem, 274, 35005-35011. The Structures of the Horseradish Peroxidase C-Ferulic Acid Complex and the Ternary Complex with Cyanide Suggest How Peroxidases Oxidize Small Phenolic Substrates. DOI:10.1074/jbc.274.49.35005. PMID:10574977.

Step 1. His42 deprotonates a water molecule which deprotonates hydrogen peroxide. The resulting anion coordinates to the Fe(III) centre of Hem350, displacing water and forming Cpd0.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
His42A hydrogen bond donor, hydrogen bond acceptor
Asn70A hydrogen bond acceptor
Arg38A hydrogen bond donor, electrostatic stabiliser
Asn70A increase basicity, electrostatic stabiliser
His42A proton acceptor

Chemical Components

proton transfer, coordination to a metal ion, decoordination from a metal ion, overall reactant used

Step 2. The O-O bond of Cpd0 undergoes heterolytic cleavage and the resulting hydroxide is protonated by His42. The porphryin ring and Fe(III) donate an electron each to the oxygen bound to the metal.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg38A electrostatic stabiliser
His42A electrostatic stabiliser
Arg38A hydrogen bond donor
His42A hydrogen bond donor
Asn70A hydrogen bond acceptor
His170A metal ligand
Asn70A increase acidity
His42A proton donor

Chemical Components

rate-determining step, heterolysis, proton transfer, electron transfer, radical formation, redox reaction, cofactor used

Step 3. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring, forming a phenolic radical and Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
His42A hydrogen bond acceptor
Arg38A hydrogen bond donor
Asn70A hydrogen bond acceptor, increase basicity, electrostatic stabiliser
His42A proton acceptor

Chemical Components

proton transfer, electron transfer, radical propagation, redox reaction, intermediate formation, overall product formed, overall reactant used, proton relay

Step 4. The oxygen of Cpd1 is protonated by water, which is in turn protonated by His42.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
His42A hydrogen bond donor
Arg38A hydrogen bond donor
Asn70A hydrogen bond acceptor, increase acidity
His42A proton donor

Chemical Components

proton transfer, proton relay

Step 5. The porphyrin ring transfers an electron to the Fe(IV) centre to give an isomer of Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
Arg38A hydrogen bond donor
His42A hydrogen bond donor, hydrogen bond acceptor
Asn70A hydrogen bond acceptor

Chemical Components

electron transfer, radical propagation, intermediate formation

Step 6. Arg38 transfers a proton to the oxygen of Cpd1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
Arg38A hydrogen bond donor
His42A hydrogen bond donor
Asn70A hydrogen bond acceptor
Arg38A proton donor

Chemical Components

proton transfer

Step 7. Upon binding of the second phenol derivative, Arg38 deprotonates Cpd2. The doubly-protonated form of Cpd2 is less stable in the presence of the phenol derivative than in the presence of the phenolic radical. Therefore when the phenolic radical product dissociates and the phenolic derivative substrate associates, there is proton transfer to form the more stable isomer of Cpd2.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
Arg38A hydrogen bond acceptor
His42A hydrogen bond acceptor
Asn70A hydrogen bond acceptor
His42A hydrogen bond donor
Arg38A proton acceptor

Chemical Components

proton transfer

Step 8. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring radical, forming a phenolic radical.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
Arg38A hydrogen bond donor
His42A hydrogen bond donor, hydrogen bond acceptor
Asn70A hydrogen bond acceptor
Asn70A increase acidity, electrostatic stabiliser
His42A proton acceptor

Chemical Components

proton transfer, electron transfer, radical propagation, native state of cofactor regenerated, overall product formed, overall reactant used, proton relay

Step 9. The metal-bound hydroxide is protonated by water, which is in turn protonated by His42.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170A metal ligand
Arg38A hydrogen bond donor
His42A hydrogen bond donor
Asn70A hydrogen bond acceptor, increase acidity
His42A proton donor

Chemical Components

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

Step 1. His42 deprotonates a water molecule which deprotonates hydrogen peroxide. The resulting anion coordinates to the Fe(III) centre of Hem350, displacing water and forming Cpd0.

Step 2. The O-O bond of Cpd0 undergoes heterolytic cleavage and the resulting hydroxide is protonated by His42. The porphryin ring and Fe(III) donate an electron each to the oxygen bound to the metal.

Step 3. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring, forming a phenolic radical and Cpd1.

Step 4. The oxygen of Cpd1 is protonated by water, which is in turn protonated by His42.

Step 5. The porphyrin ring transfers an electron to the Fe(IV) centre to give an isomer of Cpd1.

Step 6. Arg38 transfers a proton to the oxygen of Cpd1.

Step 7. Upon binding of the second phenol derivative, Arg38 deprotonates Cpd2. The doubly-protonated form of Cpd2 is less stable in the presence of the phenol derivative than in the presence of the phenolic radical. Therefore when the phenolic radical product dissociates and the phenolic derivative substrate associates, there is proton transfer to form the more stable isomer of Cpd2.

Step 8. His42 deprotonates water, which then deprotonates the phenolic derivative substrate. The phenolate transfers an electron to the porphyrin ring radical, forming a phenolic radical.

Step 9. The metal-bound hydroxide is protonated by water, which is in turn protonated by His42.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, Steven Smith, Amelia Brasnett