Chloride peroxidase (heme dependent)

 

The heme-dependent chloroperoxidase (CPO) is a ~250 residue heme-containing glycoprotein that is secreted by various fungi. It was first identified in Caldariomyces fumago where it catalyses the hydrogen peroxide-dependent chlorination of cyclopentanedione during the biosynthesis of the antibiotic caldarioymcin. As with many of the peroxidases, it also catalyses the iodination and bromination of a wide range of substrates, dehydrogenation reactions, catalyse-type reactions (facilitating the decomposition of hydrogen peroxide to oxygen and water) and P450-like oxygen insertion reactions. The capability of chloroperoxidase to perform these diverse reactions makes it one of the most versatile of all known heme proteins.

 

Reference Protein and Structure

Sequence
P04963 UniProt (1.11.1.10) IPR000028 (Sequence Homologues) (PDB Homologues)
Biological species
Leptoxyphium fumago (Fungus) Uniprot
PDB
2cpo - CHLOROPEROXIDASE (2.1 Å) PDBe PDBsum 2cpo
Catalytic CATH Domains
1.10.489.10 CATHdb (see all for 2cpo)
Cofactors
Heme b (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.11.1.10)

hydron
CHEBI:15378ChEBI
+
chloride
CHEBI:17996ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
+
alkane
CHEBI:18310ChEBI
→
water
CHEBI:15377ChEBI
+
chloroalkane
CHEBI:23128ChEBI
Alternative enzyme names: Chloroperoxidase, CPO, Vanadium haloperoxidase,

Enzyme Mechanism

Introduction

The hydrogen peroxide, once bound to the heme cofactor, undergoes self de-protonation to produce the activated species. The activated hydrogen peroxide species collapses, with concomitant movement of an electron pair to the iron centre of heme, and shuttling of a single electron out into the porphyrin ring of the cofactor. This generates the so-called compound I. The chloride ion initiates a nucleophilic attack on the oxo group of Compound I in an addition reaction which results in the donation of two electrons to the iron centre, and a single electron being transferred to the prophyrin ring. The intermediate deprotonates Glu183, which in turn deprotonates His105. His105 deprotonates Glu183, which in turn deprotonates the alkane. This initiates a nucleophilic attack upon the heme-bound hypochlorous acid in a substitution reaction.

Catalytic Residues Roles

UniProt PDB* (2cpo)
Cys50 Cys29(30)A Acts as the axial ligand to the iron of the heme cofactor. covalently attached, activator, metal ligand
Glu204, His126 Glu183(184)A, His105(106)A Acts as a general acid/base and part of a proton relay chain. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, activator, electrostatic stabiliser
Asp127 Asp106(107)A Activates His105. 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, overall reactant used, intermediate formation, heterolysis, redox reaction, cofactor used, overall product formed, bimolecular nucleophilic addition, proton relay, bimolecular nucleophilic substitution, native state of enzyme regenerated, inferred reaction step, intermediate terminated

References

  1. Wagenknecht HA et al. (1997), Chem Biol, 4, 367-372. Identification of intermediates in the catalytic cycle of chloroperoxidase. DOI:10.1016/s1074-5521(97)90127-7. PMID:9195874.
  2. Pardillo AD et al. (2015), J Phys Chem B, 119, 12590-12602. Proximal Pocket Hydrogen Bonds Significantly Influence the Mechanism of Chloroperoxidase Compound I Formation. DOI:10.1021/acs.jpcb.5b06324. PMID:26339752.
  3. Chen H et al. (2008), J Phys Chem B, 112, 9490-9500. Quantum mechanical/molecular mechanical study on the mechanisms of compound I formation in the catalytic cycle of chloroperoxidase: an overview on heme enzymes. DOI:10.1021/jp803010f. PMID:18597525.
  4. Wang X et al. (2003), J Biol Chem, 278, 7765-7774. Two-dimensional NMR study of the heme active site structure of chloroperoxidase. DOI:10.1074/jbc.M209462200. PMID:12488315.
  5. Woggon WD et al. (2001), J Inorg Biochem, 83, 289-300. Synthetic active site analogues of heme–thiolate proteins Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase. DOI:10.1016/s0162-0134(00)00175-6. PMID:11293549.
  6. Sundaramoorthy M et al. (1998), Chem Biol, 5, 461-473. Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modeling studies. DOI:10.1016/s1074-5521(98)90003-5. PMID:9751642.

Step 1. Hydrogen peroxide binds to the Fe(III) centre of the heme cofactor, displacing water. The hydrogen peroxide undergoes self de-protonation to produce the activated species.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand, activator
His105(106)A electrostatic stabiliser, hydrogen bond donor
Asp106(107)A electrostatic stabiliser, hydrogen bond acceptor
Glu183(184)A activator, hydrogen bond acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The activated hydrogen peroxide species collapses, with concomitant movement of an electron pair to the iron centre of heme, and shuttling of a single electron out into the porphyrin ring of the cofactor. This generates the so-called compound I.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand, activator
His105(106)A electrostatic stabiliser, hydrogen bond donor
Asp106(107)A electrostatic stabiliser, hydrogen bond acceptor
Glu183(184)A electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

heterolysis, redox reaction, cofactor used, overall product formed, intermediate formation

Step 3. The chloride ion initiates a nucleophilic attack on the oxo group of Compound I in an addition reaction which results in the donation of two electrons to the iron centre, and a single electron being transferred to the prophyrin ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand, activator
His105(106)A electrostatic stabiliser, hydrogen bond donor
Asp106(107)A electrostatic stabiliser, hydrogen bond acceptor
Glu183(184)A electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, redox reaction, overall reactant used, intermediate formation

Step 4. The intermediate deprotonates Glu183, which in turn deprotonates His105.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand, activator
His105(106)A hydrogen bond donor
Asp106(107)A increase acidity, hydrogen bond acceptor
Glu183(184)A hydrogen bond acceptor, hydrogen bond donor, proton relay
His105(106)A proton donor
Glu183(184)A proton donor, proton acceptor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 5. His105 deprotonates Glu183, which in turn deprotonates the alkane. This initiates a nucleophilic attack upon the heme-bound hypochlorous acid in a substitution reaction. R represents the organic portion of the substrate to be halogenated. The exact mechanism of this step is unclear and may well depend upon the nature of the R group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand, activator
His105(106)A hydrogen bond donor, hydrogen bond acceptor
Asp106(107)A increase basicity, hydrogen bond acceptor
Glu183(184)A hydrogen bond acceptor, hydrogen bond donor, proton relay
His105(106)A proton acceptor
Glu183(184)A proton donor, proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, proton relay, intermediate formation, overall product formed

Step 6. The exact mechanism by which the hydroxide is re-protonated and then dissociates from the Fe(III) centre is not entirely clear. The proton probably comes from a water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys29(30)A covalently attached, metal ligand
His105(106)A electrostatic stabiliser, hydrogen bond donor
Asp106(107)A electrostatic stabiliser, hydrogen bond acceptor
Glu183(184)A electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

proton transfer, overall product formed, native state of enzyme regenerated, inferred reaction step, intermediate terminated
Download: Image, Marvin File

Step 1. Hydrogen peroxide binds to the Fe(III) centre of the heme cofactor, displacing water. The hydrogen peroxide undergoes self de-protonation to produce the activated species.

Step 2. The activated hydrogen peroxide species collapses, with concomitant movement of an electron pair to the iron centre of heme, and shuttling of a single electron out into the porphyrin ring of the cofactor. This generates the so-called compound I.

Step 3. The chloride ion initiates a nucleophilic attack on the oxo group of Compound I in an addition reaction which results in the donation of two electrons to the iron centre, and a single electron being transferred to the prophyrin ring.

Step 4. The intermediate deprotonates Glu183, which in turn deprotonates His105.

Step 5. His105 deprotonates Glu183, which in turn deprotonates the alkane. This initiates a nucleophilic attack upon the heme-bound hypochlorous acid in a substitution reaction. R represents the organic portion of the substrate to be halogenated. The exact mechanism of this step is unclear and may well depend upon the nature of the R group.

Step 6. The exact mechanism by which the hydroxide is re-protonated and then dissociates from the Fe(III) centre is not entirely clear. The proton probably comes from a water molecule.

Products.

Contributors

Gemma L. Holliday, Craig Porter, Amelia Brasnett