Cytochrome-c peroxidase (mono-heme type)

 

Cytochrome c peroxidase (CcP) is a heme-dependent yeast mitochondrial oxidoreductase enzyme that catalyses the the reduction of hydrogen peroxide to water, using reducing equivalents ferrocytochrome c. The catalytic mechanism couples the one-electron oxidation of ferrocytochrome c to the two-electron reduction of hydrogen peroxide to water.

 

Reference Protein and Structure

Sequence
P00431 UniProt (1.11.1.5) IPR002207 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1dj1 - CRYSTAL STRUCTURE OF R48A MUTANT OF CYTOCHROME C PEROXIDASE (1.93 Å) PDBe PDBsum 1dj1
Catalytic CATH Domains
1.10.520.10 CATHdb 1.10.420.10 CATHdb (see all for 1dj1)
Cofactors
Heme b (1)
Click To Show Structure

Enzyme Reaction (EC:1.11.1.5)

iron(2+)
CHEBI:29033ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
→
iron(3+)
CHEBI:29034ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: Apocytochrome c peroxidase, Cytochrome c peroxidase, Cytochrome c-551 peroxidase, Cytochrome c-H(2)O oxidoreductase, Cytochrome peroxidase, Mesocytochrome c peroxidase azide, Mesocytochrome c peroxidase cyanate, Mesocytochrome c peroxidase cyanide,

Enzyme Mechanism

Introduction

The catalytic cycle consists of at three major steps:

1) Reaction of CcP with peroxide to form Compound I (CpdI)
CcP(Fe3+) + H2O2 => CpdI + H2O

2) One-electron reduction of CpdI by Cc(Fe2+) to Compound II (CpdII)
CpdI + Cc(Fe2+) => CpdII + Cc(Fe3+)

3) Subsequent reduction of CpdII by another Cc(Fe2+) to regenerate the resting-state CcP(Fe3+)
CpdII + Cc(Fe2+) => CcP(Fe3+) + Cc(Fe3+)

The mechanism of electron transfer is complex and not fully understood.

CcP (Fe(III); Trp) reacts with hydrogen peroxide to form a peroxy complex. Binding is facilitated by the abstraction of a proton by His52 from the peroxide. The positively charged His52 and Arg48 stabilise the transition state to produce a hydroxide anion. The distal His52 donates its proton to the hydroxide ion, releasing the first molecule of water. Heterolytic cleavage of the oxygen-oxygen bond of the bound peroxide leaves an electron-deficient 'oxene' bound to the heme iron. The oxene is stabilised by the electron transfer from the heme iron and from the porphyrin ring, producing the classic peroxidase compound I, containing an oxy-ferryl Fe(IV) porphyrin pi-cation radical species, which is stabilised by hydrogen bonding between the iron-bound oxygen and Arg48 and Trp51. The proximal Trp-191, reduces the porphyrin pi-cation radical to generate the stable form of CcP compound I (CcP-I), which retains the oxidising equivalents of peroxide as a stable oxy-ferryl heme and an indolyl cation radical at Trp 191 (Fe(IV)=O; Trp*+).

Ferrocytochrome c (Cc2+) binds to the high affinity binding site and intracomplex electron transfer reduces the Trp191 radical site in the CcP-I, producing peroxidase compound II, CcP-II(F) (Fe(IV)=O; Trp), and ferricytochrome c (Cc3+). Following the reduction of the Trp191 radical, the oxy-ferryl heme may be reduced and the Trp191 cation radical regenerated to form CcP-II(R) (Fe(III)-OH; Trp*+). It is thought that the reaction mechanism involves an equilibrium between the two forms of CcP-II. Cc3+ dissociates and a second molecule of Cc2+ binds the high affinity site. Intermolecular electron transfer from Cc2+ either reduces the Fe(IV) site in CcP-II(F) directly, or indirectly by reducing the Trp191 radical of CcP-II(R), producing a second molecule of water and the CcP/C3+ complex. The Cc3+ dissociates to regenerate the native enzyme CcP (Fe(III); Trp).

Catalytic Residues Roles

UniProt PDB* (1dj1)
His119 His52(49)A His52, with Arg48, stabilises the transition state for the heterolytic cleavage of the oxygen-oxygen bond of the bound peroxide to form CcP-I and water.
His52 acts as a base catalyst during hydrogen peroxide binding, and then an acid as it donates its proton to the hydroxide ion formed to produce water.		 proton acceptor, electrostatic stabiliser, proton donor
Arg115 Ala48(45)A Arg48, with His52, stabilises the transition state for the heterolytic cleavage of the oxygen-oxygen bond of the bound peroxide to form CcP-I and water. Arg48, with Trp51, stabilises the classical peroxidase compound I by hydrogen bond interactions between the iron-bound oxygen and both residues. electrostatic stabiliser
Trp258 Trp191(188)A Radical formation occurs when Trp is reducing the heme.
Trp191 reduces the heme porphyrin pi-cation radical (of the classic peroxidase compound I) to generate CcP-I, containing an oxy-ferryl heme and a Trp191 cation radical species (Fe(IV)=O; Trp*+).
Trp191 cation radical oxidises the first molecule of C2+, forming C3+ and CcP-II(F) (Fe(IV)=O; Trp). The native enzyme (Fe(III); Trp)is regenerated either hy the reduction of the CcP-II(F) heme directly by a second molecule of C2+, or by the reduction of CcP-II(F) by the reduced Trp191 to form CcP-II(R) (Fe(III)-OH; Trp*+), followed by the reduction of CcP-II(R) by the second molecule of C2+.		 single electron acceptor, single electron donor
*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, overall reactant used, heterolysis, overall product formed, redox reaction, electron transfer, decoordination from a metal ion

References

  1. Hirst J et al. (2000), J Biol Chem, 275, 8582-8591. Unusual Oxidative Chemistry of Nomega -Hydroxyarginine and N-Hydroxyguanidine Catalyzed at an Engineered Cavity in a Heme Peroxidase. DOI:10.1074/jbc.275.12.8582. PMID:10722697.
  2. Kathiresan M et al. (2017), Chem Sci, 8, 1152-1162. LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2. DOI:10.1039/c6sc03125k. PMID:28451256.
  3. Bernini C et al. (2014), J Phys Chem B, 118, 9525-9537. In silico spectroscopy of tryptophan and tyrosine radicals involved in the long-range electron transfer of cytochrome c peroxidase. DOI:10.1021/jp5025153. PMID:25084495.
  4. Volkov AN et al. (2011), Biochim Biophys Acta, 1807, 1482-1503. The complex of cytochrome c and cytochrome c peroxidase: the end of the road? DOI:10.1016/j.bbabio.2011.07.010. PMID:21820401.
  5. Erman JE et al. (2002), Biochim Biophys Acta, 1597, 193-220. Yeast cytochrome c peroxidase: mechanistic studies via protein engineering. DOI:10.1016/s0167-4838(02)00317-5. PMID:12044899.
  6. Mei H et al. (1996), Biochemistry, 35, 15800-15806. Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site. DOI:10.1021/bi961487k. PMID:8961943.
  7. Vitello LB et al. (1993), Biochemistry, 32, 9807-9818. Effect of arginine-48 replacement on the reaction between cytochrome c peroxidase and hydrogen peroxide. DOI:10.1021/bi00088a036. PMID:8396973.
  8. Goodin DB et al. (1991), Biochemistry, 30, 4953-4962. Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer. DOI:10.1021/bi00234a017. PMID:1645185.
  9. Geren L et al. (1991), Biochemistry, 30, 9450-9457. Photoinduced electron transfer between cytochrome c peroxidase and yeast cytochrome c labeled at Cys 102 with (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]ruthenium2+. DOI:10.1021/bi00103a009.
  10. Vitello LB et al. (1990), Biochim Biophys Acta, 1038, 90-97. Characterization of the hydrogen peroxide - enzyme reaction for two cytochrome c peroxidase mutants. DOI:10.1016/0167-4838(90)90015-8. PMID:2156573.
  11. Erman JE et al. (1989), Biochemistry, 28, 7992-7995. Detection of an oxyferryl porphyrin .pi.-cation-radical intermediate in the reaction between hydrogen peroxide and a mutant yeast cytochrome c peroxidase. Evidence for tryptophan-191 involvement in the radical site of compound I. DOI:10.1021/bi00446a004. PMID:2557891.

Step 1. His52 acts as a general base to abstract a proton from the peroxide. The peroxide then binds to the Fe3+ centre.

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Catalytic Residues Roles

Residue Roles
His52(49)A electrostatic stabiliser
Ala48(45)A electrostatic stabiliser
His52(49)A proton acceptor

Chemical Components

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

Step 2. The oxygen-oxygen bond is cleaved heterolytically leaving an electron-deficient 'oxene' bound to the heme iron. His52 donates its proton to the hydroxide to form water. The electron-deficient 'oxene' is stabilised by electron transfer from the porphyrin ring and heme iron, resulting in a oxy-ferryl Fe(IV) porphyrin pi-cation radical species.

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Catalytic Residues Roles

Residue Roles
Ala48(45)A electrostatic stabiliser
His52(49)A proton donor

Chemical Components

proton transfer, heterolysis, overall product formed, redox reaction

Step 3. Trp191 reduces the porphryin pi-cation radical to generate the stable form of CcP Compound I (Cpd-I).

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Catalytic Residues Roles

Residue Roles
Ala48(45)A electrostatic stabiliser
Trp191(188)A single electron donor

Chemical Components

electron transfer

Step 4. Cc(Fe2+) binds to the high affinity binding site and transfers an electron to Trp191, forming Compound II (CpdII) and Cc(Fe3+).

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Catalytic Residues Roles

Residue Roles
Trp191(188)A single electron acceptor

Chemical Components

electron transfer, redox reaction

Step 5. The oxy-ferryl heme is reduced by Trp191 and the radical Trp191 cation is regenerated. Oxygen accepts a proton to form a hydroxide ion.

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Catalytic Residues Roles

Residue Roles
Ala48(45)A electrostatic stabiliser
Trp191(188)A single electron donor

Chemical Components

proton transfer, electron transfer, redox reaction

Step 6. The hydroxide ion accepts another proton and a second molecule of water is produced.

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Catalytic Residues Roles

Residue Roles
Ala48(45)A electrostatic stabiliser

Chemical Components

overall product formed, decoordination from a metal ion

Step 7. Cc3+ dissociates and a second molecule of Cc2+ binds. The Trp191 radical cation is reduced and Cc3+ is formed. Cc3+ dissociates and the native state of the enzyme is restored.

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Catalytic Residues Roles

Residue Roles
Ala48(45)A electrostatic stabiliser
Trp191(188)A single electron acceptor

Chemical Components

redox reaction, overall product formed, electron transfer
Download: Image, Marvin File

Step 1. His52 acts as a general base to abstract a proton from the peroxide. The peroxide then binds to the Fe3+ centre.

Step 2. The oxygen-oxygen bond is cleaved heterolytically leaving an electron-deficient 'oxene' bound to the heme iron. His52 donates its proton to the hydroxide to form water. The electron-deficient 'oxene' is stabilised by electron transfer from the porphyrin ring and heme iron, resulting in a oxy-ferryl Fe(IV) porphyrin pi-cation radical species.

Step 3. Trp191 reduces the porphryin pi-cation radical to generate the stable form of CcP Compound I (Cpd-I).

Step 4. Cc(Fe2+) binds to the high affinity binding site and transfers an electron to Trp191, forming Compound II (CpdII) and Cc(Fe3+).

Step 5. The oxy-ferryl heme is reduced by Trp191 and the radical Trp191 cation is regenerated. Oxygen accepts a proton to form a hydroxide ion.

Step 6. The hydroxide ion accepts another proton and a second molecule of water is produced.

Step 7. Cc3+ dissociates and a second molecule of Cc2+ binds. The Trp191 radical cation is reduced and Cc3+ is formed. Cc3+ dissociates and the native state of the enzyme is restored.

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett