CPO

CPO (EC 1.11.1.10, chloroperoxidase) belongs to the the class of oxido-reductase, is a peroxidase. The systematic name of this enzyme is chloride:hydrogen-peroxide oxidoreductase. Other names in common use include chloroperoxidase, CPO, vanadium haloperoxidase. CPO catalyzes the chlorination of organic compounds that combine inorganic substrate chloride and hydrogen peroxide to produce Cl⁺, replacing protons in hydrocarbon substrate. It uses heme or vanadium as a cofactor during the reaction. CPO is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Each molecule of CPO contains a ferriprotoporphyrin IX prosthetic group that catalyzes various reactions such as halogenation, peroxidation and epoxidation. For many years, CPO has been the only peroxidase known to exhibit similar spectral properties to P450, but similar enzymes have now been found in other organisms.

Structure

The complete CPO gene encodes a 373 amino acid protein, which is processed by two proteolytic processes into mature CPO, one at the N-terminus to remove a 20 amino acid signal peptide, the other at the C- terminus to remove a 52 amino acid peptide. The tertiary structure of CPO contains 50% helices similar to other peroxidases and P450s, but the overall topology is not identical to other heme enzymes. There is similarity in the structure near the active site. Like other heme peroxidases, CPO folds into N-terminal and C-terminal domains, and the heme is sandwiched between two domains. The fold comprises 8 α-helical segments (A-H), three short 3₁₀ helices (C', D', G') and a short anti-parallel β pair. Like other heme enzymes, CPO has a proximal helix (helix A) on the heme side containing the protein axial heme ligand, and a distal helix (helix F) that provides the catalytic group required for peroxide activation. In CPO, the proximal helix A is perpendicular to the heme plane, but in peroxidase and P450, it is parallel to the heme plane.

Figure 1. Structure of CPO. (Sundaramoorthy M. 1995)

Studies have shown that Cys29 provides a thiolated heme ligand, the axial ligand residue located in the N-terminal domain of the CPO. The other two cysteines, Cys79 and Cys87, form a disulfide bond. The three prolines (Pro9, Pro230 and Pro292) have a cis conformation, in which Pro9 exhibits a classical cis-proline turn. The polypeptide in residues 26-37 surrounds the cysteine ligand (Cys29) and forms a rigid scaffolding for the interaction of iron and sulfur. The side chain of Asn33 and the backbone atoms of Ala27 form a pair of hydrogen bonds, while the side chains of Arg26 and Asp37 form hydrogen bonds with each other.

Figure 2. Stereo diagram showing the hydrogen-bonding interactions. (Sundaramoorthy M. 1995)

Catalytic Mechanism

Among all the heme peroxidases currently known, CPO catalyzes a variety of reaction types and is considered to be the most versatile enzyme. The first type of reaction is halogenation using hydrogen peroxide or other hydroperoxide as an initiator, a halide (other than fluoride) and a compound susceptible to the halogenation reaction as a substrate. The second type of reaction is similar to the first type of reaction, but the substrate is uric acid. The third type of reaction is a peroxidation reaction. CPO can catalyze the disproportionation of hydrogen peroxide, which is a catalase-specific reaction. In addition, CPO can also catalyze the P450-like monooxygenation reaction and the dehalogenation of selected halogenated aromatic compounds.

Figure 3. Reactions catalyzed by CPO. (Poulos T.L. 2014)

The resting state of the CPO contains a ferricheme center. The binding of hydrogen peroxide to the heme iron initiates the reaction, and then two electrons are transferred from the heme center to hydrogen peroxidase, resulting in the cleavage of the peroxide bond and formation of water molecules and activated heme intermediate compound I. Compound I is a common intermediate for most heme peroxidase-catalyzed reactions. The chemical structure of compound I is an oxo-ferryl porphyrin cation-radical [heme (Fe⁴⁺=O)^•+]. Depending on the nature of the protein, compound I of the heme protein can participate in different chemical reactions. In the catalase reaction, compound I interacts with the second molecule of hydrogen peroxide to form dioxygen and reduce heme iron to ferric iron. In peroxidation reaction, compound I can convert an organic molecule (HA) into a radical product (A•) and be reduced to compound II by transferring one electron to an organic substrate. The structure of the compound II is Fe^IV=O. The oxidation of a second substrate can regenerate ferric iron. In the chlorination reaction, compound I interacts with chloride ions to form a ferric complex (heme Fe³⁺-O-X) called compound X, which is unstable and easily decomposed into a resting state of the enzyme, halonium ion (X⁺) and hydroxyl ion (OH⁻), they can be further reacted to form hypohalous acid (HOX).

Figure 4. Mechanism of CPO-catalyzed reactions. (Lin J. 2012)

Application

CPO is one of the most promising heme peroxidases for synthetic applications. Optically active 2,3-epoxy alcohols are important components of synthetic biological or pharmaceutical important compounds. The sharpless asymmetrical epoxidation of allylic alcohol is one of the most versatile methods for the preparation of optically active epoxy alcohols. The epoxidation of CPO to olefins exhibits high enantioselectivity, but the enzyme does not catalyze the formation of allylic alcohols to form epoxides, but instead forms aldehydes.

References

1. Sundaramoorthy, M., Terner, J., Poulos, T.L. The crystal structure of chloroperoxidase: a heme peroxidase-cytochrome P450 functional hybrid. Structure, 1995, 3:1367-1377.
2. Lin, J. Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by sitedirected mutagenesis and isotopic labeling. FIU Electronic Theses and Dissertations, 2012.
3. Poulos, T.L. Heme enzyme structure and function. Chem Rev. 2014, 114(7): 3919–3962.
4. Kiljunen, E., Kanerva, L.T. Novel applications of chloroperoxidase: enantioselective oxidation of racemic epoxyalcohols. Tetrahedron: Asymmetry. 1999, 10: 3529–3535.