Mechanism for the reactivation of the peroxidase activity of human cyclooxygenases: investigation using phenol as a reducing cosubstrate

Abstract

It has been known for many years that the peroxidase activity of cyclooxygenase 1 and 2 (COX-1 and COX-2) can be reactivated in vitro by the presence of phenol, which serves as a reducing compound, but the underlying mechanism is still poorly understood. In the present study, we use phenol as a model compound to investigate the mechanism by which the peroxidase activity of human COXs is reactivated after each catalytic cycle. Molecular docking and quantum mechanics calculations are carried out to probe the interaction of phenol with the peroxidase site of COXs and the reactivation mechanism. It is found that the oxygen atom associated with the Fe ion in the heme group (i.e., the complex of Fe ion and porphyrin) of COXs can be removed by addition of two protons. Following its removal, phenol can readily bind inside the peroxidase active sites of the COX enzymes, and directly interact with Fe in heme to facilitate electron transfer from phenol to heme. This investigation provides theoretical evidence for several intermediates formed in the COX peroxidase reactivation cycle, thereby unveiling mechanistic details that would aid in future rational design of drugs that target the peroxidase site.

Scheme 1

A proposed mechanism for the reactivation of the catalytic cycle of the COX peroxidase by phenol. PPIX is shown as an abbreviated parallelogram, and the imidazole ring of His388 as Im. Also, PGG₂ is for prostaglandin G₂, and PGH₂ for prostaglandin H₂. The proposed mechanisms for reaction 1 and 2 are depicted beneath the main scheme. In these two reactions, two protons attack the oxygen atom of Fe=O, resulting in the formation of H₂O molecule. Detailed molecular orbital analysis of reaction 1 is shown in Scheme 2.

Figure 1

Molecular docking analysis of the binding mode of phenol in both non-ionized and ionized states inside the peroxidase active sites of human COX-1 and COX-2 with PPIX^•+Fe^IV=O and PPIX²⁺Fe^III. The protein structures are shown as solid ribbons. Carbon atoms in PPIX^•+Fe^IV=O and PPIX²⁺Fe^III are colored in green, nitrogen in blue, oxygen in red, hydrogen in white, and iron in bice. Carbon atoms in phenol are colored in yellow, oxygen in red and hydrogen in white. In addition, the dash line corresponds to the distance between phenol’s oxygen atom and iron in PPIX^•+Fe^IV=O or PPIX²⁺Fe^III.

Figure 2

Optimization of reaction between two protons and Por^•+Fe^IV=O. ²LS doublet spin state, ⁴IS quartet spin state, ⁶HS sextet spin state. Color representation of atoms: gray for carbon, white for hydrogen, red for oxygen, and blue for nitrogen.

Figure 3

Optimized structures of Por^•+Fe^IV=O, Por²⁺Fe^III and PorFe^III. ²LS doublet spin state, ⁴IS quartet spin state, ⁶HS sextet spin state. Color representation of atoms: gray for carbon, white for hydrogen, red for oxygen, and blue for nitrogen.

Scheme 2

Electron transfer from ²Por^•+Fe^IV=O to ²Por²⁺Fe^III‒OH when the first proton binds to the oxygen atom of the Fe=O group.

Scheme 3

Electron transfer from phenol ion to ²Por²⁺Fe^III forming ¹Por²⁺Fe^II and ³Por^•+Fe^III during the first reduction.

Scheme 4

The Gibbs free energy surface of the proposed cycle.

Figure 4

Molecular docking analysis of the binding interactions between phenol ion and amino acid residues in the peroxidase active site of human COX-2 in complex with PPIX²⁺Fe^III . (A) The structure of COX-2 in complex with phenol ion and PPIX²⁺Fe^III. (B) Two-dimensional (2D) interaction diagram of the docked phenol ion and key residues in the peroxidase active site of COX-2. (C) The zoom-in view of the docked phenol ion inside the peroxidase site of COX-2 with PPIX²⁺Fe^III. The protein structure is shown as solid ribbons, with different colors representing different types of the secondary structures in (A) and (C). Phenol ion is shown as sticks, with different colors representing different atomic elements. Fe^III is shown as sphere and colored in pink. All the nearby residues are shown in line, with PPIX²⁺ ring in orange, H388 in marine, Y385 in dark green, H207 in raspberry, V291 in yellow, Q203 in wheat, and L294 in light blue. All intermolecular interactions that facilitate the binding of phenol ion are shown in dash line, with metal-acceptor in gray, salt bridge in cyan, Pi-alkyl in pink, hydrogen bond in green, and Pi-Pi in magenta.

Figure 5

Molecular docking analysis of the binding mode of phenol in both non-ionized and ionized states inside the peroxidase active sites of human COX-1 and COX-2 in complex with PPIX⁺Fe^III. The protein structures are shown as solid ribbons. Carbon atoms in PPIX⁺Fe^III are colored in green, nitrogen in blue, oxygen in red, hydrogen in white, and iron in bice. Carbon atoms in phenol are colored in yellow, oxygen in red, and hydrogen in white. In addition, the dash line corresponds to the distance between phenol’s oxygen atom and iron in PPIX⁺Fe^III.

Figure 6

Molecular docking analysis of the binding interactions between phenol ion and amino acid residues in the peroxidase active site of human COX-2 in complex with PPIX^•+Fe^III . (A) The structure of COX-2 in complex with phenol ion and PPIX^•+Fe^III. (B) Two-dimensional (2D) interaction diagram of the docked phenol ion and key residues in the peroxidase active site of COX-2. (C) The zoom-in view of docked phenol ion inside the peroxidase site of COX-2 with PPIX^•+Fe^III. The protein structure is shown as solid ribbons, with different colors representing different types of the secondary structures in (A) and (C). Phenol ion is shown as sticks, with different colors representing different atomic elements. Fe^III is shown as sphere and colored in pink. All the nearby residues are shown in line, with PPIX^•+ ring in orange, H388 in marine, Y385 in dark green, H207 in raspberry, V291 in yellow, and L294 in light blue. All intermolecular interactions that facilitate the binding of phenol ion are shown in dash line, with metal-acceptor in gray, salt bridge in cyan, Pi-alkyl in pink, and Pi-Pi in magenta.

Scheme 5

Electron transfer from phenol ion to ³Por^•+Fe^III forming ²PorFe^III during the second reduction.

Figure 7

Mulliken charges of Por²⁺Fe^III and Por^•+Fe^III. ¹LS singlet spin state, ²LS doublet spin state, ³LS triplet spin state, ⁴IS quartet spin state, ⁵IS quintet spin state, ⁶HS sextet spin state, ⁷IS septet spin state. The red color is for negative charges, and green for positive charges.