Horseradish peroxidase-catalyzed oxidation of chlorophyll a with hydrogen peroxide: Characterization of the products and mechanism of the reaction

Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13²(S) and 13²(R) diastereomers of 13²-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, ¹H, and ¹³C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15²-methyl, 17³-phytyl ester of Mg-unstable chlorin was identified on the basis of its UV-vis spectrum and reactivity with diazomethane, which converted it to the 13¹,15²-dimethyl, 17³-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17³-phytyl ester of Mg-purpurin 18, which is known to form easily from the Mg-unstable chlorin. The side products also included two red components with UV-vis spectral features resembling those of pure Chl a enolate anion. Hence, the two red components were assigned to the enolate anions of Chl a and pheophytin a or, alternatively, two different complexes of the Chl a enolate ion with Triton X-100. All the above products characterized by us are included in our published free-radical allomerization mechanism of Chl a, i.e. oxidation by ground-state dioxygen. The HRP clearly accelerated the allomerization process, but it did not produce bilins, that is, open-chain tetrapyrroles, the formation of which would require oxygenolysis of the chlorin macrocycle. In this regard, our results are in discrepancy with the claim by several researchers that ‘bilirubin-like compounds’ are formed in the HRP-catalyzed oxidation of Chl a. Inspection of the likely reactions that occurred on the distal side of the heme in the active centre of HRP provided a reasonable explanation for the observed catalytic effect of the HRP on the allomerization of Chl. In the active centre of HRP, the imidazole nitrogen of His-42 was considered to play a crucial role in the C-13² deprotonation of Chl a, which resulted in the Chl a enolate ion resonance hybrid. The Chl enolate was then oxidized to the Chl 13²-radical while the HRP Compound I was reduced to Compound II. The same reactive Chl derivatives, i.e. the Chl enolate anion and the Chl 13²-radical, which are produced twice in the HRP reaction cycle, happen to be the crucial intermediates in the initial stages of the Chl allomerization mechanism.