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

Department of Chemistry, Laboratory of Organic Chemistry, A.I. Virtasen Aukio 1, University of Helsinki, Finland.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2010; 1797(5):531-42. DOI: 10.1016/j.bbabio.2010.01.017
Source: PubMed

ABSTRACT Horseradish peroxidase was verified to catalyze, without any phenol, the hydrogen peroxide oxidation of chlorophyll a (Chl a), solubilized with Triton X-100. The 13(2)(S) and 13(2)(R) diastereomers of 13(2)-hydroxyChl a were characterized as major oxidation products (ca. 60%) by TLC on sucrose, UV-vis, (1)H, and (13)C NMR spectra, as well as fast-atom bombardment MS. A minor amount of the 15(2)-methyl, 17(3)-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 13(1),15(2)-dimethyl, 17(3)-phytyl ester of Mg-purpurin 7. The side products (ca. 10%) were suggested to include the 17(3)-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-13(2) deprotonation of Chl a, which resulted in the Chl a enolate ion resonance hybrid. The Chl enolate was then oxidized to the Chl 13(2)-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 13(2)-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.

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