Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (β-hematin formation)

Abstract

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (β-hematin formation) through its oxidation. Heme when added to HRP compound II (Fe^IV = O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, Fe ^III) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1^H=14.75 G, a2^H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.