Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Ilia G. Denisov, Thomas M. Makris, Stephen G. Sligar

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Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo π-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is nonphysiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via re-arrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.

Original languageEnglish (US)
Pages (from-to)42706-42710
Number of pages5
JournalJournal of Biological Chemistry
Issue number45
StatePublished - Nov 8 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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