Abstract
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 pi-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 non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.
Highlights
Horseradish peroxidase (HRP)1 is a highly characterized heme enzyme that has historically served as a paradigm for evaluating the rules of formation of redox active heme-oxygen intermediates, including the ferryl-oxo heme complexes commonly known as “Compound I” and “Compound II.” A detailed mechanism for Compound I formation in the reaction of H2O2 with peroxidases was formulated by Poulos and Kraut on the basis of structural analysis of cytochrome c peroxidase active center [1] and has been successfully applied to other enzymes [2,3,4,5], as shown in Reaction 1
The physiological substrate H2O2 brings both protons to the active site of horseradish peroxidase (HRP), and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide
The absorption spectrum of this intermediate has a red-shifted Soret band and blue-shifted Q-bands (556 and 526 nm). This spectrum is in very good agreement with the spectrum of the (Fe3ϩP-OOHϪ) complex of heme oxygenase,2 and with the kinetically resolved spectrum of the same intermediate obtained in stopped-flow studies of the reaction of Mb mutant H64Q with H2O2 [24]
Summary
Horseradish peroxidase (HRP)1 is a highly characterized heme enzyme that has historically served as a paradigm for evaluating the rules of formation of redox active heme-oxygen intermediates, including the ferryl-oxo heme complexes commonly known as “Compound I” and “Compound II.” A detailed mechanism for Compound I formation in the reaction of H2O2 with peroxidases was formulated by Poulos and Kraut on the basis of structural analysis of cytochrome c peroxidase active center [1] and has been successfully applied to other enzymes [2,3,4,5], as shown in Reaction 1. 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.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
