Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis.
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.
Denisov
Ilia G
IG
Department of Biochemistry, Center for Biophysics and Computational Biology, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
Makris
Thomas M
TM
Sligar
Stephen G
SG
D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D005720:Gamma Rays; D006418:Heme; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D007501:Iron; D007700:Kinetics; D013053:Spectrophotometry; D013057:Spectrum Analysis
D006418:Heme; D007501:Iron; D006735:Horseradish Peroxidase