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Related Experiment Videos

Cobalt-substituted horseradish peroxidase.

M Y Wang, B M Hoffman, P F Hollenberg

    The Journal of Biological Chemistry
    |September 25, 1977
    PubMed
    Summary

    Cobalt reconstitution of horseradish peroxidase (HRP) yields a cobaltic holoenzyme. This cobaltous form (CoHRP) and its oxygenated analogue (oxy-CoHRP) allow EPR studies of the heme crevice, revealing a nitrogenous proximal ligand.

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    Area of Science:

    • Biochemistry
    • Biophysics
    • Enzymology

    Background:

    • Horseradish peroxidase (HRP) is a crucial enzyme with a ferric heme active site.
    • Reconstitution of HRP with cobalt porphyrin creates a cobaltic holoenzyme (Co3+HRP) with similar physicochemical properties to native HRP.
    • The cobaltic protein can be reduced to the cobaltous form (CoHRP), an analogue of ferroperoxidase.

    Purpose of the Study:

    • To investigate the heme crevice structure of CoHRP and oxy-CoHRP using Electron Paramagnetic Resonance (EPR) spectroscopy.
    • To explore the functional and catalytic properties of cobalt-reconstituted HRP analogues.
    • To compare the reactivity of cobalt-HRP analogues with native HRP and cytochrome P-450.

    Main Methods:

    • Cobalt reconstitution of horseradish peroxidase.
    • Reduction of cobaltic HRP to cobaltous HRP (CoHRP).
    • Oxygenation of CoHRP to form oxy-CoHRP.
    • Electron Paramagnetic Resonance (EPR) spectroscopy to analyze heme crevice structure and ligand interactions.
    • Assessment of catalytic activity with peroxidase substrates and oxidation-reduction reactions.

    Main Results:

    • The EPR spectrum of CoHRP shows a three-line 14N superhyperfine pattern, confirming a nitrogenous proximal axial ligand in the divalent state.
    • The EPR spectrum of oxy-CoHRP exhibits a large Aparallel(59Co) value of 23.2 G.
    • Co3+HRP does not catalyze classical peroxidase substrate oxidations.
    • Oxy-CoHRP participates in oxidation-reduction reactions analogous to the cytochrome P-450 catalytic cycle.

    Conclusions:

    • Cobalt reconstitution provides a valuable tool for probing the heme crevice environment in HRP analogues.
    • The proximal ligand in the reduced cobalt-HRP is unequivocally identified as a nitrogenous base.
    • Cobalt-HRP analogues, particularly oxy-CoHRP, exhibit functional similarities to cytochrome P-450, suggesting conserved mechanistic principles.

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