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Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2.
Biochemistry 1998; 37(48):16922-33B

Abstract

Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H2O2 with concurrent consumption of O2. Both adrenochrome formation and O2 consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H2O2. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O2- in the oxidation. Although SOD-mediated H2O2 formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O2- takes part in a vital reaction to form an intermediate for adrenochrome formation and O2 consumption. Generation of O2- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H2O2, both adrenochrome formation and O2 consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn2+ or spin-trap, alpha-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O2 consumption. The free radicals thus favor reduction of O2 rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe3+/Fe2+ shuttle consuming O2, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O2 to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN-, SCN-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O2- and H2O2. Once H2O2 is generated, the peroxidative cycle continues with the consumption of O2, through the intermediate formation of O2- and H2O2 which play an obligatory role in subsequent cycles of peroxidation.

Authors+Show Affiliations

Department of Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700032, India.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9836585

Citation

Adak, S, et al. "Mechanism of Horseradish Peroxidase Catalyzed Epinephrine Oxidation: Obligatory Role of Endogenous O2- and H2O2." Biochemistry, vol. 37, no. 48, 1998, pp. 16922-33.
Adak S, Bandyopadhyay U, Bandyopadhyay D, et al. Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2. Biochemistry. 1998;37(48):16922-33.
Adak, S., Bandyopadhyay, U., Bandyopadhyay, D., & Banerjee, R. K. (1998). Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2. Biochemistry, 37(48), pp. 16922-33.
Adak S, et al. Mechanism of Horseradish Peroxidase Catalyzed Epinephrine Oxidation: Obligatory Role of Endogenous O2- and H2O2. Biochemistry. 1998 Dec 1;37(48):16922-33. PubMed PMID: 9836585.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2. AU - Adak,S, AU - Bandyopadhyay,U, AU - Bandyopadhyay,D, AU - Banerjee,R K, PY - 1998/12/4/pubmed PY - 1998/12/4/medline PY - 1998/12/4/entrez SP - 16922 EP - 33 JF - Biochemistry JO - Biochemistry VL - 37 IS - 48 N2 - Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H2O2 with concurrent consumption of O2. Both adrenochrome formation and O2 consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H2O2. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O2- in the oxidation. Although SOD-mediated H2O2 formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O2- takes part in a vital reaction to form an intermediate for adrenochrome formation and O2 consumption. Generation of O2- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H2O2, both adrenochrome formation and O2 consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn2+ or spin-trap, alpha-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O2 consumption. The free radicals thus favor reduction of O2 rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe3+/Fe2+ shuttle consuming O2, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O2 to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN-, SCN-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O2- and H2O2. Once H2O2 is generated, the peroxidative cycle continues with the consumption of O2, through the intermediate formation of O2- and H2O2 which play an obligatory role in subsequent cycles of peroxidation. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9836585/Mechanism_of_horseradish_peroxidase_catalyzed_epinephrine_oxidation:_obligatory_role_of_endogenous_O2__and_H2O2_ L2 - https://dx.doi.org/10.1021/bi980899l DB - PRIME DP - Unbound Medicine ER -