Oxidation of 4-tert-Butylcatechol and Dopamine by Hydrogen Peroxide Catalysed by Horseradisch Peroxidase

AbstractThe catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-

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Titration Study of Guaiarcol Oxidation by Horseradish Peroxidase

Canadian Journal of Biochemistry ◽

10.1139/o75-090 ◽

1975 ◽

Vol 53 (6) ◽

pp. 649-657 ◽

Cited By ~ 35

Author(s):

Marius Santimone

Keyword(s):

Hydrogen Peroxide ◽

Electron Transfer ◽

Low Temperature ◽

Horseradish Peroxidase ◽

Catalytic Amount ◽

Reaction Scheme ◽

General Mechanism ◽

Compound I ◽

Compound Ii ◽

Titration Study

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.

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Horseradish peroxidase. XXIX. Reactions in water and deuterium oxide: cyanide binding, compound I formation, and reactions of compounds I and II with ferrocyanide

Canadian Journal of Chemistry ◽

10.1139/v78-468 ◽

1978 ◽

Vol 56 (22) ◽

pp. 2844-2852 ◽

Cited By ~ 52

Author(s):

H. Brian Dunford ◽

W. Donald Hewson ◽

Håkan Steiner

Keyword(s):

Hydrogen Peroxide ◽

Horseradish Peroxidase ◽

Kinetic Isotope Effect ◽

Deuterium Oxide ◽

Isotope Effects ◽

Kinetic Isotope Effects ◽

Compound I ◽

Kinetic Isotope ◽

Cyanide Binding ◽

Compound Ii

The kinetics of the reactions of hydrogen peroxide and cyanide with native horseradish peroxidase, as well as reactions of compounds I and II with ferrocyanide have been studied in ordinary water and in deuterium oxide at 25 °C and ionic strength 0.11 using a stopped-flow apparatus. Rate constants for all reactions were measured over a wide range of acidity in both solvents from which equilibrium and kinetic isotope effects were evaluated. Protonation of an ionizable group on the enzyme with a pKa value of 4.15 ± 0.05 in water inhibits the reactions with both hydrogen peroxide and cyanide. A significant kinetic isotope effect, kH/kD = 1.6 ± 0.1, was measured for compound I formation whereas no significant kinetic isotope effect was found for cyanide binding. On the basis of these findings, a partial mechanism for compound I formation is proposed in which the group of pKa 4.15 plays a crucial role. The pH dependencies of the ferrocyanide reaction in the pH interval 4.5–10.8 confirmed the role of an acid group with a pKa of 5.2 for compound I and for compound II a pKa of 8.6 and another with a value lower than that encompassed by the pH range of the study. Equilibrium isotope effects were found but no kinetic isotope effects for either the reaction of compound I or of compound II This suggests that there are no rate-limiting proton transfers in the reactions between ferrocyanide and compounds I and II of horseradish peroxidase. The only reducing substrates which exhibit positive kH/kD values possess a labile proton.

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