Horseradish peroxidase—catalyzed hydroxylation of phenol: I. Thermodynamic analysis

We analyzed the horseradish peroxidase (HRP)—catalyzed hydroxylation of phenol in the presence of dihydroxy-fumaric acid and oxygen. All of the intermediate forms of the enzyme are reviewed. The last step of hydroxylation, consisting of the production of OH^• radicals that further react on phenol, is emphasized. Possible OH^• radicals production reactions were compiled and analyzed with respect to the available thermodynamic data. Some results of electrochemical experiments were also used to choose the correct set of reactions. At the end of analysis only two reactions for producing OH^• seemed to be consistent with the thermodynamic and experimental data. Neither of these reactions involved compound III or any other intermediate form of HRP. The last step of hydroxylation was thus totally independent of the pure catalytic cycle of the enzyme. As a consequence, HRP cannot be used as an hydroxylation enzyme in place of the P₄₅₀ cytochrome, as is sometimes suggested.