Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical--enzyme complex and its reaction with nucleophiles.
Changes in the optical absorption spectrum of horseradish peroxidase, during the oxidation of o-dianisidine at pH 7.5, reveal an intermediate distinct from the previously described compounds I and II. The rate of decay of this new complex appeared to be rate limiting for the catalytic cycle, in this pH range, since imidazole, which augments the catalytic reaction, also enhanced the rate of decay of this complex. Nitrogenous compounds reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the HRP-catalyzed rate of oxidation of o-dianisidine. The activity of nitrogenous compounds, in this regard, appeared to be a function of their nucleophilicity and was sensitive to steric factors but relatively free of a deuterium solvent isotope effect. The data presented in this and in the preceding paper [Claiborne, A., & Fridovich, I. (1979) Biochemistry 18 (preceding paper in this issue)] lead to the suggestion that the nucleophile-responsive intermediate is an enzyme--dianisidine radical complex and that abstraction of the second electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.
D001560:Benzidines; D003962:Dianisidine; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide; D007093:Imidazoles; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism; D010545:Peroxides; D011485:Protein Binding; D011725:Pyridines; D013053:Spectrophotometry
D001560:Benzidines; D005609:Free Radicals; D007093:Imidazoles; D010545:Peroxides; D011725:Pyridines; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D003962:Dianisidine