Chemical and enzymic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical-enzyme complex and its reaction with nucleophiles.

Claiborne, A. and I. Fridovich (1979).
Biochemistry 18(11): 2329-2335.

Abstract:
Changes in the optical absorption spectrum of horseradish peroxidase during the oxidn. of o-dianisidine at pH 7.5 reveal an intermediate distinct from the previously described compds. 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 compds. reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the peroxidase-catalyzed rate of oxidn. of o-dianisidine. The activity of nitrogenous compds., 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. It is suggested that the nucleophile-responsive intermediate is an enzyme-dianisidine radical complex and that abstraction of the 2nd electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.