In order to investigate the nature of compounds I and I1 of horseradish peroxidase, the kinetics were studied of ferrocyanide oxidation catalyzed by these compounds which were prepared from three different oxidizing agents. The p H dependence of the apparent second-order rate constant for ferrocyanide oxidation by compound I, prepared from ethyl hydroperoxide and m-chloroperbenzoic acid, was interpreted in terms of an ionization on the enzyme with a pK, = 5.3, identical to that reported previously for hydrogen peroxide. The second-order rate constant for the compound 11-ferrocyanide reaction also showed the same p H dependence for the three oxidizing substrates. However, with more accurate results, the compound 11-ferrocyanide reaction was reinterpreted in terms of a single ionization with pK, = 8.5. The same dependence of ferrocyanide oxidation on p H suggests structurally identical active sites for compounds I and I1 prepared from the three different oxidizing substrates.Pour etudier la nature des composes I et I1 de la peroxydase de raifort, on a examine les cinetiques de l'oxydation du ferrocyanure catalysee par ces composes, qui ont ete prepares a partir de trois agents oxydants differents. L'influence du p H sur la constante de vitesse apparente du deuxieme ordre poul l'oxydation du ferrocyanure par le compose I prepare a partir de I'hydroperoxyde d'ethyle et de l'acide nz-chloroperbenzoique, a ete interpretee en fonction de I'ionisation de I'enzyme a un pK, = 5.3, identique a celle precedemrnent rapportee pour I'eau oxygenee. La constante de vitesse du second ordre pour la reaction entre le ferrocyanure et le compose 11, montre aussi la mCme relation de p H pour les trois substrats oxydants. Cependant, avec des risultats plus precis, la reaction entre le ferrocyanure et le compose I1 a ete reinterprktee par rapport a l'ionisation simple a pK, = 8.5. La mCme relation entre I'oxydation du ferrocyanure et le p H laisse penser a des sites actifs de structure identique pour les composts I et TI, prepares a partir de trois substrats oxydants differents.[Traduit par le journal]Can. J. Chern.. 51. 582 (1973) The intermediate compounds of horseradish peroxidase (HRP), that is, compound I (HRP-I) and compound PI (HRP-II), have been known for many years (1, 2). Early investigations indicated that these compounds could be formed from alkyl (3, 4) and acyl hydroperoxides (5) as well as several inorganic oxidizing agents (6). A variety of possibie structures has been proposed for these compounds (7)(8)(9)(10). Recently, it has been shown that oxygen is incorporated into compound I of ch!oroperoxidase upon its formation from m-chloroperbenzoic acid (11,12). Clearly, in addition to the electron transfer processes occurring, a portion of the peroxide must be retained at the active site upon HRP-1 formation.Although many techriiques have been applied to the study of HRP compounds (I3-17), the kinetic method, in principle, provides one of the 'Author vo whom corrsspo:.dei7ce sh~a?c% be addressed better sources of informa...

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The effect of cyanide on the steady-state oxidation of ferrocyanide by horseradish peroxidase was studied at pH values 9.00, 7.10, and 5.00. An inhibition was observed which could be explained by the binding of cyanide only to native horseradish peroxidase. Spectra in the Soret region supported these conclusions.