1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged ‘electrostatic gate’ controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.

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