The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)