The distal histidine has been proposed to play a crucial role in the reaction of peroxidases with hydroperoxide. Myoglobin (Mb), due to its close similarity with peroxidases, also reacts with various peroxides. The effect of mutation of the distal histidine on the peroxidase activity of Mb has been investigated by stopped-flow kinetics of the reaction of hydrogen peroxide with wild-type Mb and [Gly64]Mb. The results indicate kinetic and mechanistic differences in the formation of peroxide compounds from these two forms of Mb. The rate of reaction of H 2 O 2 with the wild-type Mb decreased 8Ϫ9-fold on mutation of the distal histidine to glycine ([Gly64]Mb). A second slow phase was observed for the reaction of H 2 O 2 with [Gly64]Mb, but was not observed in the corresponding reaction with wild-type Mb. It is suggested that the decrease in the rate of the reaction on mutation is due to the absence of a general acid-base catalyst. The effect of pH on the rate of reaction of H 2 O 2 with wild-type Mb and [Gly64]Mb is contrasting. While the rate of the M6 reaction decreased for wild-type Mb at higher pH, probably due to the acid-alkaline transition of Mb at higher pH, the rate of reaction was found to increase at higher pH for mutant Mb. The increase in the rate of the reaction is suggested to be due to an increase in the ionization of H 2 O 2 at higher pH, the rate-determining step being the formation of the intermediate Fe-O-O-H complex and not the subsequent step of oxo-ferryl complex formation. The thermodynamic parameters calculated from the temperature-dependent study showed that the enthalpy of binding of H 2 O 2 with Mb is positive, indicating that the process is endothermic. The apparent energy of activation of the reaction of H 2 O 2 with Mb was found to be higher than that of peroxidases, suggesting that this may be oue of the reasons for the slower rate of the reaction of H 2 O 2 with Mb compared with peroxidases.

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