Direct Examination of H₂O₂ Activation by a Heme Peroxidase

Roth, J. P.; Cramer, C. J.
J. Am. Chem. Soc. 2008, 130, 7802.

Oxygen isotope fractionation (¹⁸O vs. ¹⁶O) is applied to the reaction of a H₂O₂-utilizing enzyme for the first time. Horseradish peroxidase (HRP) has been the subject of intensive experimental and computational investigations yet questions remain as to the timing of O-O cleavage and proton transfer. New insight is afforded by the competitive ¹⁸O kinetic isotope effect (KIE) on H₂O₂ consumption determined under turnover conditions. The ¹⁸O KIE is compared to isotope effects calculated for the O-O heterolysis transition state and potential intermediates. The analysis employs complete sets of vibrational frequencies from high level density functional calculations. In addition, isotope fractionation in ¹⁸O-enriched water provides evidence for HRP-catalyzed scrambling of the ¹⁸O label into the unreacted H₂O₂. Together the results provide an unprecedented view of enzymatic H₂O₂ activation where proton transfer or some physical step is rate-limiting rather than heterolysis of the O-O bond.

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