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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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