Direct examination of H₂O₂ activation by a heme peroxidase

Described here is the application of oxygen isotope fractionation together with computational methods, to elucidate a mechanism of enzymatic H₂O₂ activation. Horseradish peroxidase (HRP) has been the subject of intensive experimental and computational studies, yet questions remain as to the reversibility of the O-O cleavage step. New insight is afforded by the competitive oxygen kinetic isotope effect (¹⁸O KIE) upon 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 using density functional theory. In addition, experiments in enriched water provide evidence for HRP-catalyzed scrambling of the ¹⁸O label into the unreacted H₂O₂. The results provide an unprecedented view of H₂O₂ activation by a heme peroxidase and challenge the assumption of rate-limiting O-O heterolysis.