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 H(2)O(2) 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 ((18)O KIE) upon H(2)O(2) consumption determined under turnover conditions. The 180 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 (18)O label into the unreacted H(2)O(2). The results provide an unprecedented view of H(2)O(2) activation by a heme peroxidase and challenge the assumption of rate-limiting O-O heterolysis.