Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first oxygen-binding globin that possesses a biologically relevant peroxidase activity. Two known isoforms of DHP, A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as the oxidant. The catalytically competent species in dehaloperoxidase appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. For DHP B, EPR studies suggested that two different radicals are present, a primary tyrosyl radical consistent with being located on either Tyr28 or Tyr38. Previously, our lab has shown that recombinant substitution of both of these tyrosines with the redox inactive residue, phenylalanine, prevented the formation of Compound ES in DHP B at pH7. However, subsequent experiments at lower pH values indicated that the formation of Compound ES is restored. Beyond the aforementioned tyrosyl residues, DHP B also contains two other tyrosines, Tyr16 and Tyr107, as wells as a tryptophan, Trp120, as possible candidates for this observation. In order to provide insight into the novel location of this protein-based radical, we have recombinantly introduced redox inactive phenylalanine at position 107 in both a single mutant (Y107F) and a triple mutant (Y28/38/107F) species to investigate the radical formation in DHP activated by hydrogen peroxide at these lower pH values under a variety of conditions using both kinetic assays with hydrogen peroxide and stopped-flow UV-visible spectroscopic methods.