Decay of Compound ES in Dehaloperoxidase-Hemoglobin

Abstract

Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that catalyzes the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low temperature EPR spectroscopy in the range of reaction time of 50 ms - 2 min at three different pH values. Two high spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES has been assigned to Tyr34, the other radical - to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. The HPLC analysis of the reaction products showed a pH dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH formed under H2O2 in the absence of substrates is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH or the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available.