Tyrosyl Radicals in Dehaloperoxidase: HOW NATURE DEALS WITH EVOLVING AN OXYGEN-BINDING GLOBIN TO A BIOLOGICALLY RELEVANT PEROXIDASE* |
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Authors: | Rania Dumarieh Jennifer D'Antonio Alexandria Deliz-Liang Tatyana Smirnova Dimitri A. Svistunenko Reza A. Ghiladi |
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Affiliation: | From the ‡Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 and ;§School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom |
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Abstract: | Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501–17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment. |
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Keywords: | Hemoglobin Iron Peroxidase Radicals Tyrosine Compound ES Compound I Compound II Dehaloperoxidase Ferryl |
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