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Tyrosyl radicals in dehaloperoxidase: how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase.

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.

SUBMITTER: Dumarieh R

PROVIDER: S-EPMC3829192 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Tyrosyl radicals in dehaloperoxidase: how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase.

Dumarieh Rania R D'Antonio Jennifer J Deliz-Liang Alexandria A Smirnova Tatyana T Svistunenko Dimitri A DA Ghiladi Reza A RA

The Journal of biological chemistry 20131006 46

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, ...[more]

PMID: 24100039

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Project description:The location of the Trp radical and the catalytic function of the [Fe(IV)═O Trp₁₉₁(•+)] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr₇₁(•) and Tyr₂₃₆(•) is independent of the [Fe(IV)═O Trp₁₉₁(•+)] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Å from the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632-1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr₇₁(•) as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248-255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665-11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557-8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.

Project description:BackgroundThe use of 3-D similarity techniques in the analysis of biological data and virtual screening is pervasive, but what is a biologically meaningful 3-D similarity value? Can one find statistically significant separation between "active/active" and "active/inactive" spaces? These questions are explored using 734,486 biologically tested chemical structures, 1,389 biological assay data sets, and six different 3-D similarity types utilized by PubChem analysis tools.ResultsThe similarity value distributions of 269.7 billion unique conformer pairs from 734,486 biologically tested compounds (all-against-all) from PubChem were utilized to help work towards an answer to the question: what is a biologically meaningful 3-D similarity score? The average and standard deviation for the six similarity measures STST-opt, CTST-opt, ComboTST-opt, STCT-opt, CTCT-opt, and ComboTCT-opt were 0.54 ± 0.10, 0.07 ± 0.05, 0.62 ± 0.13, 0.41 ± 0.11, 0.18 ± 0.06, and 0.59 ± 0.14, respectively. Considering that this random distribution of biologically tested compounds was constructed using a single theoretical conformer per compound (the "default" conformer provided by PubChem), further study may be necessary using multiple diverse conformers per compound; however, given the breadth of the compound set, the single conformer per compound results may still apply to the case of multi-conformer per compound 3-D similarity value distributions. As such, this work is a critical step, covering a very wide corpus of chemical structures and biological assays, creating a statistical framework to build upon.The second part of this study explored the question of whether it was possible to realize a statistically meaningful 3-D similarity value separation between reputed biological assay "inactives" and "actives". Using the terminology of noninactive-noninactive (NN) pairs and the noninactive-inactive (NI) pairs to represent comparison of the "active/active" and "active/inactive" spaces, respectively, each of the 1,389 biological assays was examined by their 3-D similarity score differences between the NN and NI pairs and analyzed across all assays and by assay category types. While a consistent trend of separation was observed, this result was not statistically unambiguous after considering the respective standard deviations. While not all "actives" in a biological assay are amenable to this type of analysis, e.g., due to different mechanisms of action or binding configurations, the ambiguous separation may also be due to employing a single conformer per compound in this study. With that said, there were a subset of biological assays where a clear separation between the NN and NI pairs found. In addition, use of combo Tanimoto (ComboT) alone, independent of superposition optimization type, appears to be the most efficient 3-D score type in identifying these cases.ConclusionThis study provides a statistical guideline for analyzing biological assay data in terms of 3-D similarity and PubChem structure-activity analysis tools. When using a single conformer per compound, a relatively small number of assays appear to be able to separate "active/active" space from "active/inactive" space.

Dataset Information

Tyrosyl radicals in dehaloperoxidase: how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase.

Publications

Tyrosyl radicals in dehaloperoxidase: how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase.

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