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Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

ABSTRACT: The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their ?-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.

SUBMITTER: Yu MA

PROVIDER: S-EPMC3418888 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

Yu Michelle A MA Egawa Tsuyoshi T Shinzawa-Itoh Kyoko K Yoshikawa Shinya S Guallar Victor V Yeh Syun-Ru SR Rousseau Denis L DL Gerfen Gary J GJ

Journal of the American Chemical Society 20120306 10

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values sim ...[more]

PMID: 22296274

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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.

Dataset Information

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

Publications

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

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