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

Michelle A. Yu, Tsuyoshi Egawa, Kyoko Shinzawa-Itoh, Shinya Yoshikawa, Victor Guallar, Syun-Ru Yeh, Denis L. Rousseau, Gary J. Gerfen

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H 2O 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 2O 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 β-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.

Original languageEnglish (US)
Pages (from-to)4753-4761
Number of pages9
JournalJournal of the American Chemical Society
Volume134
Issue number10
DOIs
StatePublished - Mar 14 2012

Fingerprint

Electron Spin Resonance Spectroscopy
Electron Transport Complex IV
Paramagnetic resonance
Protons
Energy conservation
Tyrosine
Oxidation
Mechanics
Enzymes
Molecular mechanics
Quantum theory
Hydrogen peroxide
Tryptophan
Hydrogen Peroxide
Tensors
Substitution reactions
Irradiation
Hydrogen
tyrosine radical

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Two tyrosyl radicals stabilize high oxidation states in cytochrome c oxidase for efficient energy conservation and proton translocation. / Yu, Michelle A.; Egawa, Tsuyoshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Guallar, Victor; Yeh, Syun-Ru; Rousseau, Denis L.; Gerfen, Gary J.

In: Journal of the American Chemical Society, Vol. 134, No. 10, 14.03.2012, p. 4753-4761.

Research output: Contribution to journalArticle

@article{35c690b5baa9470b930934bb62031757,
title = "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 2O 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 2O 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 β-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.",
author = "Yu, {Michelle A.} and Tsuyoshi Egawa and Kyoko Shinzawa-Itoh and Shinya Yoshikawa and Victor Guallar and Syun-Ru Yeh and Rousseau, {Denis L.} and Gerfen, {Gary J.}",
year = "2012",
month = "3",
day = "14",
doi = "10.1021/ja210535w",
language = "English (US)",
volume = "134",
pages = "4753--4761",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Two tyrosyl radicals stabilize high oxidation states in cytochrome c oxidase for efficient energy conservation and proton translocation

AU - Yu, Michelle A.

AU - Egawa, Tsuyoshi

AU - Shinzawa-Itoh, Kyoko

AU - Yoshikawa, Shinya

AU - Guallar, Victor

AU - Yeh, Syun-Ru

AU - Rousseau, Denis L.

AU - Gerfen, Gary J.

PY - 2012/3/14

Y1 - 2012/3/14

N2 - The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H 2O 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 2O 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 β-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.

AB - The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H 2O 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 2O 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 β-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.

UR - http://www.scopus.com/inward/record.url?scp=84858208696&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84858208696&partnerID=8YFLogxK

U2 - 10.1021/ja210535w

DO - 10.1021/ja210535w

M3 - Article

C2 - 22296274

AN - SCOPUS:84858208696

VL - 134

SP - 4753

EP - 4761

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 10

ER -