Radical formation in cytochrome c oxidase

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

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2- ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".

Original languageEnglish (US)
Pages (from-to)1295-1304
Number of pages10
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1807
Issue number10
DOIs
StatePublished - Oct 2011

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Reducing Agents
Electron Transport Complex IV
Protons
Dithionite
Quenching
Paramagnetic resonance
Equipment and Supplies
Reaction intermediates
Propionates
Electron Spin Resonance Spectroscopy
Reaction products
Hydrogen Peroxide
Oxidation-Reduction
Tensors
Proteins
Hand
Ions
Amino Acids
Molecules
Water

Keywords

  • Bioenergetics
  • Electron paramagnetic resonance
  • Peroxyl
  • Proton translocation
  • Radicals

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Radical formation in cytochrome c oxidase. / Yu, Michelle A.; Egawa, Tsuyoshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yeh, Syun-Ru; Rousseau, Denis L.; Gerfen, Gary J.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1807, No. 10, 10.2011, p. 1295-1304.

Research output: Contribution to journalArticle

Yu, Michelle A. ; Egawa, Tsuyoshi ; Shinzawa-Itoh, Kyoko ; Yoshikawa, Shinya ; Yeh, Syun-Ru ; Rousseau, Denis L. ; Gerfen, Gary J. / Radical formation in cytochrome c oxidase. In: Biochimica et Biophysica Acta - Bioenergetics. 2011 ; Vol. 1807, No. 10. pp. 1295-1304.
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N2 - The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2- ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".

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