Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Izumi Ishigami, Nadia A. Zatsepin, Masahide Hikita, Chelsie E. Conrad, Garrett Nelson, Jesse D. Coe, Shibom Basu, Thomas D. Grant, Matthew H. Seaberg, Raymond G. Sierra, Mark S. Hunter, Petra Fromme, Raimund Fromme, Syun-Ru Yeh, Denis L. Rousseau

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ∼142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Original languageEnglish (US)
Pages (from-to)8011-8016
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number30
DOIs
StatePublished - Jul 25 2017

Fingerprint

X Ray Crystallography
Electron Transport Complex IV
Carbon Monoxide
Synchrotrons
Temperature
Protons
Iron
Oxidation-Reduction
Electrons
Radiation
Mitochondrial Membranes
Heme
Artifacts
Lasers
X-Rays
heme a
Oxygen
Ligands
Light
Water

Keywords

  • Bioenergetics
  • Crystallography
  • Cytochrome c oxidase
  • Serial femtosecond crystallography
  • X-ray free electron laser

ASJC Scopus subject areas

  • General

Cite this

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature. / Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide; Conrad, Chelsie E.; Nelson, Garrett; Coe, Jesse D.; Basu, Shibom; Grant, Thomas D.; Seaberg, Matthew H.; Sierra, Raymond G.; Hunter, Mark S.; Fromme, Petra; Fromme, Raimund; Yeh, Syun-Ru; Rousseau, Denis L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 30, 25.07.2017, p. 8011-8016.

Research output: Contribution to journalArticle

Ishigami, Izumi ; Zatsepin, Nadia A. ; Hikita, Masahide ; Conrad, Chelsie E. ; Nelson, Garrett ; Coe, Jesse D. ; Basu, Shibom ; Grant, Thomas D. ; Seaberg, Matthew H. ; Sierra, Raymond G. ; Hunter, Mark S. ; Fromme, Petra ; Fromme, Raimund ; Yeh, Syun-Ru ; Rousseau, Denis L. / Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 30. pp. 8011-8016.
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abstract = "Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 {\AA}, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 {\AA}, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ∼142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 {\AA}. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.",
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T1 - Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

AU - Ishigami, Izumi

AU - Zatsepin, Nadia A.

AU - Hikita, Masahide

AU - Conrad, Chelsie E.

AU - Nelson, Garrett

AU - Coe, Jesse D.

AU - Basu, Shibom

AU - Grant, Thomas D.

AU - Seaberg, Matthew H.

AU - Sierra, Raymond G.

AU - Hunter, Mark S.

AU - Fromme, Petra

AU - Fromme, Raimund

AU - Yeh, Syun-Ru

AU - Rousseau, Denis L.

PY - 2017/7/25

Y1 - 2017/7/25

N2 - Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ∼142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

AB - Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ∼142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

KW - Bioenergetics

KW - Crystallography

KW - Cytochrome c oxidase

KW - Serial femtosecond crystallography

KW - X-ray free electron laser

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