Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

Christoph Von Ballmoos, Nathalie Gonska, Peter Lachmann, Robert B. Gennis, Pia Ädelroth, Peter Brzezinski

Research output: Contribution to journalArticle

Abstract

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

Original languageEnglish (US)
Pages (from-to)3397-3402
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number11
DOIs
StatePublished - Mar 17 2015

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Protons
Oxidoreductases
Mutation
Electron Transport Complex IV
Electrons
Thermus thermophilus
Oxidation-Reduction
Catalytic Domain
Membrane Proteins
Membranes

Keywords

  • Cytochrome aa
  • Cytochrome c oxidase
  • Electron transfer
  • Membrane protein
  • Respiration

ASJC Scopus subject areas

  • General

Cite this

Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site. / Von Ballmoos, Christoph; Gonska, Nathalie; Lachmann, Peter; Gennis, Robert B.; Ädelroth, Pia; Brzezinski, Peter.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 11, 17.03.2015, p. 3397-3402.

Research output: Contribution to journalArticle

Von Ballmoos, Christoph ; Gonska, Nathalie ; Lachmann, Peter ; Gennis, Robert B. ; Ädelroth, Pia ; Brzezinski, Peter. / Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site. In: Proceedings of the National Academy of Sciences of the United States of America. 2015 ; Vol. 112, No. 11. pp. 3397-3402.
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AU - Ädelroth, Pia

AU - Brzezinski, Peter

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N2 - The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

AB - The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

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