Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Gisela Brändén, Ashtamurthy S. Pawate, Robert B. Gennis, Peter Brzezinski

Research output: Contribution to journalArticlepeer-review


Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located ≈30 Å from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pKa of an internal proton donor, Glu-286, from ≈9.4 to > 11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pKa to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P → F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.

Original languageEnglish (US)
Pages (from-to)317-322
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number2
StatePublished - Jan 10 2006


  • Cytochrome oxidase
  • Electron transfer
  • Gating
  • Proton transfer

ASJC Scopus subject areas

  • General

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