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Controlled Uncoupling and Recoupling of Proton Pumping in Cytochrome c Oxidase

Gisela Brändén, Ashtamurthy S. Pawate, Robert B. Gennis and Peter Brzezinski
Proceedings of the National Academy of Sciences of the United States of America
Vol. 103, No. 2 (Jan. 10, 2006), pp. 317-322
Stable URL: http://www.jstor.org/stable/30048299
Page Count: 6
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Controlled Uncoupling and Recoupling of Proton Pumping in Cytochrome c Oxidase
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Abstract

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 $pK_{a}$ 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 $pK_{a}$ 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.

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