Open AccessControlled uncoupling and recoupling of proton pumping in cytochrome c oxidase
Author(s) -
Gisela Brändén,
Ashtamurthy S. Pawate,
Robert B. Gennis,
Peter Brzezinski
Publication year - 2006
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0507734103
Subject(s) - chemistry , proton , proton pump , cytochrome c oxidase , catalysis , proton transport , oxygen , electron transport chain , stereochemistry , photochemistry , enzyme , membrane , biochemistry , organic chemistry , atpase , physics , quantum mechanics
Cytochromec oxidase (Cc O) 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 Cc O (D132N/N139D double-mutant Cc O) 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 Cc O 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 Cc O 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 Cc O and the structural elements involved in this process.