Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase.
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 approximately 30 A 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 approximately 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.
Brändén
Gisela
G
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Pawate
Ashtamurthy S
AS
Gennis
Robert B
RB
Brzezinski
Peter
P
D002384:Catalysis; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013057:Spectrum Analysis
D017494:Proton Pumps; D011522:Protons; D003576:Electron Transport Complex IV