The timing of proton migration in membrane-reconstituted cytochrome c oxidase.
In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (tau(D) congruent with 200 micros, "peroxy" to "ferryl" transition) followed by a significantly slower proton release to the other side of the membrane (tau(D) congruent with 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.
Salomonsson
Lina
L
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University SE-106 91 Stockholm, Sweden.
Faxén
Kristina
K
Adelroth
Pia
P
Brzezinski
Peter
P
D003902:Detergents / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D012996:Solutions; D013997:Time Factors
D003902:Detergents; D008055:Lipids; D011522:Protons; D012996:Solutions; D003576:Electron Transport Complex IV