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Single Proteoliposomes withE. coliQuinol Oxidase: Proton Pumping without Transmembrane Leaks
Single Proteoliposomes withE. coliQuinol Oxidase: Proton Pumping without Transmembrane Leaks
- Source :
- Israel Journal of Chemistry. 57:437-445
- Publication Year :
- 2017
- Publisher :
- Wiley, 2017.
-
Abstract
- Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochrome bo3 from E. coli at single-molecule level. Liposomes with reconstituted cytochrome bo3 were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochrome bo3 in the membrane; in ?70 % of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a ?pH (in the presence of the K+ ionophore valinomycin), which was stable over at least ?800 s. No rapid changes in ?pH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E. coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li et al. J. Am. Chem. Soc. (2015) 137, 16055–16063).
- Subjects :
- 0301 basic medicine
030102 biochemistry & molecular biology
Proton
Chemistry
Ionophore
Analytical chemistry
General Chemistry
Photochemistry
Proton pump
03 medical and health sciences
Electron transfer
Valinomycin
chemistry.chemical_compound
030104 developmental biology
Membrane
Steady state (chemistry)
Electrochemical gradient
Subjects
Details
- ISSN :
- 00212148
- Volume :
- 57
- Database :
- OpenAIRE
- Journal :
- Israel Journal of Chemistry
- Accession number :
- edsair.doi...........f15cbf4527af42200eadc80cac6323ef
- Full Text :
- https://doi.org/10.1002/ijch.201600138