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Single Proteoliposomes with E.coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 4
2017 (English)In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, 437-445 p.Article in journal (Refereed) Published
Abstract [en]

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, cytochromebo(3) from E.coli at single-molecule level. Liposomes with reconstituted cytochromebo(3) 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 cytochromebo(3) in the membrane; in approximate to 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 approximate to 800s. 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 etal. J. Am. Chem. Soc. (2015) 137, 16055-16063).

Place, publisher, year, edition, pages
2017. Vol. 57, no 5, 437-445 p.
Keyword [en]
proton translocation, single molecule, proton pump, electron transfer, membrane protein
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-144709DOI: 10.1002/ijch.201600138ISI: 000401329000010OAI: oai:DiVA.org:su-144709DiVA: diva2:1127988
Available from: 2017-07-20 Created: 2017-07-20 Last updated: 2017-07-20Bibliographically approved

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Berg, JohanBrzezinski, Peter
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