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Single Proteoliposomes withE. coliQuinol Oxidase: Proton Pumping without Transmembrane Leaks

Single Proteoliposomes withE. coliQuinol Oxidase: Proton Pumping without Transmembrane Leaks

Authors :
Peter Brzezinski
Stephan Block
Fredrik Höök
Johan Berg
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).

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