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Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks
Author(s) -
Berg Johan,
Block Stephan,
Höök Fredrik,
Brzezinski Peter
Publication year - 2017
Publication title -
israel journal of chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.908
H-Index - 54
eISSN - 1869-5868
pISSN - 0021-2148
DOI - 10.1002/ijch.201600138
Subject(s) - chemistry , electrochemical gradient , valinomycin , cytochrome , cytochrome c oxidase , ionophore , proton pump , proton , membrane potential , respiratory chain , transmembrane protein , chemiosmosis , electron transport complex iv , photochemistry , biophysics , membrane , biochemistry , atp synthase , enzyme , atpase , physics , receptor , quantum mechanics , biology
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 bo 3 from E. coli at single‐molecule level. Liposomes with reconstituted cytochrome bo 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 cytochrome bo 3 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 ΔpH (in the presence of the K + ionophore valinomycin), which was stable over at least ∼800 s. No rapid changes in ΔpH (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).