Single Proteoliposomes with E.coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Berg, JohanStockholms universitet,Institutionen för biokemi och biofysik,Stockholm University (författare)

Single Proteoliposomes with E.coli Quinol Oxidase : Proton Pumping without Transmembrane Leaks

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).

Block, Stephan,1978Chalmers tekniska högskola,Chalmers University of Technology(Swepub:cth)blocks (författare)
Höök, Fredrik,1966Chalmers tekniska högskola,Chalmers University of Technology(Swepub:cth)fredrikh (författare)
Brzezinski, PeterStockholms universitet,Institutionen för biokemi och biofysik,Stockholm University(Swepub:su)brzez (författare)
Stockholms universitetInstitutionen för biokemi och biofysik (creator_code:org_t)

Av författaren/redakt...: Berg, Johan; Block, Stephan, ...; Höök, Fredrik, 1 ...; Brzezinski, Pete ...

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