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Open AccessArticle

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

1
Institute of Chemistry and Biochemistry, Emmy-Noether Group “Bionanointerfaces”, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
2
Department of Physics, Genetic Biophysics, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
3
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
4
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(19), 6981; https://doi.org/10.3390/ijms21196981
Received: 24 August 2020 / Revised: 15 September 2020 / Accepted: 18 September 2020 / Published: 23 September 2020
(This article belongs to the Special Issue Lipid-Protein and Protein-Protein Interactions in Membranes)
The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin. View Full-Text
Keywords: proton translocation; single molecule; proton pump; electron transfer; membrane protein; single enzyme fluorescence microscopy proton translocation; single molecule; proton pump; electron transfer; membrane protein; single enzyme fluorescence microscopy
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MDPI and ACS Style

Hugentobler, K.G.; Heinrich, D.; Berg, J.; Heberle, J.; Brzezinski, P.; Schlesinger, R.; Block, S. Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase. Int. J. Mol. Sci. 2020, 21, 6981.

AMA Style

Hugentobler KG, Heinrich D, Berg J, Heberle J, Brzezinski P, Schlesinger R, Block S. Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase. International Journal of Molecular Sciences. 2020; 21(19):6981.

Chicago/Turabian Style

Hugentobler, Katharina G.; Heinrich, Dorothea; Berg, Johan; Heberle, Joachim; Brzezinski, Peter; Schlesinger, Ramona; Block, Stephan. 2020. "Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase" Int. J. Mol. Sci. 21, no. 19: 6981.

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