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

Int J Mol Sci. 2020 Sep 23;21(19):6981. doi: 10.3390/ijms21196981.


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.

Keywords: electron transfer; membrane protein; proton pump; proton translocation; single enzyme fluorescence microscopy; single molecule.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Electron Transport Complex IV / chemistry*
  • Hydrogen-Ion Concentration
  • Liposomes
  • Membrane Lipids / chemistry*
  • Rhodobacter sphaeroides / enzymology*


  • Bacterial Proteins
  • Liposomes
  • Membrane Lipids
  • Electron Transport Complex IV