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Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Department of Biochemistry, University of Illinois at Urbana-Champaign.
Department of Chemistry, University of Illinois at Urbana-Champaign.
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2012 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, 640-645 p.Article in journal (Refereed) Published
Abstract [en]

The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa3- and ba3-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.

Place, publisher, year, edition, pages
Elsevier, 2012. Vol. 586, 640-645 p.
Keyword [en]
Cytochrome c oxidase; Cytochrome aa3; Cytochrome ba3; Membrane protein; Lipid; Energy transduction
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-75159DOI: 10.1016/j.febslet.2011.12.023ISI: 000301222900023OAI: oai:DiVA.org:su-75159DiVA: diva2:514659
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Membrane effects on proton transfer in cytochrome c oxidase
Open this publication in new window or tab >>Membrane effects on proton transfer in cytochrome c oxidase
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The biological membrane is composed of lipids and proteins that make up dynamic barriers around cells and organelles. Membrane-spanning proteins are involved in many key processes in the cell such as energy conversion, nerve conduction and signal transduction. These proteins interact closely with lipids as well as with other proteins in the membrane, which modulates and affects their structure and function. In the energy-conversion process, membrane-bound proton-transport proteins maintain an electrochemical proton gradient across the mitochondrial inner membrane or the cytoplasmic membrane of bacteria. This gradient is utilized for ATP synthesis or transport of ions and molecules across the membrane. Results from earlier studies have shown that proton transporters are influenced by their environment.

Here, one of these proton transporters, cytochrome c oxidase, has been purified and reconstituted into liposomes or nanodiscs and membrane effects on specific proton-transfer processes were studied. In these studies we observed that the membrane accelerated proton transfer to the surface of cytochrome c oxidase and that there is a protonic link, via a Glu residue that mediates proton transfer from the membrane surface to a proton-transfer pathway in this protein. In addition, the membrane was shown to modulate specific internal electron and proton-transfer reactions.

The results from these studies show that the membrane composition influences transmembrane transport. Consequently, our understanding of these processes requires investigation of these transporter proteins in different membrane-mimetic systems of variable and well-defined composition. Furthermore, the data show that membrane surfaces facilitate lateral proton transfer which is presumably essential for maintaining high efficiency in energy conversion. This is particular important in organisms such as alkaliphilic bacteria where the driving force of the electrochemical proton gradient, between the bulk solution on each side of the membrane is not sufficient for ATP synthesis.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University, 2012. 57 p.
Keyword
cytochrom c oxidase, lipids, membrane, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-75633 (URN)978-91-7447-482-4 (ISBN)
Public defence
2012-06-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Available from: 2012-05-11 Created: 2012-04-24 Last updated: 2012-05-15Bibliographically approved

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Näsvik Öjemyr, Lindavon Ballmoos, ChristophBrzezinski, Peter
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