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Isolation of yeast complex IV in native lipid nanodiscs
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Moscow State University, Russian Federation.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 11
2016 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, 2984-2992 p.Article in journal (Refereed) Published
Abstract [en]

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

Place, publisher, year, edition, pages
2016. Vol. 1858, no 12, 2984-2992 p.
Keyword [en]
Bioenergetics, Proton transfer, Membrane protein, Energy conservation
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-136722DOI: 10.1016/j.bbamem.2016.09.004ISI: 000388048600004PubMedID: 27620332OAI: oai:DiVA.org:su-136722DiVA: diva2:1057736
Available from: 2016-12-19 Created: 2016-12-14 Last updated: 2016-12-19Bibliographically approved

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Smirnova, Irina A.Sjöstrand, DanBjörck, MarkusSchäfer, JacobÖstbye, HenrikHögbom, MartinÄdelroth, PiaBrzezinski, Peter
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