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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: 112016 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, p. 2984-2992Article 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, p. 2984-2992
Keywords [en]
Bioenergetics, Proton transfer, Membrane protein, Energy conservation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-136722DOI: 10.1016/j.bbamem.2016.09.004ISI: 000388048600004PubMedID: 27620332OAI: oai:DiVA.org:su-136722DiVA, id: diva2:1057736
Available from: 2016-12-19 Created: 2016-12-14 Last updated: 2020-03-05Bibliographically approved
In thesis
1. Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP
Open this publication in new window or tab >>Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cytochrome c oxidase (CytcO) is the final electron acceptor of the respiratory chain. In this chain a current of electrons, derived from degradation of nutrients, along with protons, are used to reduce oxygen to water. The reaction is exergonic and the excess energy is used to pump protons across the membrane. This proton-coupled electron transfer is regulated, for example, by the membrane potential, the composition of the membrane and the ATP/ADP concentrations. 

Here, we have investigated the mechanism of this regulation. Specifically, we investigated ligand binding to CytcO in mitochondria, which provides mechanistic information about CytcO in its native environment. In addition to CytcO, a water soluble protein, flavohemoglobin (yHb) was found to bind CO and we found that it is localized in the intermembrane space (IMS). We also extracted CytcO from mitochondria without detergent using the styrene maleic acid (SMA) co-polymer. We could show that the SMA-extracted CytcO behaved similarly in its reaction with O2 and CO as CytcO in mitochondria.

In mitochondria and bacterial membranes CytcO transports charges against a transmembrane electrochemical gradient. We induced a membrane potential across sub-mitochondrial particles (SMPs) by addition of ATP and measured single CytcO turnover. Our results indicate that proton transfer, but not electron transfer, across the membrane is affected by the membrane potential.

In yeast CytcO subunit Cox13 has been shown to play a role in ATP/ADP binding to regulate activity. We have solved the structure of Cox13 using NMR and identified the residues that constitute the ATP-binding site, which is located at the C-terminus.

Finally we showed that the main proton-transfer pathways in yeast CytcO function similarly to their bacterial counterparts and that the proposed H-pathway, absent in bacteria, is not responsible for proton translocation in mitochondrial CytcO from S. cerevisiae.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm university, 2019. p. 53
Keywords
cytochrome c oxidase, charge transfer, membrane potential, membrane protein, mitochondria, ATP, proton pump
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167130 (URN)978-91-7797-624-0 (ISBN)978-91-7797-625-7 (ISBN)
Public defence
2019-05-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2019-04-15 Created: 2019-03-19 Last updated: 2020-05-13Bibliographically approved

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