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NMR structural analysis of yeast Cox13 reveals its C-terminus in interaction with ATP
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The organization of mitochondrial respiratory chain complexes into supercomplexes is vital to cellular activities. In the yeast Saccharomyces cerevisiae, Cox13 is a conserved peripheral subunit of complex IV (cytochrome c oxidase, CytcO) involved in the assembly of monomeric complex IV into supercomplexes. Here we report the solution NMR structure of a Cox13 dimer in detergent micelles. Each Cox13 monomer has three short flexible helices (SH), corresponding to the disordered regions in its homologous X-ray structure, and the dimer formation is mainly induced by the hydrophobic interaction between the sole transmembrane (TM) helix of each monomer. Furthermore, analysis of chemical shift changes upon addition of ATP reveal positions that are able to bind ATP at the conserved sites of the C-terminus with considerable conformational flexibility. From functional analysis of purified CytcO, we conclude that this ATP interaction is inhibitory of catalytic activity. Our results show the structure of an important subunit of yeast CytcO and provide structure-based insight into its ATP interaction.

Keywords [en]
ATP, dimer, membrane protein, NMR, solution structure
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biophysics
Identifiers
URN: urn:nbn:se:su:diva-167100OAI: oai:DiVA.org:su-167100DiVA, id: diva2:1296858
Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-04-05Bibliographically 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)
Opponent
Supervisors
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: 2019-03-28Bibliographically approved

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