Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
cytochrome c oxidase, charge transfer, membrane potential, membrane protein, mitochondria, ATP, proton pump
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-167130ISBN: 978-91-7797-624-0 (print)ISBN: 978-91-7797-625-7 (electronic)OAI: oai:DiVA.org:su-167130DiVA, id: diva2:1297399
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
List of papers
1. Reaction of S-cerevisiae mitochondria with ligands: Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase
Open this publication in new window or tab >>Reaction of S-cerevisiae mitochondria with ligands: Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase
Show others...
2017 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1858, no 2, p. 182-188Article in journal (Refereed) Published
Abstract [en]

Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (CytcO) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, CytcO and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with T congruent to 270 ms, which is significantly slower than observed with the cytosolic yHb (main component T congruent to 1.3 ms). The data indicate that the yHb populations in the different cell compartments differ in structure.

Keywords
Electron transfer, Cytochrome aa(3), Yeast, Membrane protein, Ligand, Kinetics, Mechanism
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-140291 (URN)10.1016/j.bbabio.2016.11.009 (DOI)000392776400010 ()27871795 (PubMedID)
Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2019-03-19Bibliographically approved
2. Isolation of yeast complex IV in native lipid nanodiscs
Open this publication in new window or tab >>Isolation of yeast complex IV in native lipid nanodiscs
Show others...
2016 (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.

Keywords
Bioenergetics, Proton transfer, Membrane protein, Energy conservation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136722 (URN)10.1016/j.bbamem.2016.09.004 (DOI)000388048600004 ()27620332 (PubMedID)
Available from: 2016-12-19 Created: 2016-12-14 Last updated: 2019-03-19Bibliographically approved
3. Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential
Open this publication in new window or tab >>Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential
2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3187Article in journal (Refereed) Published
Abstract [en]

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-160116 (URN)10.1038/s41467-018-05615-5 (DOI)000441157600022 ()30093670 (PubMedID)
Available from: 2018-09-18 Created: 2018-09-18 Last updated: 2019-03-20Bibliographically approved
4. NMR structural analysis of yeast Cox13 reveals its C-terminus in interaction with ATP
Open this publication in new window or tab >>NMR structural analysis of yeast Cox13 reveals its C-terminus in interaction with ATP
Show others...
(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
ATP, dimer, membrane protein, NMR, solution structure
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biophysics
Identifiers
urn:nbn:se:su:diva-167100 (URN)
Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-04-05Bibliographically approved
5. Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase
Open this publication in new window or tab >>Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In cytochrome c oxidase (CytcO) reduction of O2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than throughthe D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D(Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO withO2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and pumping.

Keywords
cytochrome c oxidase, electron transfer, cytochrome aa3, membrane protein, ligand, kinetics, mechanism
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167129 (URN)
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-03-20Bibliographically approved

Open Access in DiVA

Regulation of proton-coupled electron transfer in cytochrome c oxidase(1474 kB)30 downloads
File information
File name FULLTEXT01.pdfFile size 1474 kBChecksum SHA-512
70b886fc18342784ce62893babf90673f5b3dbb780cb258b3c7a6add4620ea787baad2167487ba9fe2dc7637e320005827cf11f9223a231d11f3922e0f4e1fa8
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Björck, Markus
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 30 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 231 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf