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Modulators of Saccharomyces cerevisiae cytochrome c oxidase: Implications for the regulation of mitochondrial respiration
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxidative phosphorylation in mitochondria is performed by enzyme complexes and electron carriers that reside in the inner membrane. It is now generally accepted that these respiratory enzyme complexes assemble into larger so-called supercomplexes. However, it is presently not known why, under which conditions or how these supercomplexes form.

A number of factors of particular importance for the formation of supercomplexes have been identified, such as the Respiratory supercomplex factors (Rcf1 and Rcf2) and cardiolipin. The work presented in this thesis is focused on the characterization of cytochrome c oxidase (CytcO) in mitochondria from Saccharomyces cerevisiae strains in which these components have been removed, with a particular focus on Rcf1. First, we concluded that Rcf1 has an impact on the activity and ligand binding kinetics of CytcO, which upon genetic deletion of rcf1 leads to formation of sub-populations of CytcO with different functionality. Second, we noted that the ability of CytcO to oxidize cytochrome c (cyt. c) depends on the presence of Rcf1. Further, we observed that while CytcO in wild-type mitochondria displayed differences in the oxidation kinetics of cyt. c from horse heart or S. cerevisiae, with the Δrcf1 mitochondria these differences were lost. This observation suggested that Rcf1 interacts with cyt. c. Furthermore, the data showed that in CytcO from Δrcf1 mitochondria heme a3 was altered while heme a was intact.

Using proteo-liposomes of different lipid composition and size we also investigated the influence of lipid head groups on the coupled activity of a quinol oxidase and ATP-synthase. Specifically, we addressed the question if protons are transferred between proton “producers” and “consumers” via lateral proton transfer along the membrane surface or via bulk water. Our data supported the principle of lateral proton transfer.

Lastly, we characterized the ligand binding of yeast flavohemoglobin and concluded that the flavohemoglobin has a population that resides in the intermembrane space of mitochondria, not only in matrix and cytosol as previously suggested.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018. , p. 48
Keywords [en]
cytochrome c oxidase, cytochrome c, OXPHOS, membrane protein, kinetics, ligand-binding, electron transfer, Rcf1, respiratory supercomplexes, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-161515ISBN: 978-91-7797-457-4 (print)ISBN: 978-91-7797-456-7 (electronic)OAI: oai:DiVA.org:su-161515DiVA, id: diva2:1259361
Public defence
2018-12-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2018-11-20 Created: 2018-10-29 Last updated: 2018-11-13Bibliographically approved
List of papers
1. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis
Open this publication in new window or tab >>Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 24113Article in journal (Refereed) Published
Abstract [en]

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-130172 (URN)10.1038/srep24113 (DOI)000374219300001 ()27063297 (PubMedID)
Available from: 2016-05-11 Created: 2016-05-09 Last updated: 2018-10-31Bibliographically approved
2. Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae
Open this publication in new window or tab >>Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae
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2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 31, p. E4476-E4485Article in journal (Refereed) Published
Abstract [en]

The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinolcytochrome c reductase; cyt. bc(1)) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc(1). In the present study, we have investigated the kinetics of ligand binding, the singleturn-over reaction of CytcO with O-2, and the linked cyt. bc(1)-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1 Delta and rcf2 Delta, respectively). The data show that in the rcf1 Delta and rcf2 Delta strains, in a significant fraction of the population, ligand binding occurs over a time scale that is similar to 100-fold faster (tau congruent to 100 mu s) than observed with the wild-type mitochondria (tau congruent to 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc(1)-CytcO supercomplex. Furthermore, in the rcf1 Delta and rcf2 Delta strains, the single-turnover reaction of CytcO with O-2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electrontransfer bridge from cyt. bc(1) to CytcO via a tightly bound cyt. c. We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc(1) and CytcO.

Keywords
cytochrome c oxidase, electron transfer, membrane protein, cytochrome aa(3), cytochrome bc(1)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-133376 (URN)10.1073/pnas.1601196113 (DOI)000380586600010 ()
Available from: 2016-09-09 Created: 2016-09-06 Last updated: 2018-10-31Bibliographically approved
3. Modulation of O-2 reduction in Saccharomyces cerevisiae mitochondria
Open this publication in new window or tab >>Modulation of O-2 reduction in Saccharomyces cerevisiae mitochondria
2017 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 24, p. 4049-4055Article in journal (Refereed) Published
Abstract [en]

Respiratory supercomplex factor (Rcf) 1 is a membrane-bound protein that modulates the activity of cytochrome c oxidase (CytcO) in Saccharomycescerevisiae mitochondria. To investigate this regulatory mechanism, we studied the interactions of CytcO with potassium cyanide (KCN) upon removal of Rcf Delta. While the addition of KCN to the wild-type mitochondria results in a full reduction of heme a, with the rcf Delta mitochondria, a significant fraction remains oxidized. Upon addition of ascorbate in the presence of O-2 and KCN, the reduction level of hemes a and b was a factor of similar to 2 larger with the wild-type than with the rcf Delta mitochondria. These data indicate that turnover of CytcO was less blocked in rcf Delta than in the wild-type mitochondria, suggesting that Rcf Delta modulates the structure of the catalytic site.

Keywords
cytochrome aa(3), cytochrome c oxidase, electron transfer, membrane protein, respiratory supercomplex factor, Saccharomy cescerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-152520 (URN)10.1002/1873-3468.12918 (DOI)000418825700008 ()29171870 (PubMedID)
Available from: 2018-02-05 Created: 2018-02-05 Last updated: 2018-10-31Bibliographically approved
4. 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
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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: 2018-10-31Bibliographically approved

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