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Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae
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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Number of Authors: 52016 (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.

Place, publisher, year, edition, pages
2016. Vol. 113, no 31, p. E4476-E4485
Keywords [en]
cytochrome c oxidase, electron transfer, membrane protein, cytochrome aa(3), cytochrome bc(1)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-133376DOI: 10.1073/pnas.1601196113ISI: 000380586600010OAI: oai:DiVA.org:su-133376DiVA, id: diva2:967788
Available from: 2016-09-09 Created: 2016-09-06 Last updated: 2018-10-31Bibliographically approved
In thesis
1. Modulators of Saccharomyces cerevisiae cytochrome c oxidase: Implications for the regulation of mitochondrial respiration
Open this publication in new window or tab >>Modulators of Saccharomyces cerevisiae cytochrome c oxidase: Implications for the regulation of mitochondrial respiration
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
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:nbn:se:su:diva-161515 (URN)978-91-7797-457-4 (ISBN)978-91-7797-456-7 (ISBN)
Public defence
2018-12-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Available from: 2018-11-20 Created: 2018-10-29 Last updated: 2018-11-13Bibliographically approved

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