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Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria fulfill a plethora of functions, including harboring metabolic pathways and converting energy stored in metabolites into ATP, the common energy source of the cell. This last function is performed by the oxidative phosphorylation system, consisting of the respiratory chain and the ATP synthase. Electrons are channeled through the complexes of the respiratory chain, while protons are translocated across the inner mitochondrial membrane. This process establishes an electrochemical gradient, which is used by the ATP synthase to generate ATP. The subunits of two of the respiratory chain complexes, the bc1 complex and the cytochrome c oxidase, are encoded by two genetic origins, the nuclear and the mitochondrial genome. Therefore, the assembly of these complexes needs to be coordinated and highly regulated.

Several proteins are involved in the biogenesis of the bc1 complex. Amongst these proteins, the Cbp3-Cbp6 complex was shown to regulate translation and assembly of the bc1 complex subunit cytochrome b. In this work, we established a homology model of yeast Cbp3. Using a site-specific crosslink approach, we identified binding sites of Cbp3 to its obligate binding partner Cbp6 and its client, cytochrome b, enabling a deeper insight in the molecular mechanisms of bc1 complex biogenesis. 

The bc1 complex and the cytochrome c oxidase form macromolecular structures, called supercomplexes. The detailed assembly mechanisms and functions of these structures remain to be solved. Two proteins, Rcf1 and Rcf2, were identified associating with supercomplexes in the yeast Saccharomyces cerevisiae. Our studies demonstrate that, while Rcf1 has a minor effect on supercomplex assembly, its main function is to modulate cytochrome c oxidase activity. We show that cytochrome c oxidase is present in three structurally different populations. Rcf1 is needed to maintain the dominant population in a functionally active state. In absence of Rcf1, the abundance of a population with an altered active site is increased. We propose that Rcf1 is needed, especially under a high work load of the respiratory chain, to maintain the function of cytochrome c oxidase.

This thesis aims to unravel molecular mechanisms of proteins involved in biogenesis and functionality of respiratory chain complexes to enable a deeper understanding. Dysfunctional respiratory chain complexes lead to severe disease, emphasizing the importance of this work.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 75
Keywords [en]
respiratory chain, bc1 complex, cytochrome c oxidase, Cbp3, Rcf1, Rcf2, respiratory supercomplexes, biogenesis, mitochondria, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-175276ISBN: 978-91-7797-839-8 (print)ISBN: 978-91-7797-840-4 (electronic)OAI: oai:DiVA.org:su-175276DiVA, id: diva2:1361702
Public defence
2019-12-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2019-11-13 Created: 2019-10-16 Last updated: 2019-11-04Bibliographically approved
List of papers
1. Structural basis for Cbp3 interaction with newly synthesized cytochrome b during mitochondrial respiratory chain assembly
Open this publication in new window or tab >>Structural basis for Cbp3 interaction with newly synthesized cytochrome b during mitochondrial respiratory chain assembly
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2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 45, p. 16663-16671Article in journal (Refereed) Published
Abstract [en]

Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.

Keywords
respiratory chain, complex III, assembly factor, mitochondrial translation, protein assembly, membrane biogenesis, protein crosslinking, ubiquinol-cytochrome c chaperone domain, structural biology, electron transfer chain
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-171513 (URN)10.1074/jbc.RA119.010483 (DOI)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-12-12Bibliographically approved
2. Rcf1 modulates cytochrome c oxidase activity especially under energy-demanding conditions
Open this publication in new window or tab >>Rcf1 modulates cytochrome c oxidase activity especially under energy-demanding conditions
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175274 (URN)
Available from: 2019-10-16 Created: 2019-10-16 Last updated: 2019-10-21Bibliographically approved
3. Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae
Open this publication in new window or tab >>Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae
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2018 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 9, p. 699-704Article in journal (Refereed) Published
Abstract [en]

Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc1) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O2-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.

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-158696 (URN)10.1016/j.bbabio.2018.05.004 (DOI)000442708200009 ()
Available from: 2018-08-14 Created: 2018-08-14 Last updated: 2019-10-16Bibliographically approved
4. Regulation of cytochrome c oxidase activity by modulation of the catalytic site
Open this publication in new window or tab >>Regulation of cytochrome c oxidase activity by modulation of the catalytic site
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 11397Article in journal (Refereed) Published
Abstract [en]

The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158697 (URN)10.1038/s41598-018-29567-4 (DOI)000440144400014 ()
Available from: 2018-08-14 Created: 2018-08-14 Last updated: 2019-10-16Bibliographically approved

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