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Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
2018 (engelsk)Inngår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 9, s. 699-704Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2018. Vol. 1859, nr 9, s. 699-704
Emneord [en]
Cytochrome c oxidase, Electron transfer, Cytochrome aa3, Membrane protein, Ligand, Kinetics, Mechanism
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-158696DOI: 10.1016/j.bbabio.2018.05.004ISI: 000442708200009OAI: oai:DiVA.org:su-158696DiVA, id: diva2:1238576
Tilgjengelig fra: 2018-08-14 Laget: 2018-08-14 Sist oppdatert: 2019-10-16bibliografisk kontrollert
Inngår i avhandling
1. Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
Åpne denne publikasjonen i ny fane eller vindu >>Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The enzyme complexes of the respiratory chain are organized in supramolecular assemblies, so-called respiratory supercomplexes. In the yeast Saccharomyces cerevisiae, these supercomplexes consist of two copies of complex III (bc1 complex) and one or two copies of complex IV (cytochrome c oxidase, CytcO). Several factors, including lipids and small proteins, have been identified to facilitate or stabilize this organization.

Respiratory supercomplex factor (Rcf) 1 interacts with CytcO. In this work, we show that in the native S. cerevisiae mitochondrial membrane several forms of CytcO co-exist. Intact CytcO shows spectral and functional properties similar to those of CytcOs from other organisms characterized earlier. A second population displayed a lower midpoint potential of heme a3 as well as accelerated ligand binding, suggesting structural differences around the catalytic site. Severe structural changes of the catalytic site and the overall structure of the enzyme were found in a third population of CytcO. The fraction of the structurally altered CytcO increased upon removal of Rcf1. Here, a mechanism is proposed in which Rcf1 regulates function of the CytcO by altering the catalytic site so that electron transfer between heme a and heme a3 is slowed, resulting in a more exergonic O2-ligand binding. This scenario would in turn increase heat production on the expense of the proton electrochemical gradient.

Rcf1 was further shown to facilitate electron transfer from the bc1 complex to CytcO in a supercomplex by interacting with the electron carrier cytochrome c (cyt. c).

In addition, we purified and structurally and functionally characterized the supercomplex of Mycobacterium smegmatis, which contains a membrane-anchored cyt. c as a subunit of the bcc1 complex.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 76
Emneord
Cytochrome c oxidase, Electron transfer, Membrane protein, Ligand, Kinetics, Mechanism, Rcf1, Cytochrome c, Respiratory supercomplex, Cryo-electron microscopy
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-158733 (URN)978-91-7797-370-6 (ISBN)978-91-7797-371-3 (ISBN)
Disputas
2018-09-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Tilgjengelig fra: 2018-09-05 Laget: 2018-08-14 Sist oppdatert: 2018-08-29bibliografisk kontrollert
2. Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
Åpne denne publikasjonen i ny fane eller vindu >>Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 75
Emneord
respiratory chain, bc1 complex, cytochrome c oxidase, Cbp3, Rcf1, Rcf2, respiratory supercomplexes, biogenesis, mitochondria, Saccharomyces cerevisiae
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-175276 (URN)978-91-7797-839-8 (ISBN)978-91-7797-840-4 (ISBN)
Disputas
2019-12-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (engelsk)
Opponent
Veileder
Merknad

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

Tilgjengelig fra: 2019-11-13 Laget: 2019-10-16 Sist oppdatert: 2019-11-04bibliografisk kontrollert

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