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Regulation of cytochrome c oxidase activity by modulation of the catalytic site
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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2018. Vol. 8, article id 11397
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-158697DOI: 10.1038/s41598-018-29567-4ISI: 000440144400014OAI: oai:DiVA.org:su-158697DiVA, id: diva2:1238583
Available from: 2018-08-14 Created: 2018-08-14 Last updated: 2019-10-16Bibliographically approved
In thesis
1. Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
Open this publication in new window or tab >>Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 76
Keywords
Cytochrome c oxidase, Electron transfer, Membrane protein, Ligand, Kinetics, Mechanism, Rcf1, Cytochrome c, Respiratory supercomplex, Cryo-electron microscopy
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158733 (URN)978-91-7797-370-6 (ISBN)978-91-7797-371-3 (ISBN)
Public defence
2018-09-28, 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 3: Manuscript. Paper 4: Manuscript.

Available from: 2018-09-05 Created: 2018-08-14 Last updated: 2018-08-29Bibliographically approved
2. Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
Open this publication in new window or tab >>Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
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
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:nbn:se:su:diva-175276 (URN)978-91-7797-839-8 (ISBN)978-91-7797-840-4 (ISBN)
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

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