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Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018. , s. 76
Nyckelord [en]
Cytochrome c oxidase, Electron transfer, Membrane protein, Ligand, Kinetics, Mechanism, Rcf1, Cytochrome c, Respiratory supercomplex, Cryo-electron microscopy
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-158733ISBN: 978-91-7797-370-6 (tryckt)ISBN: 978-91-7797-371-3 (digital)OAI: oai:DiVA.org:su-158733DiVA, id: diva2:1238792
Disputation
2018-09-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.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

Tillgänglig från: 2018-09-05 Skapad: 2018-08-14 Senast uppdaterad: 2020-05-11Bibliografiskt granskad
Delarbeten
1. Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae
Öppna denna publikation i ny flik eller fönster >>Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae
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2018 (Engelska)Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 9, s. 699-704Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
Cytochrome c oxidase, Electron transfer, Cytochrome aa3, Membrane protein, Ligand, Kinetics, Mechanism
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-158696 (URN)10.1016/j.bbabio.2018.05.004 (DOI)000442708200009 ()
Tillgänglig från: 2018-08-14 Skapad: 2018-08-14 Senast uppdaterad: 2019-10-16Bibliografiskt granskad
2. Regulation of cytochrome c oxidase activity by modulation of the catalytic site
Öppna denna publikation i ny flik eller fönster >>Regulation of cytochrome c oxidase activity by modulation of the catalytic site
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2018 (Engelska)Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 11397Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-158697 (URN)10.1038/s41598-018-29567-4 (DOI)000440144400014 ()
Tillgänglig från: 2018-08-14 Skapad: 2018-08-14 Senast uppdaterad: 2019-10-16Bibliografiskt granskad
3. A membrane-bound anchor for cytochrome c in S. cerevisiae
Öppna denna publikation i ny flik eller fönster >>A membrane-bound anchor for cytochrome c in S. cerevisiae
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-158698 (URN)
Tillgänglig från: 2018-08-14 Skapad: 2018-08-14 Senast uppdaterad: 2020-01-06Bibliografiskt granskad
4. Biochemical and structural characterization of a superoxide dismutase-containing respiratory supercomplex from Mycobacterium smegmatis
Öppna denna publikation i ny flik eller fönster >>Biochemical and structural characterization of a superoxide dismutase-containing respiratory supercomplex from Mycobacterium smegmatis
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-158717 (URN)
Tillgänglig från: 2018-08-14 Skapad: 2018-08-14 Senast uppdaterad: 2020-03-05Bibliografiskt granskad

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