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Protein and lipid interactions within the respiratory chain: Studies using membrane-mimetic systems
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-2930-801X
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Energy conversion from nutrients to ATP is a vital process in cells. The process, called oxidative phosphorylation (OXPHOS) is performed by a combination of membrane-bound proteins. These proteins have been studied in great detail in the past, however much is still unknown about how they interact with each other. Studying the OXPHOS proteins in their native environment can be difficult due to the complexity of living cells. By isolating parts of the OXPHOS system and inserting them into membrane-mimetic systems it is possible to investigate their functions in a controlled environment.

In the work presented here, we co-reconstituted several of these proteins into liposomes made from synthetic lipids. We demonstrated production of ATP at steady-state conditions with the ATP synthase, driven by proton pumping by cytochrome bo3. Introduction of anionic lipids decreased the coupled activity and we could correlate this effect to weaker interactions between ATP synthase and cytochrome bo3 in the membrane. We also reconstituted cytochrome c oxidase (CytcO) from Saccharomyces cerevisiae with Respiratory supercomplex factor 1 (Rcf1) into liposomes and submitochondrial particles (SMPs). Loss of Rcf1 has previously been found to result in a lower CytcO activity. We found that activity could be restored upon co-reconstitution of CytcO with Rcf1, but only after unfolding and re-folding of the latter, which shows that Rcf1 can adopt two configurations in the membrane.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 67
Keywords [en]
Cytochrome c oxidase, ATP synthase, Bioenergetics, membrane-mimetics, Rcf1, liposomes, oxidative phosphorylation, lipids, protein-protein interactions
National Category
Biochemistry and Molecular Biology Other Chemistry Topics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-173617ISBN: 978-91-7797-845-9 (print)ISBN: 978-91-7797-846-6 (electronic)OAI: oai:DiVA.org:su-173617DiVA, id: diva2:1354915
Public defence
2019-11-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

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

Available from: 2019-10-30 Created: 2019-09-26 Last updated: 2019-11-27Bibliographically approved
List of papers
1. Mimicking respiratory phosphorylation using purified enzymes
Open this publication in new window or tab >>Mimicking respiratory phosphorylation using purified enzymes
2016 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1857, no 4, p. 321-331Article in journal (Refereed) Published
Abstract [en]

The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP x s(-1) x enzyme(-1). We have also used the novel system to study the phenomenon of mild uncoupling as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Keywords
ATP synthesis, Respiratory chain, Liposomes, Mild uncoupling, Ionophore, Lateral proton diffusion
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-129914 (URN)10.1016/j.bbabio.2015.12.007 (DOI)000372675600001 ()26707617 (PubMedID)
Available from: 2016-05-03 Created: 2016-05-03 Last updated: 2019-09-30Bibliographically approved
2. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis
Open this publication in new window or tab >>Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 24113Article in journal (Refereed) Published
Abstract [en]

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-130172 (URN)10.1038/srep24113 (DOI)000374219300001 ()27063297 (PubMedID)
Available from: 2016-05-11 Created: 2016-05-09 Last updated: 2019-09-26Bibliographically approved
3. The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
Open this publication in new window or tab >>The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 2926Article in journal (Refereed) Published
Abstract [en]

We have investigated the effect of lipid composition on interactions between cytochrome bo(3) and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d congruent to 100 nm) or giant (d congruent to 10 mu m) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be similar to 80 nm.

Keywords
Bioenergetics, Biophysical chemistry, Membrane biophysics
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-144786 (URN)10.1038/s41598-017-02836-4 (DOI)000402879200011 ()
Available from: 2017-07-14 Created: 2017-07-14 Last updated: 2019-09-30Bibliographically approved
4. Activation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1
Open this publication in new window or tab >>Activation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In S. cerevisiae the transmembrane protein Respiratory Supercomplex Factor 1 (Rcf1) is involved in formation of the cytochrome c oxidase - bc1 supercomplex. It has also been suggested to mediate electron transfer between the two respiratory enzymes via interactions with cytochrome c. Removal of Rcf1 results in decreased CytcO activity as well as a decrease in the fraction of supercomplexes. The Rcf1 protein can presumably be found as both a monomer and dimer in the membrane. A structure of the latter has been determined using NMR. In this study, we show that co-reconstitution of purified Rcf1 with CytcO from a rcf1Δ strain in liposomes yielded an increase in the CytcO activity. Also, reconstitution of Rcf1 in sub-mitochondrial particles from the rcf1Δ strain yielded an increase in the CytcO activity. However, the increased activity was only observed when the Rcf1 protein was fully unfolded and then refolded in the presence of a membrane. Collectively, the data indicate that Rcf1 can be reconstituted in a membrane as a dimer, but the protein can interact with and reactivate CytcO only in the monomeric form.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-173584 (URN)
Available from: 2019-09-26 Created: 2019-09-26 Last updated: 2019-09-26Bibliographically approved

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