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A membrane-bound anchor for cytochrome c 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.
Visa övriga samt affilieringar
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-158698OAI: oai:DiVA.org:su-158698DiVA, id: diva2:1238641
Tillgänglig från: 2018-08-14 Skapad: 2018-08-14 Senast uppdaterad: 2020-01-06Bibliografiskt granskad
Ingår i avhandling
1. Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
Öppna denna publikation i ny flik eller fönster >>Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria
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
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:nbn:se:su:diva-158733 (URN)978-91-7797-370-6 (ISBN)978-91-7797-371-3 (ISBN)
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
2. Proton transfer across and along biological membranes
Öppna denna publikation i ny flik eller fönster >>Proton transfer across and along biological membranes
2020 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Proton-transfer reactions belong to the most prevalent reactions in the biosphere and make life on Earth possible, as they are central to energy conversion. In most known organisms, protons are translocated from one side of a membrane to the other, which generates an electrochemical gradient that drives ATP synthesis. Both the membranes and the proteins that are involved in these processes are vital components of energy-conversion machineries. This thesis presents and discusses proton transfer at surfaces of membranes and proteins, as well as proton translocation across membranes via enzymes.

In the first work, we developed a single-enzyme approach to study proton translocation by the proton pump cytochrome bo3 (cyt. bo3). The generated proton gradients were stable as long as substrate (electrons, oxygen) was available. Individual cyt. bo3 could generate proton gradients of ∼2 pH units, which correspond to the measured electrochemical gradient in Escherichia coli cells.

When acidic and basic amino acids are in close proximity to each other on a protein surface, their individual Coulomb cages can merge to form a proton antenna that enables fast proton transfer to specific groups. To investigate how the function of a proton pump is affected by structural changes in a proton antenna, close to a proton uptake pathway, we characterized the function and structure of genetic variants of cytochrome c oxidase (CytcO). When a Glu, located about 10 Å from the first residue of the D-pathway, was replaced by a non-protonatable residue (Ala) the proton pumping efficiency decreased by more than half compared to the wild-type enzyme. The proton-uptake kinetics was also altered in this variant.

Cardiolipin (CL) is found in membranes where ATP is generated. This phospholipid alters the membrane structure and binds a variety of proteins including all complexes that take part in oxidative phosphorylation. To investigate the role of CL in proton-transfer reactions on the surface of membranes we used fluorescence correlation spectroscopy to study inner mitochondrial membranes from Saccharomyces cerevisiae. The protonation rate at wild-type membranes was about 50% of that measured with membranes prepared from mitochondria lacking CL. The protonation rate on the surface of small unilamellar vesicles (SUVs) decreased by about a factor of three when DOPC-SUVs were supplemented with 20% CL. Furthermore, phosphate buffer titrations with SUVs showed that CL can act as a local proton buffer in a membrane.

The respiratory supercomplex factor 1 (Rcf1) has been suggested to facilitate direct electron transfer from the bc1 complex to CytcO by bridging the enzymes and binding cytochrome c (cyt. c) to a flexible domain of Rcf1. We investigated biding of cyt. c to Rcf1 reconstituted into different membrane environments. The apparent KD of the binding between cyt. c and DOPC-liposomes was almost five times lower when Rcf1 was present in the vesicles. Moreover, the apparent KD between cyt. c and liposome reconstituted CytcO was about nine times lower for CytcO isolated from a wild-type strain compared to a Rcf1-lacking strain.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. s. 93
Nyckelord
biological membranes, cardiolipin, cytochrome bo3, cytochrome c oxidase, energy conversion, fluorescence correlation spectroscopy (FCS), localized coupling, mitochondria, proton transfer, Rcf1, respiration, single-enzyme measurement
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-177422 (URN)978-91-7797-941-8 (ISBN)978-91-7797-942-5 (ISBN)
Disputation
2020-02-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, 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: 2020-01-29 Skapad: 2020-01-06 Senast uppdaterad: 2020-05-25Bibliografiskt granskad

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Sjöholm, JohannesSchäfer, JacobZhou, ShuRydström Lundin, CamillaBerg, JohanÄdelroth, PiaBrzezinski, Peter
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Institutionen för biokemi och biofysik
Biokemi och molekylärbiologi

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