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The role of cardiolipin in lateral proton transfer along inner mitochondrial membranes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Keywords [en]
cardiolipin, protonation kinetics, mitochondria, cytochrome c oxidase, fluorescence correlation spectroscopy
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-177420OAI: oai:DiVA.org:su-177420DiVA, id: diva2:1382876
Available from: 2020-01-06 Created: 2020-01-06 Last updated: 2020-01-23Bibliographically approved
In thesis
1. Proton transfer across and along biological membranes
Open this publication in new window or tab >>Proton transfer across and along biological membranes
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 93
Keywords
biological membranes, cardiolipin, cytochrome bo3, cytochrome c oxidase, energy conversion, fluorescence correlation spectroscopy (FCS), localized coupling, mitochondria, proton transfer, Rcf1, respiration, single-enzyme measurement
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-177422 (URN)978-91-7797-941-8 (ISBN)978-91-7797-942-5 (ISBN)
Public defence
2020-02-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, 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: 2020-01-29 Created: 2020-01-06 Last updated: 2020-01-23Bibliographically approved

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Berg, JohanSjöholm, JohannesBrzezinski, Peter
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