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Membrane effects on proton transfer in cytochrome c oxidase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The biological membrane is composed of lipids and proteins that make up dynamic barriers around cells and organelles. Membrane-spanning proteins are involved in many key processes in the cell such as energy conversion, nerve conduction and signal transduction. These proteins interact closely with lipids as well as with other proteins in the membrane, which modulates and affects their structure and function. In the energy-conversion process, membrane-bound proton-transport proteins maintain an electrochemical proton gradient across the mitochondrial inner membrane or the cytoplasmic membrane of bacteria. This gradient is utilized for ATP synthesis or transport of ions and molecules across the membrane. Results from earlier studies have shown that proton transporters are influenced by their environment.

Here, one of these proton transporters, cytochrome c oxidase, has been purified and reconstituted into liposomes or nanodiscs and membrane effects on specific proton-transfer processes were studied. In these studies we observed that the membrane accelerated proton transfer to the surface of cytochrome c oxidase and that there is a protonic link, via a Glu residue that mediates proton transfer from the membrane surface to a proton-transfer pathway in this protein. In addition, the membrane was shown to modulate specific internal electron and proton-transfer reactions.

The results from these studies show that the membrane composition influences transmembrane transport. Consequently, our understanding of these processes requires investigation of these transporter proteins in different membrane-mimetic systems of variable and well-defined composition. Furthermore, the data show that membrane surfaces facilitate lateral proton transfer which is presumably essential for maintaining high efficiency in energy conversion. This is particular important in organisms such as alkaliphilic bacteria where the driving force of the electrochemical proton gradient, between the bulk solution on each side of the membrane is not sufficient for ATP synthesis.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University , 2012. , 57 p.
Keyword [en]
cytochrom c oxidase, lipids, membrane, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-75633ISBN: 978-91-7447-482-4 (print)OAI: oai:DiVA.org:su-75633DiVA: diva2:517518
Public defence
2012-06-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2012-05-11 Created: 2012-04-24 Last updated: 2012-05-15Bibliographically approved
List of papers
1. Lateral proton transfer between the membrane and a membrane protein.
Open this publication in new window or tab >>Lateral proton transfer between the membrane and a membrane protein.
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 10, 2173-9 p.Article in journal (Refereed) Published
Abstract [en]

Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (approximately 7 x 1010 s(-1) M(-1), compared to the expected value of (1-2) x 10(10) s(-1) M(-1)), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to approximately 3 x 10(13) s(-1) M(-1), which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-36116 (URN)10.1021/bi8022152 (DOI)000264059500012 ()19166299 (PubMedID)
Available from: 2010-01-21 Created: 2010-01-21 Last updated: 2017-12-12Bibliographically approved
2. Functional interactions between membrane-bound transporters and membranes
Open this publication in new window or tab >>Functional interactions between membrane-bound transporters and membranes
2010 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 36, 15763-15767 p.Article in journal (Refereed) Published
Abstract [en]

One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated protein-protein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the two-dimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.

Keyword
cytochrome aa(3), electron transfer, energy transduction, membrane protein, respiration
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-50181 (URN)10.1073/pnas.1006109107 (DOI)000281637800027 ()
Note
authorCount :4Available from: 2010-12-29 Created: 2010-12-21 Last updated: 2017-12-11Bibliographically approved
3. The membrane modulates internal proton transfer in cytochrome c oxidase
Open this publication in new window or tab >>The membrane modulates internal proton transfer in cytochrome c oxidase
Show others...
2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, 1092-1100 p.Article in journal (Refereed) Published
Abstract [en]

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O2 reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pKa in the pH dependence profile of the proton-uptake rate. This pKa change resulted in decreased proton-uptake rates in the pH range of 6.5–9.5, which is explained in terms of lowering of the pKa of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal CuA–heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-75164 (URN)10.1021/bi201795c (DOI)000300132900005 ()
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
4. Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer
Open this publication in new window or tab >>Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer
Show others...
2012 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, 640-645 p.Article in journal (Refereed) Published
Abstract [en]

The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa3- and ba3-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
Cytochrome c oxidase; Cytochrome aa3; Cytochrome ba3; Membrane protein; Lipid; Energy transduction
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-75159 (URN)10.1016/j.febslet.2011.12.023 (DOI)000301222900023 ()
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved

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