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The membrane modulates internal proton transfer in cytochrome c oxidase
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
2012 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, s. 1092-1100Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2012. Vol. 51, s. 1092-1100
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-75164DOI: 10.1021/bi201795cISI: 000300132900005OAI: oai:DiVA.org:su-75164DiVA, id: diva2:514664
Tillgänglig från: 2012-04-10 Skapad: 2012-04-10 Senast uppdaterad: 2022-02-24Bibliografiskt granskad
Ingår i avhandling
1. Membrane effects on proton transfer in cytochrome c oxidase
Öppna denna publikation i ny flik eller fönster >>Membrane effects on proton transfer in cytochrome c oxidase
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Department of Biochemistry and Biophysics, Stockholm University, 2012. s. 57
Nyckelord
cytochrom c oxidase, lipids, membrane, proton transfer
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-75633 (URN)978-91-7447-482-4 (ISBN)
Disputation
2012-06-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2012-05-11 Skapad: 2012-04-24 Senast uppdaterad: 2022-02-24Bibliografiskt granskad
2. Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase
Öppna denna publikation i ny flik eller fönster >>Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In cellular respiration, electron transfer from the breakdown of foodstuff is coupled to the formation of an electrochemical proton gradient. This is accomplished through proton translocation by respiratory complexes, and the proton gradient is subsequently used e.g. to drive ATP production. Consequently, proton- and electron-transfer reactions through the hydrophobic interior of membrane proteins are central to cellular respiration. In this thesis, proton- and electron transfer through an aa3-type terminal oxidase, cytochrome c oxidase (CytcO) from Rhodobacter sphaeroides, have been studied with the aim of understanding the molecular proton-transfer machinery of this proton pump.

In the catalytic site of CytcO the electrons combine with protons and the terminal electron acceptor O2 to form water in an exergonic reaction that drives proton pumping. Therefore, CytcO must transfer both protons that are pumped and protons for the oxygen chemistry through its interior. This is done through its two proton-transfer pathways, termed the D pathway and the K pathway. Our studies have shown that the protons pumped during oxidation of CytcO are taken through the D pathway, and that this process does not require a functional K pathway. Furthermore, our data suggests that the K pathway is used for charge compensation of electron transfer to the catalytic site, but only in the A2  P3 state transition. Our data also show that the water molecules identified in the crystal structures of CytcO play an important role in proton transfer through the D pathway. Finally, the effects of liposome reconstitution of CytcO on D-pathway proton transfer were investigated. The results suggest that the membrane modulates the rates of proton transfer through the D pathway, and also influences the extent of electron transfer between redox-active sites CuA and heme a.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. s. 63
Nyckelord
membrane protein, respiration, redox reaction, cytochrome aa3, cytochrome c oxidase
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-108335 (URN)978-91-7447-967-6 (ISBN)
Disputation
2014-11-28, Magneli hall, Chemical Practice Laboratory, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2014-11-06 Skapad: 2014-10-21 Senast uppdaterad: 2022-02-23Bibliografiskt granskad

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Näsvik Öjemyr, Lindavon Ballmoos, ChristophSvahn, EmelieBrzezinski, Peter

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