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Active Transport of Ions across Biomembranes: A Kinetic Study of Cytochrome c Oxidase Reconstituted into Phospholipid Vesicles
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ion transport across membranes is of uttermost importance for us. It is the foundation for signaling of various kinds e.g. in the nerve-system. Furthermore, energy from photosynthesis and metabolism is conserved in electrochemical gradients across membranes, maintained by ion pumps. In this thesis I discuss mechanisms of how protons and other ions are translocated across biomembranes against their concentration gradients. I have studied one specific proton pump, cytochrome c oxidase (CytcO) and in the summary I also compare CytcO with two other pumps for which a wealth of structural and functional information has recently been obtained. The data in the articles presented in this thesis support a model were proton pumping can be achieved without simultaneous oxidation of heme a or electron transfer (paper I); where a proton is transferred to the catalytic site before the pump site is protonated (paper IV); and where proton release is preceded by a conformational change (paper II). These observations could be explained by a model involving a conformational change of the pump element, recently proposed from our laboratory1. Furthermore the results from the papers in this thesis show that proton uptake precedes proton release in D2O (paper II). The kinetics of electron transfers linked to proton pumping is solely determined by the pH on the N-side of the membrane (paper III). Finally Zn2+ added on the P-side of the membrane inhibits a specific reaction step (paper IV). In the three pumps described here conformational changes, modulating ion affinities, and the opening and closing of gates, seem to be involved in driving the ions across the membrane.

1. Brzezinski, P. & Larsson, G. (2003) Biochim. Biophys. Acta 1605, 1-13.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2007. , 55 p.
Keyword [en]
cytochrome c oxidase, electron transfer, proton transfer, proton pumping, kinetic isotop effect, ion pumping, flow flash
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-6806ISBN: 978-91-7155-413-0 (print)OAI: oai:DiVA.org:su-6806DiVA: diva2:197088
Public defence
2007-05-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:30
Opponent
Supervisors
Available from: 2007-05-04 Created: 2007-04-26Bibliographically approved
List of papers
1. A Mechanistic Principle for Proton Pumping by Cytochrome c Oxidase
Open this publication in new window or tab >>A Mechanistic Principle for Proton Pumping by Cytochrome c Oxidase
2005 In: Nature, ISSN 0028-0836, Vol. 437, no 7056, 286-9 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24242 (URN)
Note
Part of urn:nbn:se:su:diva-6806Available from: 2007-05-04 Created: 2007-04-26Bibliographically approved
2. The Timing of Proton Migration in Membrane-Reconstituted Cytochrome c Oxidase
Open this publication in new window or tab >>The Timing of Proton Migration in Membrane-Reconstituted Cytochrome c Oxidase
2005 In: Proc. Natl. Acad. Sci. USA, ISSN 0027-8424, Vol. 102, no 49, 17624-17629 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24243 (URN)
Note
Part of urn:nbn:se:su:diva-6806Available from: 2007-05-04 Created: 2007-04-26Bibliographically approved
3. The Inside pH Determines Rates of Electron and Proton Transfer in Vesicle-Reconstituted Cytochrome c Oxidase
Open this publication in new window or tab >>The Inside pH Determines Rates of Electron and Proton Transfer in Vesicle-Reconstituted Cytochrome c Oxidase
2007 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 5, 381-386 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

Keyword
Rhodobacter sphaeroides; Proton pumping; Cytochrome aa3; Respiration; Kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-24244 (URN)10.1016/j.bbabio.2007.02.023 (DOI)
Note
Part of urn:nbn:se:su:diva-6806Available from: 2007-05-04 Created: 2007-04-26 Last updated: 2010-07-29Bibliographically approved
4. Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.
Open this publication in new window or tab >>Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.
2006 In: Biochimica et Biophysica Acta (BBA) - Bioenergetics, ISSN 0005-2728, Vol. 1757, no 5-6, 388-394 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24245 (URN)
Note
Part of urn:nbn:se:su:diva-6806Available from: 2007-05-04 Created: 2007-04-26Bibliographically approved

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