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Proton pumping by an inactive structural variant of cytochrome c oxidase:
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 140, 6-11 p.Article in journal (Refereed) Published
Abstract [en]

The aa3-type cytochrome c oxidases (CytcOs) from e.g. Rhodobacter sphaeroides and Paracoccus denitrificans harbor two proton-transfer pathways. The K pathway is used for proton uptake upon reduction of the CytcO, while the D pathway is used after binding of O2 to the catalytic site. The aim of the present study was to determine whether or not CytcO in which the K pathway is blocked (by e.g. the Lys362Met replacement) is capable of pumping protons. The process can not be studied using conventional assays because the O2-reduction activity is too low when the K pathway is blocked. Consequently, proton pumping with a blocked K pathway has not been demonstrated directly. Here, the Lys362Met and Ser299Glu structural variants were reconstituted in liposomes and allowed to (slowly) become completely reduced. Then, the reaction with O2 was studied with μs time resolution after flash photolysis of a blocking CO ligand bound to heme a3. The data show that with both the inactive Lys362Met and partly active Ser299Glu variants proton release occurred with the same time constants as with the wild-type oxidase, i.e. ~ 200 μs and ~ 3 ms, corresponding in time to formation of the ferryl and oxidized states, respectively. Thus, the data show that the K pathway is not required for proton pumping, suggesting that D and K pathways operate independently of each other after binding of O2 to the catalytic site.

Place, publisher, year, edition, pages
2014. Vol. 140, 6-11 p.
Keyword [en]
Electron transfer, Membrane protein, Electrochemical potential, redox reaction, Cytochrome aa3
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-108334DOI: 10.1016/j.jinorgbio.2014.06.016ISI: 000342608900002OAI: oai:DiVA.org:su-108334DiVA: diva2:757091
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase
Open this publication in new window or tab >>Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 63 p.
Keyword
membrane protein, respiration, redox reaction, cytochrome aa3, cytochrome c oxidase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-108335 (URN)978-91-7447-967-6 (ISBN)
Public defence
2014-11-28, Magneli hall, Chemical Practice Laboratory, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2014-11-06 Created: 2014-10-21 Last updated: 2014-11-18Bibliographically approved

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