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Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in 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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 17, 4929-4935 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase couples electron transfer from cytochrome C to 02 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with 02, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.

Place, publisher, year, edition, pages
2008. Vol. 47, no 17, 4929-4935 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-25429DOI: 10.1021/bi7024707ISI: 000255164700008OAI: oai:DiVA.org:su-25429DiVA: diva2:199694
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Experimental studies of proton translocation reactions in biological systems: Electrogenic events in heme-copper oxidases
Open this publication in new window or tab >>Experimental studies of proton translocation reactions in biological systems: Electrogenic events in heme-copper oxidases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O2 to H2O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the aa3-type oxidases, to which cytochrome c oxidase (CytcO) belongs, which uses energy released in the O2-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO2. Although as exergonic as O2-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3 oxidases and the NORs. The cbb3 oxidases have been shown to pump protons with nearly the same efficiency as the aa3 oxidases, despite low sequence similarity.

This thesis is focused on measurements of membrane potential generating reactions during catalysis in the CytcO and the cbb3 oxidase from Rhodobacter sphaeroides, and the NOR from Paracoccus denitrificans, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in CytcO showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of CytcO. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in cbb3 oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O2-reduction.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 62 p.
Keyword
heme-copper oxidase, cytochrome c oxidase, nitric oxide reductase, cbb3-type oxidase, proton pumping, uncoupling, charge-transfer, electrogenic event, flow-flash
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-8147 (URN)978-91-7155-712-4 (ISBN)
Public defence
2008-09-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2008-09-04 Created: 2008-09-04Bibliographically approved
2. 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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