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Choreography of a proton pump: Studies of charge-transfer reactions in cytochrome c oxidase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the last step of cellular respiration, electrons from metabolites are transferred to molecular oxygen, mediated by the enzyme complexes of the respiratory chain. Some of these enzyme complexes couple these redox reactions to formation of an electrochemical proton gradient across the cell membrane. The proton gradient is used e.g. by ATP synthase to drive synthesis of ATP. 

The terminal enzyme complex in the respiratory chain, cytochrome c oxidase (CytcO), catalyses reduction of O2 to water. In this process it contributes to maintaining the electrochemical proton gradient by two separate mechanisms: (i) by uptake of electrons and protons from the opposite sides of the membrane (for O2 reduction to water). (ii) by proton pumping across the membrane. Protons used in the O2 reduction, as well as protons that are pumped, are taken up through two different proton-uptake pathways, the D and the K pathways. In addition, a third proton-transfer pathway has been suggested for the mitochondrial CytcOs, namely the H pathway. So far, the molecular mechanism by which CytcO pumps protons has not been determined. 

In this work we have studied proton- and electron-transfer reactions in aa3-type CytcOs, with the aim of understanding the functional design of the proton-pumping machinery in CytcO. First, we studied structural variants of CytcO from the bacterium Rhodobacter (R.) sphaeroides, where an amino-acid at position 425, previously shown to undergo redox-induced conformational changes, was substituted. The results point to a link between redox-induced structural changes and intramolecular proton-transfer rates through the D pathway. Second, we studied the electron distribution in the “activated” oxidized (OH) state of CytcO, by using an electrostatic complex of CytcO and cytochrome c. We also investigated electron-transfer reactions linked to proton pumping in structural variants of CytcO from R. sphaeroides and the yeast Saccharomyces (S.) cerevisiae, with mutations in the proposed D and H proton-uptake pathways. The data indicate that the S. cerevisiae mitochondrial CytcO uses the D pathway for proton uptake and pumping as the R. sphaeroides CytcO. Lastly, we studied reactions linked to proton uptake and pumping in structural variants of CytcO from R. sphaeroides with alterations in both proton-uptake pathways. The data elucidated the mechanism of proton transfer and gating in CytcO.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 78
Keywords [en]
heme-copper oxidase, cytochrome c oxidase, respiration, proton pump, electron transfer, proton transfer, proton-transfer pathway, midpoint potential, proton-pumping mechanism
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-173568ISBN: 978-91-7797-855-8 (print)ISBN: 978-91-7797-856-5 (electronic)OAI: oai:DiVA.org:su-173568DiVA, id: diva2:1354641
Public defence
2019-11-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2019-10-16 Created: 2019-09-25 Last updated: 2019-10-08Bibliographically approved
List of papers
1. Structural Changes and Proton Transfer in Cytochrome c Oxidase
Open this publication in new window or tab >>Structural Changes and Proton Transfer in Cytochrome c Oxidase
2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12047Article in journal (Refereed) Published
Abstract [en]

In cytochrome c oxidase electron transfer from cytochrome c to O-2 is linked to transmembrane proton pumping, which contributes to maintaining a proton electrochemical gradient across the membrane. The mechanism by which cytochrome c oxidase couples the exergonic electron transfer to the endergonic proton translocation is not known, but it presumably involves local structural changes that control the alternating proton access to the two sides of the membrane. Such redox-induced structural changes have been observed in X-ray crystallographic studies at residues 423-425 ( in the R. sphaeroides oxidase), located near heme a. The aim of the present study is to investigate the functional effects of these structural changes on reaction steps associated with proton pumping. Residue Ser425 was modified using site-directed mutagenesis and time-resolved spectroscopy was used to investigate coupled electron-proton transfer upon reaction of the oxidase with O2. The data indicate that the structural change at position 425 propagates to the D proton pathway, which suggests a link between redox changes at heme a and modulation of intramolecular proton-transfer rates.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-120910 (URN)10.1038/srep12047 (DOI)000360145400001 ()
Available from: 2015-09-24 Created: 2015-09-18 Last updated: 2019-09-30Bibliographically approved
2. The electron distribution in the activated state of cytochrome c oxidase
Open this publication in new window or tab >>The electron distribution in the activated state of cytochrome c oxidase
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 7502Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase catalyzes reduction of O-2 to H2O at a catalytic site that is composed of a copper ion and heme group. The reaction is linked to translocation of four protons across the membrane for each O-2 reduced to water. The free energy associated with electron transfer to the catalytic site is unequal for the four electron-transfer events. Most notably, the free energy associated with reduction of the catalytic site in the oxidized cytochrome c oxidase (state O) is not sufficient for proton pumping across the energized membrane. Yet, this electron transfer is mechanistically linked to proton pumping. To resolve this apparent discrepancy, a high-energy oxidized state (denoted O-H) was postulated and suggested to be populated only during catalytic turnover. The difference between states O and O-H was suggested to be manifested in an elevated midpoint potential of Cu-B in the latter. This proposal predicts that one-electron reduction of cytochrome c oxidase after its oxidation would yield re-reduction of essentially only Cu-B. Here, we investigated this process and found similar to 5% and similar to 6% reduction of heme a(3) and Cu-B, respectively, i.e. the apparent redox potentials for heme a(3) and CuB are lower than that of heme a.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-157778 (URN)10.1038/s41598-018-25779-w (DOI)000431955100007 ()29760451 (PubMedID)
Available from: 2018-07-27 Created: 2018-07-27 Last updated: 2019-09-30Bibliographically approved
3. Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase
Open this publication in new window or tab >>Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In cytochrome c oxidase (CytcO) reduction of O2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than throughthe D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D(Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO withO2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and pumping.

Keywords
cytochrome c oxidase, electron transfer, cytochrome aa3, membrane protein, ligand, kinetics, mechanism
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167129 (URN)
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-09-25Bibliographically approved
4. Proton transfer in uncoupled variants of cytochrome c oxidase
Open this publication in new window or tab >>Proton transfer in uncoupled variants of cytochrome c oxidase
Show others...
(English)Manuscript (preprint) (Other academic)
Keywords
electron transfer, proton transfer, cytochrome aa3, membrane protein, proton pumping, mechanism
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-173514 (URN)
Available from: 2019-09-25 Created: 2019-09-25 Last updated: 2019-09-30Bibliographically approved

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