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Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
membrane protein, respiration, redox reaction, cytochrome aa3, cytochrome c oxidase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-108335ISBN: 978-91-7447-967-6 (print)OAI: oai:DiVA.org:su-108335DiVA: diva2:757251
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
List of papers
1. Proton pumping by an inactive structural variant of cytochrome c oxidase:
Open this publication in new window or tab >>Proton pumping by an inactive structural variant of cytochrome c oxidase:
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.

Keyword
Electron transfer, Membrane protein, Electrochemical potential, redox reaction, Cytochrome aa3
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-108334 (URN)10.1016/j.jinorgbio.2014.06.016 (DOI)000342608900002 ()
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
2. Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase
Open this publication in new window or tab >>Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-25429 (URN)10.1021/bi7024707 (DOI)000255164700008 ()
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2017-12-13Bibliographically approved
3. Intricate Role of Water in Proton Transport through Cytochrome c Oxidase
Open this publication in new window or tab >>Intricate Role of Water in Proton Transport through Cytochrome c Oxidase
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2010 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, no 45, 16225-16239 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O-2 to H2O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200lle, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200lle and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P-R -> F and F -> O transitions (P-R is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O-2, F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P-R -> F transition is Shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-51337 (URN)10.1021/ja107244g (DOI)000284202200066 ()
Note
authorCount :7Available from: 2011-01-11 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
4. The membrane modulates internal proton transfer in cytochrome c oxidase
Open this publication in new window or tab >>The membrane modulates internal proton transfer in cytochrome c oxidase
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2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, 1092-1100 p.Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-75164 (URN)10.1021/bi201795c (DOI)000300132900005 ()
Available from: 2012-04-10 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved

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