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Structural elements involved in protein-mediated proton transfer: Implications from studies of cytochrome c oxidase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Martin Högbom, Peter Brzezinski)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proton transfer is one of the most common reactions in biological systems. During energy conversion inside a cell, proton transfer is crucial to maintain an electrochemical proton gradient across the cell membrane. This gradient is in turn used to e.g. produce ATP, the energy currency of the cell. One of the key components of the build-up of this gradient is cytochrome c oxidase. This membrane-bound enzyme catalyzes the reduction of molecular oxygen to water, using protons and electrons, and in the process protons are pumped across the membrane. All protons used during oxygen reduction and those that are pumped, are transferred via hydrophilic pathways inside the hydrophobic interior of the enzyme. One of these pathways, called the D pathway, is used to transfer protons both to the catalytic site and towards a pump site. It is yet not fully understood how these proton-transfer reactions are timed, coupled and controlled.

 

This thesis is focused on studies of proton-transfer reactions through the D pathway in variants of cytochrome c oxidase that lack the ability to pump protons. The results suggest that changes in pKa values of key residues, as well as structural changes inside the pathway, can explain the non-pumping phenotypes. The results have led us to propose that an internal proton shuttle (Glu286I) can adopt two different conformations that are in equilibrium with each other, and that this equilibrium is altered in non-pumping variants of cytochrome c oxidase. We also observed that proton transfer through the D pathway could occur with the same rate as in the wild-type enzyme even when one of the key residues (Asp132I) is absent. This result contradicts previous assumptions that acidic residues must be present at an orifice of proton pathways. We therefore suggest that this specific residue could have an additional role, e.g. as a selectivity filter that excludes all ions except protons from entering the pathway.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2013. , 62 p.
Keyword [en]
cytochrome c oxidase, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-85934ISBN: 978-91-7447-565-4 (print)OAI: oai:DiVA.org:su-85934DiVA: diva2:585584
Public defence
2013-02-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Accepted; Paper 3: Manuscript

Available from: 2013-01-24 Created: 2013-01-10 Last updated: 2013-03-15Bibliographically approved
List of papers
1. Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase
Open this publication in new window or tab >>Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase
2010 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, 710-23 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c2+ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O2. In this reaction part of the free energy available from O2 reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O2. Our understanding of the molecular design of the machinery that couples O2 reduction to proton pumping in oxidases has greatly benefited from studies of so called "uncoupled" structural variants of the oxidases. In these uncoupled oxidases the catalytic O2-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O2 is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pKa of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pKa presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pKa and uncoupling of proton pumping from O2 reduction.

Keyword
proton, Glu, oxygen
National Category
Natural Sciences
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-51707 (URN)10.1016/j.bbabio.2010.02.020 (DOI)000279663200015 ()20184858 (PubMedID)
Available from: 2011-01-12 Created: 2011-01-12 Last updated: 2017-12-11Bibliographically approved
2. Proton Uptake and pK(a) Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase
Open this publication in new window or tab >>Proton Uptake and pK(a) Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase
Show others...
2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 5, 827-836 p.Article in journal (Other academic) Published
Abstract [en]

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O-2 to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located similar to 10 angstrom from the protein surface, is particularly sensitive to mutations that uncouple the O-2 reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-85919 (URN)10.1021/bi301597a (DOI)000314675800007 ()
Available from: 2013-01-10 Created: 2013-01-10 Last updated: 2017-12-06Bibliographically approved
3. The role of acidic residues at the orifice of a proton pathway in cytochrome c oxidase
Open this publication in new window or tab >>The role of acidic residues at the orifice of a proton pathway in cytochrome c oxidase
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-85922 (URN)
Available from: 2013-01-10 Created: 2013-01-10 Last updated: 2013-01-10
4. Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects
Open this publication in new window or tab >>Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects
Show others...
2011 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 9, 1083-1094 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa(3) CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O(2) reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a pump site and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the HID kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from 02 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F -> O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P -> F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F -> O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

Keyword
Respiration, Electron transfer, Cytochrome aa(3), Membrane protein, Electrostatics, Energy transduction
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-68035 (URN)10.1016/j.bbabio.2011.03.012 (DOI)000293550300008 ()
Note

authorCount :6

Available from: 2012-01-03 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved

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