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Experimental studies of proton translocation reactions in biological systems: Electrogenic events in heme-copper oxidases
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
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 [en]
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: urn:nbn:se:su:diva-8147ISBN: 978-91-7155-712-4 (print)OAI: oai:DiVA.org:su-8147DiVA: diva2:199699
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
List of papers
1. pH dependence of voltage changes associated with intramolecular electron and proton transfer reactions in cytochrome c oxidase
Open this publication in new window or tab >>pH dependence of voltage changes associated with intramolecular electron and proton transfer reactions in cytochrome c oxidase
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-25428 (URN)
Note
Part of urn:nbn:se:su:diva-8147Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2010-01-13Bibliographically 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. Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway
Open this publication in new window or tab >>Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway
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2008 In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1777, no 7-8, 897-903 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25430 (URN)000257696400048 ()
Note
Part of urn:nbn:se:su:diva-8147Available from: 2008-09-04 Created: 2008-09-04Bibliographically approved
4. Plasticity of proton pathway structure and water coordination in cytochrome c oxidase
Open this publication in new window or tab >>Plasticity of proton pathway structure and water coordination in cytochrome c oxidase
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 20, 15148-15158 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-boundproton pump. One of the proton transfer pathways of the enzyme,the D pathway, used for the transfer of both substrate and pumpedprotons, accommodates a network of hydrogen-bonded water moleculesthat span the distance between an aspartate (Asp132), near theprotein surface, and glutamate Glu286, which is an internalproton donor to the catalytic site. To investigate how changesin the environment around Glu286 affect the mechanism of protontransfer through the pathway, we introduced a non-hydrogen-bonding(Ala) or an acidic residue (Asp) at position Ser197 (S197A orS197D), located 7 Å from Glu286. Although Ser197 is hydrogen-bondedto a water molecule that is part of the D pathway "proton wire,"replacement of the Ser by an Ala did not affect the proton transferrate. In contrast, the S197D mutant CytcO displayed a turnoveractivity of 35% of that of the wild-type CytcO, and the O2 reductionreaction was not linked to proton pumping. Instead, a fractionof the substrate protons was taken from the positive ("incorrect")side of the membrane. Furthermore, the pH dependence of theproton transfer rate was altered in the mutant CytcO. The resultsindicate that there is plasticity in the water coordinationof the proton pathway, but alteration of the electrostatic potentialwithin the pathway results in uncoupling of the proton translocationmachinery.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25431 (URN)10.1074/jbc.M700348200v1 (DOI)000246589000054 ()
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2017-12-13Bibliographically approved
5. A pathway for protons in nitric oxide reductase from Paracoccus denitrificans
Open this publication in new window or tab >>A pathway for protons in nitric oxide reductase from Paracoccus denitrificans
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2007 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1767, no 5, 362-373 p.Article in journal (Refereed) Published
Abstract [en]

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e(-) + 2H(+) -> N2O + H2O) as part of the denitriffication process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O-2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O-2, we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O-2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O-2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O-2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O-2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

Keyword
proton transfer, electron transfer, proteoliposomes, flow-flash, non-heme iron, nitric oxide, oxygen, homology modeling, sequence alignments
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-25432 (URN)10.1016/j.bbabio.2007.03.006 (DOI)000246654200003 ()
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2017-12-13Bibliographically approved
6. Substrate control of vectorial proton transfer in a heme-copper oxidase
Open this publication in new window or tab >>Substrate control of vectorial proton transfer in a heme-copper oxidase
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-25433 (URN)
Note
Part of urn:nbn:se:su:diva-8147Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2010-01-13Bibliographically approved

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