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Plasticity of proton pathway structure and water coordination 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.
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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.

Place, publisher, year, edition, pages
2007. Vol. 282, no 20, 15148-15158 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-25431DOI: 10.1074/jbc.M700348200v1ISI: 000246589000054OAI: oai:DiVA.org:su-25431DiVA: diva2:199696
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
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Available from: 2008-09-04 Created: 2008-09-04Bibliographically approved

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