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Intricate role of water in proton transport through cytochrome c oxidase
Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
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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 O2 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 (Ser200Ile, 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 Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the PR → F and F → O transitions (PR is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O2, F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the PR → F transition is shown to be essentially pH independent up to pH 12 (i.e., the apparent pKa of Glu286 is increased from 9.4 by at least 3 pKa 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. © 2010 American Chemical Society.

Place, publisher, year, edition, pages
2010. Vol. 132, no 45, 16225-16239 p.
Keyword [en]
Controls Electron-Transfer, Heme-Copper Oxidases, Valence-Bond Model, Rhodobacter-Sphaeroides, D-Pathway, Paracoccus-Denitrificans, Computer-Simulation, Coupled Proton, Biomolecular Systems, Charge-Transfer
National Category
Chemical Sciences
URN: urn:nbn:se:kth:diva-149706DOI: 10.1021/ja107244gISI: 000284202200066ScopusID: 2-s2.0-78449239941OAI: diva2:741214
Swedish Research Council

QC 20140827

Available from: 2014-08-27 Created: 2014-08-26 Last updated: 2014-08-27Bibliographically approved

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Lepp, Håkan
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