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Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant
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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2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 1, 73-83 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples' the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa<sub>3</sub>), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O<sub>2</sub> reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains ∼1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton transtocation by CytcO.

Place, publisher, year, edition, pages
2008. Vol. 47, no 1, 73-83 p.
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-11268ISI: 000252024100008PubMedID: 18052347OAI: oai:DiVA.org:su-11268DiVA: diva2:177787
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Cytochrome c Oxidase dysfunction in cancer: Exploring the molecular mechanisms
Open this publication in new window or tab >>Cytochrome c Oxidase dysfunction in cancer: Exploring the molecular mechanisms
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mutations in genes encoding the mitochondrial enzyme Cytochrome c Oxidase (CytcO) have lately been found in connection to various types of cancer. Some mutations result in substitutions of highly conserved amino-acid residues. As CytcO is an essential enzyme in oxidative phosphorylation, the substitutions are likely to have deleterious effects on the cellular energy metabolism. There is, however, a lack of data on the functional consequences of the pathogenic substitutions. In the publications on which this thesis is based, we investigated the effects of the substitutions on a molecular level. This was done using the validated bacterial model organism Rhodobacter sphaeroides which has a CytcO that is both structurally and functionally similar to the mammalian CytcO. For the functional studies, we used spectroscopic techniques to investigate the overall activity of the enzyme as well as the proton-pumping efficiency and the internal proton and electron transfers. We found that most of the CytcO substitutions observed in connection to cancer, resulted in a decreased catalytic activity. The impaired activity was due to defects in specific electron- or proton-transfer processes. Moreover, in several cases the substitutions resulted in an impaired proton-pumping activity. This thesis deals with the relevance of using R. sphaeroides CytcO as a model system for investigating human disease, as well as the possible links between the defective enzyme and the development of cancer.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 65 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-65303 (URN)978-91-7447-424-4 (ISBN)
Public defence
2012-02-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2012-01-12 Created: 2011-12-06 Last updated: 2012-01-05Bibliographically approved

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