Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cytochrome c Oxidase dysfunction in cancer: Exploring the molecular mechanisms
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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: urn:nbn:se:su:diva-65303ISBN: 978-91-7447-424-4 (print)OAI: oai:DiVA.org:su-65303DiVA: diva2:462163
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
List of papers
1. Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant
Open this publication in new window or tab >>Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant
Show others...
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.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-11268 (URN)000252024100008 ()18052347 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-13Bibliographically approved
2. A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O-2-reduction activity
Open this publication in new window or tab >>A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O-2-reduction activity
2010 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1797, no 5, 550-556 p.Article in journal (Refereed) Published
Abstract [en]

In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa(3)) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of similar to 5, while reprotonation of the Glu from solution was slowed by a factor of similar to 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells. 

Keyword
Respiratory chain, Electron transfer, Mitochondrial disease, Cytochrome aa(3), Mitochondria, Deficiency, mtDNA
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-50683 (URN)10.1016/j.bbabio.2010.01.027 (DOI)000276621100004 ()
Note
authorCount :4Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2017-12-11Bibliographically approved
3. A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase
Open this publication in new window or tab >>A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase
2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 9, 3402-3407 p.Article in journal (Refereed) Published
Abstract [en]

An increasing number of cancer types have been found to be linked to specific mutations in the mitochondrial DNA, which result in specific structural changes of the respiratory enzyme complexes. In this study, we have investigated the effect of 2 such mutations identified in colon cancer patients, leading to the amino acid substitutions Ser458Pro and Gly125Asp in subunit I of cytochrome c oxidase (complex IV) [Greaves et al. (2006) Proc Natl Acad Sci USA 103:714-719]. We introduced these mutations in Rhodobacter sphaeroides, which carries an oxidase that serves as a model of the mitochondrial counterpart. The lack of expression of the former variant indicates that the amino acid substitution results in severely altered overall structure of the enzyme. The latter mutation (Gly171Asp in the bacterial oxidase) resulted in a structurally intact enzyme, but with reduced activity (approximately 30%), mainly due to slowed reduction of the redox site heme a. Furthermore, even though the Gly171Asp CytcO pumps protons, an intrinsic proton leak was identified, which would lead to a decreased overall energy-conversion efficiency of the respiratory chain, and would also perturb transport processes such as protein, ion, and metabolite trafficking. Furthermore, the specific leak may act to alter the balance between the electrical and chemical components of the proton electrochemical gradient.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-34712 (URN)10.1073/pnas.0811450106 (DOI)000263844100076 ()19218458 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
4. Functional effects of mutations in cytochrome c oxidase related to prostate cancer
Open this publication in new window or tab >>Functional effects of mutations in cytochrome c oxidase related to prostate cancer
2011 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 10, 1336-1341 p.Article in journal (Refereed) Published
Abstract [en]

A number of missense mutations in subunit I of cytochrome c oxidase (CytcO) have previously been linked to prostate cancer (Petros et al. (2005) PNAS, 102, 719). To investigate the effects of these mutations at the molecular level, in the present study we prepared four different structural variants of the bacterial Rhodobacter sphaeroides CytcO (cytochrome aa3), each carrying one amino-acid residue replacement corresponding to the following substitutions identified in the above-mentioned study: Asn11Ser, Ala122Thr, Ala341Ser and Val380Ile (residues Asn25, Ser168, Ala384 and Val423 in the R. sphaeroides oxidase). This bacterial CytcO displays essentially the same structural and functional characteristics as those of the mitochondrial counterpart. We investigated the overall activity, proton pumping and internal electron- and proton- transfer reactions in the structural variants. The results show that the turnover activities of the mutant CytcOs were reduced by at most a factor of two. All variants pumped protons, but in Ser168Thr, Ala384Ser and Val423Ile we observed slight internal proton leaks. In all structural variants the internal electron equilibrium was slightly shifted away from the catalytic site at high pH (10), resulting in a slower observed ferryl to oxidized transition. Even though the effects of the mutations were relatively modest, the results suggest that they destabilize the proton-gating machinery. Such effects could be manifested in the presence of a transmembrane electrochemical gradient resulting in less efficient energy conservation.

Keyword
Respiration, Proton pumping, Electron transfer, Cytochrome aa3, Mitochondria, Membrane protein
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-65338 (URN)10.1016/j.bbabio.2011.02.005 (DOI)000294513600011 ()
Available from: 2011-12-15 Created: 2011-12-07 Last updated: 2017-12-08Bibliographically approved

Open Access in DiVA

fulltext(3002 kB)3714 downloads
File information
File name FULLTEXT01.pdfFile size 3002 kBChecksum SHA-512
ccfda434855e9efa6fe7bb0ccff76eb57722630186033ee2ebb544b36870cca5c6486c3386674247c82e9af0830e24c82477ed72eb36757ee7de321acee73d72
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Namslauer, Ida
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 3714 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 291 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf