On the antiporter-like subunits of respiratory chain Complex I - implications for the evolution and coupling mechanism of the NADH:quinone oxidoreductase enzyme complex

Cecilie Mathiesen

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Complex I or NADH:quinone oxidoreductase is the largest enzyme complex, but the least understood energy coupling site in the respiratory chain of mitochondria and bacteria. No high-resolution structural information exists for this enzyme and the molecular mechanism that couples electron transfer and proton pumping is not understood. In mammals, about 50% of the mitochondrial DNA is encoding protein subunits of Complex I, and thus there is a strong correlation between defect Complex I and various degenerative diseases. Learning more about Complex I is thus important both for basic science and for medicine.

Complex I contains three large membrane-spanning subunits, NuoL, NuoM and NuoN, that are homologous to one particular class of Na+/H+ antiporters and therefore are likely to harbor important components of the proton translocation machinery. MrpA and MrpD belong to this class of Na+/H+ antiporters and are found in a gene cluster containing seven genes, mrpA-G. In this work the transmembrane topology of the antiporter-like subunits was determined and their phylogenetic relationship investigated. In the analysis MrpA and MrpD formed distinct branches where NuoL grouped with MrpA and NuoM/N with MrpD. The result suggest that there are functional differences between the MrpA/NuoL and MrpD/NuoM proteins. The remaining proteins encoded by the mrp gene cluster were analyzed by PSI-BLAST, a bioinformatical search tool that more efficiently detect distant homologies. MrpC was found to be a NuoK homologue, and thus we conclude that NuoK, NuoL and NuoM where recruited together to Complex I, from the antiporter module MrpC, MrpA and MrpD. A functional difference between MrpA and MrpD was confirmed using Bacillus subtilis mrpA and mrpD deletion strains that were more salt and pH sensitive than wild type cells, but to a different extent.

Recent work by Julia Steubers group at ETH Zürich showed that Complex I from Escherichia coli could translocate Na+, but this has hitherto not been regarded as a general property of proton pumping Complex I. By expressing the Complex I subunits NuoL and NuoM from the alpha-proteobacteria Rhodobacter capsulatus in the B. subtilis deletion strains we have demonstrated that these subunits are capable of Na+ translocation in vivo. Since R. capsulatus Complex I is more closely related to mitochondrial Complex I than to the E. coli enzyme, it is very likely that all Complex I enzymes have the ability to translocate Na+ as well as H+.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date2003 May 23
Publisher
Print ISBNs91-7422-020-9
Publication statusPublished - 2003

Bibliographical note

Defence details

Date: 2003-05-23
Time: 10:30
Place: Kemicentrum, Sal A, Getingevägen 60, Lund

External reviewer(s)

Name: Friedrich, Thorsten
Title: Prof.
Affiliation: Albert-Ludwigs-Universitaet, Institut für Org. Chemie und Biochemie, Freiburg, Federal Republic of Germany.

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Article: I. Transmembrane topology of the NuoL, M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters.Mathiesen, C. and Hägerhäll, C. Biochim. Biophys Acta (2002). 1556:121-132.

Article: II. The "antiporter module" of respiratory chain Complex I includes the MrpC/NuoK subunit - a revision of the modular evolution scheme. Mathiesen, C. and Hägerhäll, C. (2003). FEBS-Letters, submitted.

Article: III. The Complex I subunits NuoL and NuoM from Rhodobacter capsulatus exhibit Na+ translocation ability in vivo.Mathiesen, C. and Hägerhäll, C. (2003). Manuscript.

Article: IV. Expression and purification of his-tagged NuoL subunit from Rhodobacter capsulatus Complex I in Escherichia coli.Mathiesen, C., Eek, M. and Hägerhäll, C. (2003). Manuscript.

Subject classification (UKÄ)

  • Biological Sciences

Keywords

  • Metabolism
  • Biokemi
  • metabolism
  • Biochemistry
  • mrp
  • electron transport
  • respiration
  • membrane protein

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