Developing mouse complex I as a model system: structure, function and implications in mitochondrial diseases.
type: CreativeWork
description: Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the ‘active’ and ‘de-active’ states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, …
abstract: Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the ‘active’ and ‘de-active’ states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, …
author: Agip, Ahmed-Noor
language: en
keywords: Mitochondria, Complex I, Membrane protein, CryoEM, Mitochondrial diseases, Proton pump
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c2379a3a-14ef-4fa3-a8e7-4ec76cf20fa9/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2018-10-20
date modified: 2026-07-22T22:24:11Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.30568