Investigating the rate-limiting step of mitochondrial complex I catalysis
type: CreativeWork
description: Respiratory complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in metabolism and is the least understood protein in the mitochondrial electron transfer chain (ETC). It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to the translocation of four protons across the inner mitochondrial membrane, contributing to the proton motive force (PMF) used to synthesise ATP. Although structural knowledge of complex I is now extensive, the mechanism by which it couples the redox energy for proton translocation remains unclear. First, the rate-limiting step of catalysis in complex I was investigated from the point of view of the proton. Using the proteoliposomes system, the pH and solvent isotope dependence of kinetic parameters of purified bovine and *Yarrowia lipolytica* complex I mutants were measured under a range of conditions. I find complex I robustly displays a solvent kinetic isotope effect (KIE), signifying that the rate-limiting step involves a proton transfer. This isotopic sensitive step is dependent on Q-chain length, Q binding-site mutations, and Qconcentration, but not dependent on Δp, suggesting that this step is Q-reduction. Proton inventory experiments suggest that a single proton is transferred in the rate-limiting step, and that complex I is rate-limited by a combination of a proton transfer step and an isotopically insensitive step, which was assigned as the product release of ubiquinol. Next, the role of conserved charged residues in the central axis were investigated using the *Paracoccus denitrificans* model system. Mutants of residues involved in the energy propagation pathway and subunit hydration channel gating were evaluated using solvent isotope effect and proton pumping experiments. These mutations all exhibited a greater isotope dependence than WT, showing that proton pumping steps have become robustly rate-limiting. Then, experimental results were evaluated against computational and mechanistic proposals, to iden…
abstract: Respiratory complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in metabolism and is the least understood protein in the mitochondrial electron transfer chain (ETC). It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to the translocation of four protons across the inner mitochondrial membrane, contributing to the proton motive force (PMF) used to synthesise ATP. Although structural knowledge of complex I is now extensive, the mechanism by which it couples the redox energy for proton translocation remains unclear. First, the rate-limiting step of catalysis in complex I was investigated from the point of view of the proton. Using the proteoliposomes system, the pH and solvent isotope dependence of kinetic parameters of purified bovine and *Yarrowia lipolytica* complex I mutants were measured under a range of conditions. I find complex I robustly displays a solvent kinetic isotope effect (KIE), signifying that the rate-limiting step involves a proton transfer. This isotopic sensitive step is dependent on Q-chain length, Q binding-site mutations, and Qconcentration, but not dependent on Δp, suggesting that this step is Q-reduction. Proton inventory experiments suggest that a single proton is transferred in the rate-limiting step, and that complex I is rate-limited by a combination of a proton transfer step and an isotopically insensitive step, which was assigned as the product release of ubiquinol. Next, the role of conserved charged residues in the central axis were investigated using the *Paracoccus denitrificans* model system. Mutants of residues involved in the energy propagation pathway and subunit hydration channel gating were evaluated using solvent isotope effect and proton pumping experiments. These mutations all exhibited a greater isotope dependence than WT, showing that proton pumping steps have become robustly rate-limiting. Then, experimental results were evaluated against computational and mechanistic proposals, to iden…
author: Choy, Man Nok
language: eng
keywords: Alternative oxidase, bos taurus, complex I, enzyme kinetics, kinetic isotope effect, mitochondria, NADH dehydrogenase 1, NADH dehydrogenase 2, paracoccus denitrificans, proteoliposomes, proton inventory, rate-limiting step, solvent isotope effect, ubiquinone, yarrowia lipolytica
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/7e2572e7-09ae-4742-be1a-737b2f0d9ea1/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2023-09-30
date modified: 2026-07-22T22:24:00Z
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.107220