---
_id: '8340'
abstract:
- lang: eng
text: Mitochondria are sites of oxidative phosphorylation in eukaryotic cells. Oxidative
phosphorylation operates by a chemiosmotic mechanism made possible by redox-driven
proton pumping machines which establish a proton motive force across the inner
mitochondrial membrane. This electrochemical proton gradient is used to drive
ATP synthesis, which powers the majority of cellular processes such as protein
synthesis, locomotion and signalling. In this thesis I investigate the structures
and molecular mechanisms of two inner mitochondrial proton pumping enzymes, respiratory
complex I and transhydrogenase. I present the first high-resolution structure
of the full transhydrogenase from any species, and a significantly improved structure
of complex I. Improving the resolution from 3.3 Å available previously to up to
2.3 Å in this thesis allowed us to model bound water molecules, crucial in the
proton pumping mechanism. For both enzymes, up to five cryo-EM datasets with different
substrates and inhibitors bound were solved to delineate the catalytic cycle and
understand the proton pumping mechanism. In transhydrogenase, the proton channel
is gated by reversible detachment of the NADP(H)-binding domain which opens the
proton channel to the opposite sites of the membrane. In complex I, the proton
channels are gated by reversible protonation of key glutamate and lysine residues
and breaking of the water wire connecting the proton pumps with the quinone reduction
site. The tight coupling between the redox and the proton pumping reactions in
transhydrogenase is achieved by controlling the NADP(H) exchange which can only
happen when the NADP(H)-binding domain interacts with the membrane domain. In
complex I, coupling is achieved by cycling of the whole complex between the closed
state, in which quinone can get reduced, and the open state, in which NADH can
induce quinol ejection from the binding pocket. On the basis of these results
I propose detailed mechanisms for catalytic cycles of transhydrogenase and complex
I that are consistent with a large amount of previous work. In both enzymes, conformational
and electrostatic mechanisms contribute to the overall catalytic process. Results
presented here could be used for better understanding of the human pathologies
arising from deficiencies of complex I or transhydrogenase and could be used to
develop novel therapies.
acknowledged_ssus:
- _id: EM-Fac
acknowledgement: 'I acknowledge the support of IST facilities, especially the Electron
Miscroscopy facility for providing training and resources. Special thanks also go
to cryo-EM specialists who helped me to collect the data present here: Dr Valentin
Hodirnau (IST Austria), Dr Tom Heuser (IMBA, Vienna), Dr Rebecca Thompson (Uni.
of Leeds) and Dr Jirka Nováček (CEITEC). This work has been supported by iNEXT,
project number 653706, funded by the Horizon 2020 programme of the European Union.
This project has received funding from the European Union’s Horizon 2020 research
and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 665385.'
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Domen
full_name: Kampjut, Domen
id: 37233050-F248-11E8-B48F-1D18A9856A87
last_name: Kampjut
citation:
ama: Kampjut D. Molecular mechanisms of mitochondrial redox-coupled proton pumping
enzymes. 2020. doi:10.15479/AT:ISTA:8340
apa: Kampjut, D. (2020). Molecular mechanisms of mitochondrial redox-coupled
proton pumping enzymes. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8340
chicago: Kampjut, Domen. “Molecular Mechanisms of Mitochondrial Redox-Coupled Proton
Pumping Enzymes.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8340.
ieee: D. Kampjut, “Molecular mechanisms of mitochondrial redox-coupled proton pumping
enzymes,” Institute of Science and Technology Austria, 2020.
ista: Kampjut D. 2020. Molecular mechanisms of mitochondrial redox-coupled proton
pumping enzymes. Institute of Science and Technology Austria.
mla: Kampjut, Domen. Molecular Mechanisms of Mitochondrial Redox-Coupled Proton
Pumping Enzymes. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8340.
short: D. Kampjut, Molecular Mechanisms of Mitochondrial Redox-Coupled Proton Pumping
Enzymes, Institute of Science and Technology Austria, 2020.
date_created: 2020-09-07T18:42:23Z
date_published: 2020-09-09T00:00:00Z
date_updated: 2023-09-07T13:26:17Z
day: '09'
ddc:
- '572'
degree_awarded: PhD
department:
- _id: LeSa
doi: 10.15479/AT:ISTA:8340
ec_funded: 1
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date_updated: 2021-09-11T22:30:04Z
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language:
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month: '09'
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oa_version: None
page: '242'
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
publication_identifier:
isbn:
- 978-3-99078-008-4
issn:
- 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
related_material:
record:
- id: '6848'
relation: part_of_dissertation
status: public
status: public
supervisor:
- first_name: Leonid A
full_name: Sazanov, Leonid A
id: 338D39FE-F248-11E8-B48F-1D18A9856A87
last_name: Sazanov
orcid: 0000-0002-0977-7989
title: Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes
type: dissertation
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
year: '2020'
...