Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes

D. Kampjut, Molecular Mechanisms of Mitochondrial Redox-Coupled Proton Pumping Enzymes, IST Austria, 2020.

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Thesis | Published | English
Department
Series Title
IST Austria Thesis
Abstract
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.
Publishing Year
Date Published
2020-09-09
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.
Acknowledged SSUs
Page
242
eISSN
IST-REx-ID

Cite this

Kampjut D. Molecular Mechanisms of Mitochondrial Redox-Coupled Proton Pumping Enzymes. IST Austria; 2020. doi:10.15479/AT:ISTA:8340
Kampjut, D. (2020). Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes. IST Austria. https://doi.org/10.15479/AT:ISTA:8340
Kampjut, Domen. Molecular Mechanisms of Mitochondrial Redox-Coupled Proton Pumping Enzymes. IST Austria, 2020. https://doi.org/10.15479/AT:ISTA:8340.
D. Kampjut, Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes. IST Austria, 2020.
Kampjut D. 2020. Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes, IST Austria, 242p.
Kampjut, Domen. Molecular Mechanisms of Mitochondrial Redox-Coupled Proton Pumping Enzymes. IST Austria, 2020, doi:10.15479/AT:ISTA:8340.
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2020-09-14
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2021-09-10
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2020-09-08
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