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The respiratory supercomplex from C. glutamicum
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-3328-763x
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-0144-2463
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 92022 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 30, no 3, p. 338-349Article in journal (Refereed) Published
Abstract [en]

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

Place, publisher, year, edition, pages
2022. Vol. 30, no 3, p. 338-349
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-203453DOI: 10.1016/j.str.2021.11.008ISI: 000766494300005PubMedID: 34910901OAI: oai:DiVA.org:su-203453DiVA, id: diva2:1650555
Available from: 2022-04-07 Created: 2022-04-07 Last updated: 2024-04-04Bibliographically approved
In thesis
1. Role of respiratory supercomplexes: Electronic connection between complexes III and IV
Open this publication in new window or tab >>Role of respiratory supercomplexes: Electronic connection between complexes III and IV
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the final step of cellular respiration, electrons are transferred through the respiratory chain to reduce molecular oxygen to water. The energy released in this chain is used to maintain a proton electrochemical gradient across the cell membrane, which is used, for example, by the ATP synthase to produce ATP. The enzyme complexes of the respiratory chain are known to organize in supramolecular assemblies, so-called respiratory supercomplexes.

In this work we investigated the functional significance of respiratory supercomplexes consisting of complexes III and IV in mitochondria. By combining structural and kinetic studies we showed that at the commonly assumed "physiological" ionic strength of 150 mM monovalent salt, the water-soluble cyt. c associates with the negatively charged surface of III2-IV1-2 supercomplexes in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. The data showed that one cyt. c diffuses in 2D, between complexes III and IV, indicating a kinetic advantage of forming supercomplexes. These studies also showed different relative orientation of the individual complexes in the supercomplexes from the two yeast species, indicating that 2D diffusion is a general mechanism, not limited to a specific relative orientation of complexes III and IV. 

More recent data in the literature indicate that a more realistic mimic of intracellular conditions is a monovalent salt concentration of 20 mM. We showed that under these conditions two cyt. c molecules bind simultaneously to the supercomplex. This result further supports a kinetic advantage of forming supercomplexes.

We also determined the cryo-EM structure of the obligate III2-IV2 supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure revealed an electronic connection between complexes III and IV by a di-heme cyt. cc subunit. The structure also showed that complexes III and IV are structurally intertwined and strongly connected with unique features conserved in the phylum actinobacteria. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2023. p. 72
Keywords
electron transfer, cytochrome c oxidase, cytochrome bc1, respiratory supercomplex, bioenergetics, membrane protein
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-216393 (URN)978-91-8014-294-6 (ISBN)978-91-8014-295-3 (ISBN)
Public defence
2023-06-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2023-05-10 Created: 2023-04-14 Last updated: 2023-04-27Bibliographically approved
2. Respiration in Actinobacteria: Structure, function and inhibition of the III2IV2 supercomplex
Open this publication in new window or tab >>Respiration in Actinobacteria: Structure, function and inhibition of the III2IV2 supercomplex
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The final step of aerobic respiration, oxidative phosphorylation, combines the activities of the electron transport chain and of ATP synthase. The electron transport chain is composed of membrane-bound energy transducers, which are organized in supramolecular assemblies known as respiratory supercomplexes. 

In this work we determined the cryo-EM structure of the obligate III2IV2 supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure shows that the individual complexes are intertwined and that the electron transfer between them occurs via a di-heme cc subunit instead of via soluble cytochrome c. The structure reveals additional features that distinguish the supercomplex from its canonical counterpart. These are a cytoplasmic QcrB loop that occludes the proton-entry point of the complex IV D-pathway, and an FeS cluster in a fixed position. These characteristics are conserved among actinobacteria. 

With the goal to elucidate the structure-function relationship for complexes III and IV in actinobacteria, we also investigated electron and proton transfer kinetics of an obligate respiratory supercomplex from Mycobacterium smegmatis, which is a model organism for Mycobacterium tuberculosis. The results show that the sequence of reactions involved in electron transfer in complex IV is similar to that observed in other A1-type oxidases, but the F to O transition of the catalytic cycle is slower than that reported for canonical complex IV. We also observed that reaction steps previously shown to display pH dependence in canonical complex IV were pH independent in Mycobacterium smegmatis. In addition, proton uptake kinetics through the D-pathway of complex IV were altered with no proton uptake during the F to O step. These findings can be attributed to the presence of the QcrB loop and point towards a possible unique regulatory mechanism for mycobacterial supercomplexes.

As the mycobacterial supercomplex is a promising drug target for tuberculosis treatment, we studied its interaction with the drug candidate Telacebec and the metabolite of an already approved drug, lansoprazole sulfide. We determined the cryo-EM structures of the III2IV2 supercomplex with Telacebec and with lansoprazole sulfide bound in the QP site of the QcrB subunit of complex III. In both structures the inhibitor replaces the natural substrate menaquinol in the inner position of the QP binding pocket and makes multiple interactions with the QcrA and QcrB subunits of complex III. Multiple turnover assays showed that this binding mode inhibits the supercomplex of Mycobacterium smegmatis. Results from our in silico studies show that lansoprazole sulfide is likely to bind to the supercomplex of Mycobacterium tuberculosis in a similar way as was observed for Mycobacterium smegmatis.

 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2024. p. 78
Keywords
bioenergetics, structural biology, electron transport chain, respiratory supercomplex, electron transfer, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-227926 (URN)978-91-8014-747-7 (ISBN)978-91-8014-748-4 (ISBN)
Public defence
2024-05-17, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius Väg 16 B, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2024-04-24 Created: 2024-04-04 Last updated: 2024-04-12Bibliographically approved

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Moe, AgnesKról, SylwiaSjöstrand, DanHögbom, MartinBrzezinski, Peter

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