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Respiration in Actinobacteria: Structure, function and inhibition of the III2IV2 supercomplex
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
bioenergetics, structural biology, electron transport chain, respiratory supercomplex, electron transfer, proton transfer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-227926ISBN: 978-91-8014-747-7 (print)ISBN: 978-91-8014-748-4 (electronic)OAI: oai:DiVA.org:su-227926DiVA, id: diva2:1848598
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
List of papers
1. The respiratory supercomplex from C. glutamicum
Open this publication in new window or tab >>The respiratory supercomplex from C. glutamicum
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2022 (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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-203453 (URN)10.1016/j.str.2021.11.008 (DOI)000766494300005 ()34910901 (PubMedID)
Available from: 2022-04-07 Created: 2022-04-07 Last updated: 2024-04-04Bibliographically approved
2. Electron and proton transfer in the M. smegmatis III2IV2 supercomplex
Open this publication in new window or tab >>Electron and proton transfer in the M. smegmatis III2IV2 supercomplex
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2022 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1863, no 7, article id 148585Article in journal (Refereed) Published
Abstract [en]

The M. smegmatis respiratory III2IV2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e/s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, CuA, displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII.

Keywords
Cytochrome c oxidase, Cytochrome bc 1, Actinobacteria, Membrane protein, Bioenergetics and oxidative phosphorylation, Respiratory chain
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-207909 (URN)10.1016/j.bbabio.2022.148585 (DOI)000829443100003 ()35753381 (PubMedID)2-s2.0-85132963263 (Scopus ID)
Available from: 2022-08-23 Created: 2022-08-23 Last updated: 2024-04-04Bibliographically approved
3. Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)
Open this publication in new window or tab >>Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)
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2021 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e71959Article in journal (Refereed) Published
Abstract [en]

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.

Keywords
Mycobacterium smegmatis, telacebec (Q203), cryoEM, respiration, tuberculosis, structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-198837 (URN)10.7554/eLife.71959 (DOI)000709344600001 ()34590581 (PubMedID)2-s2.0-85116880110 (Scopus ID)
Available from: 2021-11-25 Created: 2021-11-25 Last updated: 2024-04-04Bibliographically approved
4. Inhibition mechanism of potential anti-tuberculosis compound lansoprazole sulfide
Open this publication in new window or tab >>Inhibition mechanism of potential anti-tuberculosis compound lansoprazole sulfide
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-227927 (URN)
Available from: 2024-04-03 Created: 2024-04-03 Last updated: 2024-04-04

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Król, Sylwia

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