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Electron and proton transfer in the M. smegmatis III2IV2 supercomplex
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-5574-9383
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-0853-6785
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Number of Authors: 52022 (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.

Place, publisher, year, edition, pages
2022. Vol. 1863, no 7, article id 148585
Keywords [en]
Cytochrome c oxidase, Cytochrome bc 1, Actinobacteria, Membrane protein, Bioenergetics and oxidative phosphorylation, Respiratory chain
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-207909DOI: 10.1016/j.bbabio.2022.148585ISI: 000829443100003PubMedID: 35753381Scopus ID: 2-s2.0-85132963263OAI: oai:DiVA.org:su-207909DiVA, id: diva2:1689512
Available from: 2022-08-23 Created: 2022-08-23 Last updated: 2024-04-04Bibliographically approved
In thesis
1. Prokaryotic respiratory supercomplexes: Studies of interactions between complexes III and IV
Open this publication in new window or tab >>Prokaryotic respiratory supercomplexes: Studies of interactions between complexes III and IV
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Respiratory processes for cellular energy conversion are carried out by the membrane-associated enzymes of the electron transfer chain (ETC). In recent years there has been emerging data showing that the members of the ETC organize into higher-level assemblies called supercomplexes (SCs) whose functional relevance is not yet fully understood. SCs composed of complexes III (cytochrome (cyt.) bc1 complex) and IV (cyt. c oxidase) are the most common. The small electron-carrier protein cyt. c shuttles electrons between complexes III and IV. In mitochondria-like ETCs cyt. c is present only in a soluble form, while in some bacteria it has additional membrane-anchored analogs or is fused to complex III forming the cyt. cc subunit, as in actinobacteria.

We determined the structure of the obligate III/IV SC from actinobacterium Mycobacterium (M.) smegmatis with cryo-electron microscopy. The structure showed that the distances between the co-factors of the SC are short enough for electron transfer with the catalytically relevant rates. Complexes III and IV within the SC were intertwined. In particular, the entrance to the D-proton pathway of complex IV was shielded by a loop of the QcrB subunit of complex III, possibly influencing proton uptake characteristics. Furthermore, superoxide dismutase was shown to be an integral part of the M. smegmatis SC, which might have a functional role in the energy conservation by the SC.

With the goal to unravel the structure-function relationships between complexes III and IV in the actinobacterial SCs, we investigated the charge transfer kinetics in SCs on a single-turnover time scale. Using time-resolved spectroscopic techniques we have determined the rates of electron and proton transfer upon oxidation of reduced SCs of M. smegmatis and another actinobacterium Corynebacterium glutamicum. Electron transfer from cyt. cc in complex III to the primary redox center CuA in complex IV was not rate-limiting for the SC turnover. In contrast to the canonical complex IV, certain reaction steps in SC were not pH-dependent and proton uptake kinetics through the D-pathway of complex IV was altered showing much slower kinetics. This suggests that the QcrB loop of complex III, which blocks the entrance to the D-pathway, modulates the kinetics of proton uptake in complex IV. 

In another study, we showed the existence of a non-obligate supercomplex in the alfa-proteobacterium Rhodobacter (R.) sphaeroides. This SC was purified and characterized biochemically. We concluded that complexes III and IV interact via the membrane-anchored version of cyt. c (MA-cyt. c), which is expressed in the bacterium in addition to the soluble variant. MA-cyt. c most likely plays a central role in forming the SC in R. sphaeroides by functionally connecting complexes III and IV.

In addition to being an electron shuttle, in eukaryotes cyt. c participates in apoptosis. We investigated the consequences of anchoring the cyt. c to the membrane, similar to MA-cyt. c in R. sphaeroides, in a single-cell eukaryote Saccharomyces cerevisiae, thereby not allowing the release of cyt. c from the intermembrane space of mitochondria during the induced apoptosis.

The work presented in this thesis increases our understanding of the general function-structure relationships of respiratory SCs and might have applications in potential drug development.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University, 2023. p. 76
Keywords
bioenergetics, membrane protein, bacterial respiration, electron transport chain, supercomplex, cytochrome c oxidase, membrane-anchored cytochrome c, cytochrome bc1 complex, electron transfer, proton transfer, time-resolved spectroscopy, apoptosis
National Category
Biochemistry and Molecular Biology Structural Biology Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-220612 (URN)978-91-8014-484-1 (ISBN)978-91-8014-485-8 (ISBN)
Public defence
2023-10-20, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2023-09-27 Created: 2023-09-05 Last updated: 2023-09-20Bibliographically 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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Król, SylwiaFedotovskaya, OlgaHögbom, MartinÄdelroth, PiaBrzezinski, Peter

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