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Moe, A., Ädelroth, P., Brzezinski, P. & Öjemyr, L. N. (2023). Cryo-EM structure and function of S. pombe complex IV with bound respiratory supercomplex factor. Communications Chemistry, 6(1), Article ID 32.
Open this publication in new window or tab >>Cryo-EM structure and function of S. pombe complex IV with bound respiratory supercomplex factor
2023 (English)In: Communications Chemistry, E-ISSN 2399-3669, Vol. 6, no 1, article id 32Article in journal (Refereed) Published
Abstract [en]

Fission yeast Schizosaccharomyces pombe serves as model organism for studying higher eukaryotes. We combined the use of cryo-EM and spectroscopy to investigate the structure and function of affinity purified respiratory complex IV (CIV) from S. pombe. The reaction sequence of the reduced enzyme with O-2 proceeds over a time scale of mu s-ms, similar to that of the mammalian CIV. The cryo-EM structure of CIV revealed eleven subunits as well as a bound hypoxia-induced gene 1 (Hig1) domain of respiratory supercomplex factor 2 (Rcf2). These results suggest that binding of Rcf2 does not require the presence of a CIII-CIV supercomplex, i.e. Rcf2 is a component of CIV. An AlphaFold-Multimer model suggests that the Hig1 domains of both Rcf1 and Rcf2 bind at the same site of CIV suggesting that their binding is mutually exclusive. Furthermore, the differential functional effect of Rcf1 or Rcf2 is presumably caused by interactions of CIV with their different non-Hig1 domain parts. Fission yeast Schizosaccharomyces pombe shares many characteristics with higher eukaryotes. Here, the authors investigate the structure and function of respiratory complex IV from S. pombe, reveal the subunit arrangements and the reaction sequence of O-2 reduction.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-215933 (URN)10.1038/s42004-023-00827-3 (DOI)000935924300002 ()36797353 (PubMedID)2-s2.0-85148369497 (Scopus ID)
Available from: 2023-03-29 Created: 2023-03-29 Last updated: 2023-03-29Bibliographically approved
Liang, Y., Plourde, A., Bueler, S. A., Liu, J., Brzezinski, P., Vahidi, S. & Rubinstein, J. L. (2023). Structure of mycobacterial respiratory complex I. Proceedings of the National Academy of Sciences of the United States of America, 120(13), Article ID e2214949120.
Open this publication in new window or tab >>Structure of mycobacterial respiratory complex I
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2023 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, no 13, article id e2214949120Article in journal (Refereed) Published
Abstract [en]

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found in low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the “orphan” two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox-sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel, comparable to ubiquinone in other structures and suggesting a conserved quinone binding mechanism. 

Keywords
membrane protein, structure, respiration, complex, mycobacteria
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-220458 (URN)10.1073/pnas.2214949120 (DOI)001001349500006 ()36952383 (PubMedID)2-s2.0-85151043571 (Scopus ID)
Available from: 2023-08-29 Created: 2023-08-29 Last updated: 2023-08-29Bibliographically approved
Di Trani, J. M., Gheorghita, A. A., Turner, M., Brzezinski, P., Ädelroth, P., Vahidi, S., . . . Rubinstein, J. L. (2023). Structure of the bc1–cbb3 respiratory supercomplex from Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences of the United States of America, 120(40), Article ID e2307093120.
Open this publication in new window or tab >>Structure of the bc1cbb3 respiratory supercomplex from Pseudomonas aeruginosa
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2023 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, no 40, article id e2307093120Article in journal (Refereed) Published
Abstract [en]

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc1) and one of the organism’s terminal oxidases, cytochrome cbb3, which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c4 and a single heme cytochrome c5. Together, these proteins allow electron transfer from ubiquinol in cytochrome bc1 to oxygen in cytochrome cbb3. We also present evidence that different isoforms of cytochrome cbb3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc1cbb3 SCs also exist in other bacterial pathogens.

Keywords
cryoEM, electron transport chain, Pseudomonas aeruginosa, respiratory supercomplexes, structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-223001 (URN)10.1073/pnas.2307093120 (DOI)001106489600002 ()37751552 (PubMedID)2-s2.0-85172675336 (Scopus ID)
Available from: 2023-10-26 Created: 2023-10-26 Last updated: 2023-12-19Bibliographically approved
Król, S., Fedotovskaya, O., Högbom, M., Ädelroth, P. & Brzezinski, P. (2022). Electron and proton transfer in the M. smegmatis III2IV2 supercomplex. Biochimica et Biophysica Acta - Bioenergetics, 1863(7), Article ID 148585.
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: 2023-09-05Bibliographically approved
Di Trani, J. M., Liu, Z., Whitesell, L., Brzezinski, P., Cowen, L. E. & Rubinstein, J. L. (2022). Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III. Structure, 30(1), 129-138
Open this publication in new window or tab >>Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III
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2022 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 30, no 1, p. 129-138Article in journal (Refereed) Published
Abstract [en]

Electron transfer between respiratory complexes drives transmembrane proton translocation, which powers ATP synthesis and membrane transport. The homodimeric respiratory complex III (CIII2) oxidizes ubiquinol to ubiquinone, transferring electrons to cytochrome c and translocating protons through a mechanism known as the Q cycle. The Q cycle involves ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We determined structures of Candida albicans CIII2 by cryoelectron microscopy (cryo-EM), revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-201895 (URN)10.1016/j.str.2021.08.006 (DOI)000742485500001 ()34525326 (PubMedID)
Available from: 2022-02-09 Created: 2022-02-09 Last updated: 2022-02-25Bibliographically approved
Di Trani, J. M., Moe, A., Riepl, D., Saura, P., Kaila, V. R. I., Brzezinski, P. & Rubinstein, J. L. (2022). Structural basis of mammalian complex IV inhibition by steroids. Proceedings of the National Academy of Sciences of the United States of America, 119(30), Article ID e2205228119.
Open this publication in new window or tab >>Structural basis of mammalian complex IV inhibition by steroids
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2022 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, no 30, article id e2205228119Article in journal (Refereed) Published
Abstract [en]

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Keywords
electron transport chain, complex IV, cryo-EM, kinetics, molecular simulations
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-209783 (URN)10.1073/pnas.2205228119 (DOI)000839026200016 ()35858451 (PubMedID)
Available from: 2022-09-30 Created: 2022-09-30 Last updated: 2022-09-30Bibliographically approved
Moe, A., Kovalova, T., Król, S., Yanofsky, D. J., Bott, M., Sjöstrand, D., . . . Brzezinski, P. (2022). The respiratory supercomplex from C. glutamicum. Structure, 30(3), 338-349
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: 2023-04-14Bibliographically approved
Moe, A., Di Trani, J., Rubinstein, J. L. & Brzezinski, P. (2021). Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex. Proceedings of the National Academy of Sciences of the United States of America, 118(11), Article ID e2021157118.
Open this publication in new window or tab >>Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex
2021 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 11, article id e2021157118Article in journal (Refereed) Published
Abstract [en]

Energy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport, which maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred from respiratory complexes III to IV (CIII and CIV) by water-soluble cytochrome (cyt.) c. In Saccharomyces cerevisiae and some other organisms, these complexes assemble into larger CIII2CIV1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex cyt. c-mediated QH(2):O-2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. c show the positively charged cyt. c bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. c must diffuse in three dimensions, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by two-dimensional (2D) diffusion of cyt. c. This mechanism enables the CIII2CIV1/2 supercomplex to increase QH(2):O-2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.

Keywords
electron transfer, cytochrome c oxidase, cytochrome bc(1), bioenergetics, mitochondria
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-193203 (URN)10.1073/pnas.2021157118 (DOI)000629635100059 ()33836592 (PubMedID)
Available from: 2021-05-19 Created: 2021-05-19 Last updated: 2023-04-14Bibliographically approved
Fedotovskaya, O., Albertsson, I., Nordlund, G., Hong, S., Gennis, R. B., Brzezinski, P. & Ädelroth, P. (2021). Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides. Biochimica et Biophysica Acta - Bioenergetics, 1862(8), Article ID 148433.
Open this publication in new window or tab >>Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides
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2021 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1862, no 8, article id 148433Article in journal (Refereed) Published
Abstract [en]

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc1 and aa3-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O2 reduction activity of the cyt. bc1-aa3 supercomplex. The potential role of the membrane-anchored cyt. cy as a component in supercomplexes was also investigated.

Keywords
Bioenergetics, Electron transfer, Cytochrome bc(1) complex, aa(3)-type cytochrome c oxidase, Respiratory supercomplex, flow-flash
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-195701 (URN)10.1016/j.bbabio.2021.148433 (DOI)000656848200004 ()33932366 (PubMedID)
Available from: 2021-08-25 Created: 2021-08-25 Last updated: 2023-09-05Bibliographically approved
Zhou, S., Pettersson, P., Björck, M. L., Dawitz, H., Brzezinski, P., Mäler, L. & Ädelroth, P. (2021). NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP. BMC Biology, 19(1), Article ID 98.
Open this publication in new window or tab >>NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP
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2021 (English)In: BMC Biology, E-ISSN 1741-7007, Vol. 19, no 1, article id 98Article in journal (Refereed) Published
Abstract [en]

Background: Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker's yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex.

Results: Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility.

Conclusions: Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Keywords
ATP, Membrane protein, NMR, Solution structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-194982 (URN)10.1186/s12915-021-01036-x (DOI)000655036700002 ()33971868 (PubMedID)
Available from: 2021-07-28 Created: 2021-07-28 Last updated: 2024-01-17Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-3860-4988

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