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Blomberg, M. R. A. & Ädelroth, P. (2024). Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases. Journal of Inorganic Biochemistry, 255, Article ID 112534.
Open this publication in new window or tab >>Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases
2024 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 255, article id 112534Article in journal (Refereed) Published
Abstract [en]

The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O2 molecule has bound to the diferrous diiron active site and before the O–O bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both N–N bond formation and N–O bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual O–O bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O2 reduction reaction in the family of heme‑copper oxidases.

Keywords
Oxygen reduction, Flavodiiron proteins, Density functional calculations, Energy profiles, Reaction mechanisms
National Category
Biochemistry and Molecular Biology Theoretical Chemistry
Identifiers
urn:nbn:se:su:diva-229312 (URN)10.1016/j.jinorgbio.2024.112534 (DOI)001218439500001 ()38552360 (PubMedID)2-s2.0-85189009335 (Scopus ID)
Available from: 2024-05-21 Created: 2024-05-21 Last updated: 2024-05-21Bibliographically approved
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
Kahle, M., Appelgren, S., Elofsson, A., Carroni, M. & Ädelroth, P. (2023). Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins. BMC Biology, 21, Article ID 47.
Open this publication in new window or tab >>Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins
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2023 (English)In: BMC Biology, E-ISSN 1741-7007, Vol. 21, article id 47Article in journal (Refereed) Published
Abstract [en]

Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.

Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.

Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Keywords
Iron, Nitric oxide reductase, cNOR, VWA, AAA+, ATPase, FeB, Protein remodeling, nor accessory genes, MoxR, Cryo-EM, AlphaFold
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-215801 (URN)10.1186/s12915-023-01546-w (DOI)000940793200001 ()36855050 (PubMedID)2-s2.0-85149153731 (Scopus ID)
Available from: 2023-03-29 Created: 2023-03-29 Last updated: 2024-01-17Bibliographically approved
Blomberg, M. R. A. & Ädelroth, P. (2023). Reduction of Nitric Oxide to Nitrous Oxide in Flavodiiron Proteins: Catalytic Mechanism and Plausible Intermediates. ACS Catalysis, 13(3), 2025-2038
Open this publication in new window or tab >>Reduction of Nitric Oxide to Nitrous Oxide in Flavodiiron Proteins: Catalytic Mechanism and Plausible Intermediates
2023 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 13, no 3, p. 2025-2038Article in journal (Refereed) Published
Abstract [en]

The flavin dependent nonheme diiron proteins comprise a family of enzymes, which can act as scavengers for both molecular oxygen and nitric oxide. The reduction of nitric oxide to nitrous oxide and water in flavodiiron proteins (FDPs) has been studied both experimentally and computationally, but the reaction mechanism is far from well understood. From experiments, it is known that two NO molecules can bind to the reduced active site, forming an observable diferrous dinitrosyl complex. A main question has been whether nitrous oxide can be formed directly from the diferrous dinitrosyl complex or if further reduction and/or protonation is needed to make this step feasible. Experiments have shown that nitrous oxide can be formed in a deflavinated form of the enzyme, indicating that further reduction is not needed. In the present study, hybrid density functional theory calculations are performed on a cluster model of the Thermotoga maritima FDP active site. We show that nitric oxide can be reduced to nitrous oxide and water using a direct coupling mechanism, i.e., without further additions to the reduced active site. The diferrous dinitrosyl complex can form an unstable N-N bridging hyponitrite intermediate, which can rotate into an N-O bond bridging hyponitrite with a low barrier. From this intermediate, the N-O bond cleavage leading to release of nitrous oxide is energetically feasible. An energy profile for the entire catalytic cycle of such a direct coupling mechanism is presented, and it is shown that the suggested mechanism agrees with data on FDP variants. Finally, an energy profile for the entire process starting with the fully reduced enzyme turning over four NO equivalents is constructed. This energy profile suggests explanations to experimentally observed states, such as the dihydroxyl form of the fully oxidized diferric state, and the difference with respect to returning to the original oxidized state after NO reduction between the flavinated and the deflavinated form of the enzyme.

Keywords
NO reduction, flavodiiron proteins, density functional calculations, energy profiles, reaction mechanisms
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-215535 (URN)10.1021/acscatal.2c04932 (DOI)000922250000001 ()
Available from: 2023-03-16 Created: 2023-03-16 Last updated: 2023-03-16Bibliographically 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: 2024-04-04Bibliographically approved
Petrik, I. D., Davydov, R., Kahle, M., Sandoval, B., Dwaraknath, S., Ädelroth, P., . . . Lu, Y. (2021). An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network. Biochemistry, 60(4), 346-355
Open this publication in new window or tab >>An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network
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2021 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 60, no 4, p. 346-355Article in journal (Refereed) Published
Abstract [en]

Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bd oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O2 binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-191796 (URN)10.1021/acs.biochem.0c00852 (DOI)000618081900010 ()33464878 (PubMedID)
Available from: 2021-04-27 Created: 2021-04-27 Last updated: 2022-02-25Bibliographically 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
Zhou, S., Pettersson, P., Huang, J., Brzezinski, P., Pomès, R., Mäler, L. & Ädelroth, P. (2021). NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2. Structure, 29(3), 275-283
Open this publication in new window or tab >>NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2
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2021 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 29, no 3, p. 275-283Article in journal (Refereed) Published
Abstract [en]

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc(1) and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble alpha helices and five putative transmembrane (TM) alpha helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation.

Keywords
charge zipper, protein-lipid interactions, molecular dynamics, mitochondria, Hig protein, membrane protein, solution structure
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-193384 (URN)10.1016/j.str.2020.08.008 (DOI)000629154700009 ()32905793 (PubMedID)
Available from: 2021-05-25 Created: 2021-05-25 Last updated: 2022-02-25Bibliographically approved
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