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Gonska, Nathalie
Publications (7 of 7) Show all publications
Gonska, N., Young, D., Yuki, R., Okamoto, T., Hisano, T., Antonyuk, S., . . . Ädelroth, P. (2018). Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme. Scientific Reports, 8, Article ID 3637.
Open this publication in new window or tab >>Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme
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2018 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 8, article id 3637Article in journal (Refereed) Published
Abstract [en]

Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O-2-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic. Here, we focus on another type of NOR, qNOR (quinol-dependent). Recombinant qNOR from Neisseria meningitidis, a human pathogen, purified from Escherichia coli, showed high catalytic activity and spectroscopic properties largely similar to cNORs. However, in contrast to cNOR, liposome-reconstituted qNOR showed respiratory control ratios above two, indicating that NO reduction by qNOR was electrogenic. Further, we determined a 4.5 angstrom crystal structure of the N. meningitidis qNOR, allowing exploration of a potential proton transfer pathway from the cytoplasm by mutagenesis. Most mutations had little effect on the activity, however the E-498 variants were largely inactive, while the corresponding substitution in cNOR was previously shown not to induce significant effects. We thus suggest that, contrary to cNOR, the N. meningitidis qNOR uses cytoplasmic protons for NO reduction. Our results allow possible routes for protons to be discussed.

Keywords
Oxidoreductases, X-ray crystallography
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-154847 (URN)10.1038/s41598-018-21804-0 (DOI)000426045700058 ()29483528 (PubMedID)
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2022-09-15Bibliographically approved
von Ballmoos, C., Smirnova, I., Poiana, F., Gonska, N., Chang, H.-Y., Gennis, R. B., . . . Ädelroth, P. (2017). Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus. Israel Journal of Chemistry, 57(5), 424-436
Open this publication in new window or tab >>Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus
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2017 (English)In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, p. 424-436Article in journal (Refereed) Published
Abstract [en]

The ba(3) cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K-B pathway. It was previously shown that the ba(3) oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K-B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located approximate to 23 angstrom below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (approximate to 26 angstrom below the active site, approximate to 16 angstrom from Tyr-248) in subunit II (E15(II)Q). Even though the overall catalytic turnover in these two variants is similar and very low (<1% of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15(II) is the only essentially crucial residue of the K-B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site.

Keywords
heme-copper oxidases, cytochrome c oxidase, proton transfer, electron transfer, membrane protein, respiration, redox reaction, metalloprotein, cytochrome aa(3), cytochrome cbb(3)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-144708 (URN)10.1002/ijch.201600136 (DOI)000401329000009 ()
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2022-02-28Bibliographically approved
Gonska, N. (2017). Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases. (Doctoral dissertation). Stockholm: Department of Biochemistry and Biophysics, Stockholm University
Open this publication in new window or tab >>Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic.

Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction.

The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. p. 66
Keywords
heme-copper oxidase, cytochrome c oxidase, membrane protein, respiration, electron transfer, proton transfer, redox reaction, metalloprotein, non-heme iron, cytochrome ba3, flow-flash, carbon monoxide, liposome, respiratory control ratio
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147267 (URN)978-91-7649-986-3 (ISBN)978-91-7649-987-0 (ISBN)
Public defence
2017-11-09, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

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

Available from: 2017-10-17 Created: 2017-09-20 Last updated: 2022-02-28Bibliographically approved
Poiana, F., von Ballmoos, C., Gonska, N., Blomberg, M. R. A., Ädelroth, P. & Brzezinski, P. (2017). Splitting of the O-O bond at the heme-copper catalytic site of respiratory oxidases. Science Advances, 3(6), Article ID e1700279.
Open this publication in new window or tab >>Splitting of the O-O bond at the heme-copper catalytic site of respiratory oxidases
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2017 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 3, no 6, article id e1700279Article in journal (Refereed) Published
Abstract [en]

Heme-copper oxidases catalyze the four-electron reduction of O-2 to H2O at a catalytic site that is composed of a heme group, a copper ion (Cu-B), and a tyrosine residue. Results from earlier experimental studies have shown that the O-O bond is cleaved simultaneously with electron transfer from a low-spin heme (heme a/b), forming a ferryl state (P-R; Fe4+= O2-, Cu-B(2+)-OH-). We show that with the Thermus thermophilus ba(3) oxidase, at low temperature (10 degrees C, pH 7), electron transfer from the low-spin heme b to the catalytic site is faster by a factor of similar to 10 (tau congruent to 11 mu s) than the formation of the P-R ferryl (t. 110 ms), which indicates that O-2 is reduced before the splitting of the O-O bond. Application of density functional theory indicates that the electron acceptor at the catalytic site is a high-energy peroxy state [Fe3+-O--O-(H+)], which is formed before the P-R ferryl. The rates of heme b oxidation and P-R ferryl formation were more similar at pH 10, indicating that the formation of the high-energy peroxy state involves proton transfer within the catalytic site, consistent with theory. The combined experimental and theoretical data suggest a general mechanism for O-2 reduction by heme-copper oxidases.

National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147192 (URN)10.1126/sciadv.1700279 (DOI)000406370700061 ()
Available from: 2017-09-20 Created: 2017-09-20 Last updated: 2022-03-23Bibliographically approved
von Ballmoos, C., Gonska, N., Lachmann, P., Gennis, R. B., Ädelroth, P. & Brzezinski, P. (2015). Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site. Proceedings of the National Academy of Sciences of the United States of America, 112(11), 3397-3402
Open this publication in new window or tab >>Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site
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2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 11, p. 3397-3402Article in journal (Refereed) Published
Abstract [en]

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O-2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba(3) oxidase where a putative pump site was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O-2 reduction. The results from our studies show that proton uptake to the pump site (time constant similar to 65 mu s in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of similar to 1.2 ms was slowed to similar to 8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba(3) cytochrome c oxidase.

Keywords
cytochrome c oxidase, membrane protein, respiration, cytochrome aa(3), electron transfer
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116611 (URN)10.1073/pnas.1422434112 (DOI)000351060000072 ()25733886 (PubMedID)
Note

AuthorCount:6;

Available from: 2015-04-30 Created: 2015-04-22 Last updated: 2022-02-23Bibliographically approved
Salomonsson, L., Reimann, J., Tosha, T., Krause, N., Gonska, N., Shiro, Y. & Ädelroth, P. (2012). Proton transfer in the quinol dependent nitric oxide reductase from geobacillus stearothermophilus during reduction of oxygen. Biochimica et Biophysica Acta - Bioenergetics, 1817(10), 1914-1920
Open this publication in new window or tab >>Proton transfer in the quinol dependent nitric oxide reductase from geobacillus stearothermophilus during reduction of oxygen
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2012 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 10, p. 1914-1920Article in journal (Refereed) Published
Abstract [en]

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this gNOR and oxygen. Our results show that, like some cNORs, the C. stearothermophilus gNOR is capable of 02 reduction with a turnover of similar to 3 electrons s(-1) at 40 degrees C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) gNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between gNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in gNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Keywords
Heme-copper oxidase, Proton transfer pathway, Non-heme iron, Flow-flash, Carbon monoxide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-81832 (URN)10.1016/j.bbabio.2012.04.007 (DOI)000307918200028 ()
Note

AuthorCount:7;

Available from: 2012-11-07 Created: 2012-11-01 Last updated: 2022-02-24Bibliographically approved
Gonska, N., Young, D. R., Yuki, R., Okamoto, T., Antonyuk, S., Hasnain, S. S., . . . Ädelroth, P. Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme.
Open this publication in new window or tab >>Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacterial nitric oxide reductases (NORs) catalyse the reduction of two NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the host. Although NORs are members of the heme-copper oxidase superfamily, and thus relatives of proton-pumping O2-reducing enzymes, the best studied NORs, cNORs (cytochrome c dependent), were found to be non-electrogenic.

Here, we focus on another type of NOR, qNOR (quinol-dependent). qNOR from Neisseria meningitidis, a human pathogen, was expressed in Escherichia coli and purified as a stable and highly active NO reductase. Spectroscopic and metal analysis of the purified qNOR showed properties largely similar to those in cNORs. Furthermore, the liposome-reconstituted qNOR showed respiratory control ratios consistently above 2, indicative of an electrogenic reaction. We also exchanged residues in a putative proton pathway leading from the cytoplasm to the active site, but there were no significant effects on either turnover rates or electrogenicity. However, the exchange of a glutamate close to the active site (E-498) yielded drastic effects on turnover. We thus suggest that the N. meningitidis qNOR uses cytoplasmic protons, but that the pathway is rather wide and redundant, narrowing around the glutamate-498.

Keywords
heme-copper oxidase, liposome, proton transfer, respiratory control ratio
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147210 (URN)
Available from: 2017-09-20 Created: 2017-09-20 Last updated: 2022-02-28Bibliographically approved
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