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Meszaros, Livia S.
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Land, H., Ceccaldi, P., Meszaros, L. S., Lorenzi, M., Redman, H. J., Senger, M., . . . Berggren, G. (2019). Discovery of novel [FeFe]-hydrogenases for biocatalytic H-2-production. Chemical Science, 10(43), 9941-9948
Öppna denna publikation i ny flik eller fönster >>Discovery of novel [FeFe]-hydrogenases for biocatalytic H-2-production
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2019 (Engelska)Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 10, nr 43, s. 9941-9948Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

A new screening method for [FeFe]-hydrogenases is described, circumventing the need for specialized expression conditions as well as protein purification for initial characterization. [FeFe]-hydrogenases catalyze the formation and oxidation of molecular hydrogen at rates exceeding 10(3) s(-1), making them highly promising for biotechnological applications. However, the discovery of novel [FeFe]-hydrogenases is slow due to their oxygen sensitivity and dependency on a structurally unique cofactor, complicating protein expression and purification. Consequently, only a very limited number have been characterized, hampering their implementation. With the purpose of increasing the throughput of [FeFe]-hydrogenase discovery, we have developed a screening method that allows for rapid identification of novel [FeFe]-hydrogenases as well as their characterization with regards to activity (activity assays and protein film electrochemistry) and spectroscopic properties (electron paramagnetic resonance and Fourier transform infrared spectroscopy). The method is based on in vivo artificial maturation of [FeFe]-hydrogenases in Escherichia coli and all procedures are performed on either whole cells or non-purified cell lysates, thereby circumventing extensive protein purification. The screening was applied on eight putative [FeFe]-hydrogenases originating from different structural sub-classes and resulted in the discovery of two new active [FeFe]-hydrogenases. The [FeFe]-hydrogenase from Solobacterium moorei shows high H-2-gas production activity, while the enzyme from Thermoanaerobacter mathranii represents a hitherto uncharacterized [FeFe]-hydrogenase sub-class. This latter enzyme is a putative sensory hydrogenase and our in vivo spectroscopy study reveals distinct differences compared to the well established H-2 producing HydA1 hydrogenase from Chlamydomonas reinhardtii.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-397949 (URN)10.1039/c9sc03717a (DOI)000496188700033 ()
Forskningsfinansiär
EU, Europeiska forskningsrådet, 714102Vetenskapsrådet, 621-2014-5670
Tillgänglig från: 2020-01-02 Skapad: 2020-01-02 Senast uppdaterad: 2020-01-02Bibliografiskt granskad
Meszaros, L. S., Nemeth, B., Esmieu, C., Ceccaldi, P. & Berggren, G. (2018). InVivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases. Angewandte Chemie International Edition, 57(10), 2596-2599
Öppna denna publikation i ny flik eller fönster >>InVivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases
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2018 (Engelska)Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 57, nr 10, s. 2596-2599Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

EPR spectroscopy reveals the formation of two different semi-synthetic hydrogenases invivo. [FeFe] hydrogenases are metalloenzymes that catalyze the interconversion of molecular hydrogen and protons. The reaction is catalyzed by the H-cluster, consisting of a canonical iron-sulfur cluster and an organometallic [2Fe] subsite. It was recently shown that the enzyme can be reconstituted with synthetic cofactors mimicking the composition of the [2Fe] subsite, resulting in semi-synthetic hydrogenases. Herein, we employ EPR spectroscopy to monitor the formation of two such semi-synthetic enzymes in whole cells. The study provides the first spectroscopic characterization of semi-synthetic hydrogenases invivo, and the observation of two different oxidized states of the H-cluster under intracellular conditions. Moreover, these findings underscore how synthetic chemistry can be a powerful tool for manipulation and examination of the hydrogenase enzyme under invivo conditions.

Ort, förlag, år, upplaga, sidor
WILEY-V C H VERLAG GMBH, 2018
Nyckelord
[FeFe] hydrogenase, artificial enzymes, EPR spectroscopy, metalloenzymes
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-348975 (URN)10.1002/anie.201710740 (DOI)000426252400010 ()29334424 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 21-2014-5670Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 213-2014-880EU, Europeiska forskningsrådet, 714102
Tillgänglig från: 2018-05-03 Skapad: 2018-05-03 Senast uppdaterad: 2019-09-18Bibliografiskt granskad
Magnuson, A., Raleiras, P., Meszaros, L. S., Khanna, N., Miranda, H., Ho, F. M., . . . Styring, S. (2018). Sustainable photobiological hydrogen production via protein engineering of cyanobacterial hydrogenases. Paper presented at 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, MAR 18-22, 2018, New Orleans, USA.. Abstract of Papers of the American Chemical Society, 255
Öppna denna publikation i ny flik eller fönster >>Sustainable photobiological hydrogen production via protein engineering of cyanobacterial hydrogenases
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2018 (Engelska)Ingår i: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Artikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Ort, förlag, år, upplaga, sidor
Washington, D.C.: American Chemical Society (ACS), 2018
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-368928 (URN)000435539900440 ()
Konferens
255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, MAR 18-22, 2018, New Orleans, USA.
Anmärkning

Meeting Abstract: 443

Tillgänglig från: 2018-12-11 Skapad: 2018-12-11 Senast uppdaterad: 2018-12-11Bibliografiskt granskad
Khanna, N., Esmieu, C., Meszaros, L. S., Lindblad, P. & Berggren, G. (2017). In vivo activation of an [FeFe] hydrogenase using synthetic cofactors. Energy & Environmental Science, 10(7), 1563-1567
Öppna denna publikation i ny flik eller fönster >>In vivo activation of an [FeFe] hydrogenase using synthetic cofactors
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2017 (Engelska)Ingår i: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 10, nr 7, s. 1563-1567Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

[FeFe] hydrogenases catalyze the reduction of protons, and oxidation of hydrogen gas, with remarkable efficiency. The reaction occurs at the H-cluster, which contains an organometallic [2Fe] subsite. The unique nature of the [2Fe] subsite makes it dependent on a specific set of maturation enzymes for its biosynthesis and incorporation into the apo-enzyme. Herein we report on how this can be circumvented, and the apo-enzyme activated in vivo by synthetic active site analogues taken up by the living cell.

Nationell ämneskategori
Kemi
Identifikatorer
urn:nbn:se:uu:diva-330015 (URN)10.1039/c7ee00135e (DOI)000405279900003 ()
Forskningsfinansiär
Vetenskapsrådet, 621-2014-5670Forskningsrådet Formas, 213-2014-880Energimyndigheten, 11674-5Knut och Alice Wallenbergs Stiftelse, 2011.0067Wenner-Gren Stiftelserna
Anmärkning

Correction in: ENERGY & ENVIRONMENTAL SCIENCE, Volume: 11, Issue: 11, Pages: 3321-3321, DOI: 10.1039/c8ee90054j

Tillgänglig från: 2017-10-11 Skapad: 2017-10-11 Senast uppdaterad: 2019-01-17Bibliografiskt granskad
Kovacs, D., Lu, X., Meszaros, L. S., Ott, M., Andres, J. & Borbas, K. E. (2017). Photophysics of Coumarin and Carbostyril-Sensitized Luminescent Lanthanide Complexes: Implications for Complex Design in Multiplex Detection. Journal of the American Chemical Society, 139(16), 5756-5767
Öppna denna publikation i ny flik eller fönster >>Photophysics of Coumarin and Carbostyril-Sensitized Luminescent Lanthanide Complexes: Implications for Complex Design in Multiplex Detection
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2017 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, nr 16, s. 5756-5767Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Luminescent lanthanide (Ln(III)) complexes with coumarin or carbostyril antennae were synthesized and their photophysical properties evaluated using steady-state and time-resolved UV-vis spectroscopy. Ligands bearing distant hydroxycoumarin-derived antennae attached through triazole linkers were modest sensitizers for Eu(III) and Tb(III), whereas ligands with 7-amidocarbostyrils directly linked to the coordination site could reach good quantum yields for multiple Ln(III), including the visible emitters Sm(III) and Dy(III), and the near-infrared emitters Nd(III) and Yb(III). The highest lanthanide-centered luminescence quantum yields were 35% (Tb), 7.9% (Eu), 0.67% (Dy), and 0.18% (Sm). Antennae providing similar luminescence intensities with 2-4 Ln-emitters were identified. Photoredox quenching of the carbostyril antenna excited states was observed for all Eu(III)-complexes and should be sensitizing in the case of Yb(III); the scope of the process extends to Ln(III) for which it has not been seen previously, specifically Dy(III) and Sm(III). The proposed process is supported by photophysical and electrochemical data. A FRET-type mechanism was identified in architectures with both distant and close antennae for all of the Lns. This mechanism seems to be the only sensitizing one at long distance and probably contributes to the sensitization at shorter distances along with the triplet pathway. The complexes were nontoxic to either bacterial or mammalian cells. Complexes of an ester-functionalized ligand were taken up by bacteria in a concentration-dependent manner. Our results suggest that the effects of FRET and photoredox quenching should be taken into consideration when designing luminescent Ln complexes. These results also establish these Ln(III)-complexes for multiplex detection beyond the available two-color systems.

Ort, förlag, år, upplaga, sidor
AMER CHEMICAL SOC, 2017
Nationell ämneskategori
Kemi Teknik och teknologier
Identifikatorer
urn:nbn:se:uu:diva-322722 (URN)10.1021/jacs.6b11274 (DOI)000400321500029 ()28388066 (PubMedID)
Forskningsfinansiär
Vetenskapsrådet, 2013-4655Stiftelsen Olle Engkvist Byggmästare
Tillgänglig från: 2017-05-29 Skapad: 2017-05-29 Senast uppdaterad: 2019-04-16Bibliografiskt granskad
Raleiras, P., Khanna, N., Miranda, H., Meszaros, L. S., Krassen, H., Ho, F., . . . Styring, S. (2016). Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme. Energy & Environmental Science, 9(2), 581-594
Öppna denna publikation i ny flik eller fönster >>Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme
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2016 (Engelska)Ingår i: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 9, nr 2, s. 581-594Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 produces hydrogen via nitrogenase in heterocysts upon onset of nitrogen-fixing conditions. N. punctiforme expresses concomitantly the uptake hydrogenase HupSL, which oxidizes hydrogen in an effort to recover some of the reducing power used up by nitrogenase. Eliminating uptake activity has been employed as a strategy for net hydrogen production in N. punctiforme (Lindberg et al., Int. J. Hydrogen Energy, 2002, 27, 1291-1296). However, nitrogenase activity wanes within a few days. In the present work, we modify the proximal iron-sulfur cluster in the hydrogenase small subunit HupS by introducing the designed mutation C12P in the fusion protein f-HupS for expression in E. coli (Raleiras et al., J. Biol. Chem., 2013, 288, 18345-18352), and in the full HupSL enzyme for expression in N. punctiforme. C12P f-HupS was investigated by EPR spectroscopy and found to form a new paramagnetic species at the proximal cluster site consistent with a [4Fe-4S] to [3Fe-4S] cluster conversion. The new cluster has the features of an unprecedented mixed-coordination [3Fe-4S] metal center. The mutation was found to produce stable protein in vitro, in silico and in vivo. When C12P HupSL was expressed in N. punctiforme, the strain had a consistently higher hydrogen production than the background [capital Delta]hupSL mutant. We conclude that the increase in hydrogen production is due to the modification of the proximal iron-sulfur cluster in HupS, leading to a turn of the electron flow in the enzyme.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2016
Nationell ämneskategori
Kemi
Identifikatorer
urn:nbn:se:uu:diva-271302 (URN)10.1039/C5EE02694F (DOI)000369744500023 ()
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse, 2011.0067EU, FP7, Sjunde ramprogrammet, 317184Energimyndigheten, 11674-5
Tillgänglig från: 2016-01-07 Skapad: 2016-01-07 Senast uppdaterad: 2017-12-01Bibliografiskt granskad
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