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Németh, Brigitta
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Publications (5 of 5) Show all publications
Nemeth, B., Esmieu, C., Redman, H. J. & Berggren, G. (2019). Monitoring H-cluster assembly using a semi-synthetic HydF protein. Dalton Transactions, 48(18), 5978-5986
Open this publication in new window or tab >>Monitoring H-cluster assembly using a semi-synthetic HydF protein
2019 (English)In: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, Vol. 48, no 18, p. 5978-5986Article in journal (Refereed) Published
Abstract [en]

The [FeFe] hydrogenase enzyme interconverts protons and molecular hydrogen with remarkable efficiency. The reaction is catalysed by a unique metallo-cofactor denoted as the H-cluster containing an organometallic dinuclear Fe component, the [2Fe] subsite. The HydF protein delivers a precursor of the [2Fe] subsite to the apo-[FeFe] hydrogenase, thus completing the H-cluster and activating the enzyme. Herein we generate a semi-synthetic form of HydF by loading it with a synthetic low valent dinuclear Fe complex. We show that this semi-synthetic protein is practically indistinguishable from the native protein, and utilize this form of HydF to explore the mechanism of H-cluster assembly. More specifically, we show that transfer of the precatalyst from HydF to the hydrogenase enzyme results in the release of CO, underscoring that the pre-catalyst is a four CO species when bound to HydF. Moreover, we propose that an electron transfer reaction occurs during H-cluster assembly, resulting in an oxidation of the [2Fe] subsite with concomitant reduction of the [4Fe4S] cluster present on the HydF protein.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Theoretical Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390520 (URN)10.1039/c8dt04294b (DOI)000472449300013 ()30632592 (PubMedID)
Funder
Swedish Research Council, 621-2014-5670Swedish Research Council Formas, 213-2014-880EU, European Research Council, 714102
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-09-18Bibliographically approved
Németh, B. (2019). The birth of the hydrogenase: Studying the mechanism of [FeFe] hydrogenase maturation. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>The birth of the hydrogenase: Studying the mechanism of [FeFe] hydrogenase maturation
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The [FeFe] hydrogenases are ancient metalloenzymes that catalyse the reversible interconversion between protons, electrons and molecular hydrogen. Despite the large structural variability within the [FeFe] hydrogenase family, the active site, the so called “H-cluster” is present in every representative. The H-cluster is composed by a four cysteine coordinated [4Fe4S] cluster, ligated via a shared cysteine to a biologically unique [2Fe] subsite decorated with CO and CN ligands and an azadithiolate bridging ligand. The biosynthesis of the [2Fe] subsite requires a maturation machinery, composed of at least three maturase enzymes, denoted HydG, HydE, and HydF. HydE and HydG are members of the radical SAM enzyme family, and are responsible for the construction of a pre-catalyst on HydF. This pre-catalyst is finally transferred from HydF to HydA, where it becomes part of the H-cluster.

Recently, a pioneer study combined synthetic chemistry and biochemistry in order to create semi-synthetic HydF proteins. Synthetic mimics of the [2Fe] subsite were introduced to HydF, and this resulting semi-synthetic HydF was used to activate the unmatured hydrogenase (apo-HydA). This technique ushered in a new era in [FeFe] hydrogenase research.

This thesis work is devoted to a deeper understanding of H-cluster formation and [FeFe] hydrogenase maturation, and this process is studied using standard molecular biological and biochemical techniques, and EPR, FTIR, XAS and GEMMA spectroscopic techniques combined with this new type of chemistry mentioned above. EPR spectroscopy was employed to verify the construction of a semi-synthetic [FeFe] hydrogenase inside living cells. The addition of a synthetic complex to cell cultures expressing apo-HydA resulted in a rhombic EPR signal, attributable to an Hox-like species. Moreover, the assembly mechanism of the H-cluster was probed in vitro using XAS, EPR, and FTIR spectroscopy. We verified with all three techniques that the Hox-CO state is formed on a time-scale of seconds, and this state slowly turns into the catalytically active Hox via release of a CO ligand. Furthermore, a semi-synthetic form of the HydF protein from Clostridium acetobutylicum was prepared and characterized in order to prove that such semi-synthetic forms of HydF are biologically relevant. Finally,GEMMA measurements were performed to elucidate the quaternary structure of the HydF-HydA interaction, revealing that dimeric HydF is interacting with a monomeric HydA. However, mutant HydF proteins were prepared, lacking the dimerization (as well as its GTPase) domain, and these severely truncated forms of HydF was found to still retain the capacity to both harbor the pre-catalyst as well as transferring it to apo-HydA. These observations highlight the multi-functionality of HydF, where different domains are critical in different steps of the maturation, that is the dimerization and GTPase domain are rather involved in pre-catalyst assembly rather than its transfer to apo-HydA.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 78
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1856
Keywords
HydF, HydA, [FeFe] hydrogenase maturation, semi-synthetic enzymes
National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-393261 (URN)978-91-513-0754-1 (ISBN)
Public defence
2019-11-06, Å4101, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 62120145670Swedish Research Council Formas, 2132014880
Available from: 2019-10-11 Created: 2019-09-18 Last updated: 2019-11-12
Tian, H., Nemeth, B., Berggren, G. & Tian, L. (2018). Hydrogen evolution by a photoelectrochemical cell based on a Cu2O-ZnO-[FeFe] hydrogenase electrode. Journal of Photochemistry and Photobiology A: Chemistry, 366, 27-33
Open this publication in new window or tab >>Hydrogen evolution by a photoelectrochemical cell based on a Cu2O-ZnO-[FeFe] hydrogenase electrode
2018 (English)In: Journal of Photochemistry and Photobiology A: Chemistry, ISSN 1010-6030, E-ISSN 1873-2666, Vol. 366, p. 27-33Article in journal (Refereed) Published
Abstract [en]

A Cu2O-ZnO-hydrogenase photocathode possessed enzyme/semiconductor junction has been constructed by immobilizing a biological protein catalyst, hydrogenase-CrHydA1 enzyme on the ZnO protected Cu2O electrode. With light illumination, a photocurrent of 0.8 mA/cm2 at 0.15 V vs. RHE was obtained and hydrogen was successfully detected from the photocathode in photoelectrochemical measurements with Faradaic efficiency of ca. 1%. The construction as well as the stability of the system are also reported. The result shows that this biohybrid photocathode is capable of photocatalytic proton reduction under mild conditions.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-371529 (URN)10.1016/j.jphotochem.2018.01.035 (DOI)000452577600005 ()
Funder
Stiftelsen Olle Engkvist Byggmästare, 2015/456J. Gust. Richert stiftelse, 2016-00231Swedish Energy Agency, 111674-8Swedish Research Council, 621-2014-5670Swedish Research Council Formas, 213-2014-880Wenner-Gren Foundations
Available from: 2018-12-21 Created: 2018-12-21 Last updated: 2019-01-16Bibliographically approved
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
Open this publication in new window or tab >>InVivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases
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2018 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 57, no 10, p. 2596-2599Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
[FeFe] hydrogenase, artificial enzymes, EPR spectroscopy, metalloenzymes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-348975 (URN)10.1002/anie.201710740 (DOI)000426252400010 ()29334424 (PubMedID)
Funder
Swedish Research Council, 21-2014-5670Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 213-2014-880EU, European Research Council, 714102
Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2019-09-18Bibliographically approved
Németh, B., Land, H., Magnuson, A., Hofer, A. & Berggren, G.Studying [FeFe] hydrogenase maturation and the nature of the HydF-HydA interaction.
Open this publication in new window or tab >>Studying [FeFe] hydrogenase maturation and the nature of the HydF-HydA interaction
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-393550 (URN)
Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2019-09-26Bibliographically approved
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