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Studying [FeFe] hydrogenase maturation and the nature of the HydF-HydA interaction
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Biophysical and Bioinorganic Chemistry)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.ORCID iD: 0000-0003-3073-5641
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Institutionen för medicinsk kemi och biofysik, Umeå Universitet .
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-393550OAI: oai:DiVA.org:uu-393550DiVA, id: diva2:1354016
Available from: 2019-09-24 Created: 2019-09-24 Last updated: 2019-09-26Bibliographically approved
In thesis
1. The birth of the hydrogenase: Studying the mechanism of [FeFe] hydrogenase maturation
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)
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Supervisors
Funder
Swedish Research Council, 62120145670Swedish Research Council Formas, 2132014880
Available from: 2019-10-11 Created: 2019-09-18 Last updated: 2019-11-12

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Németh, BrigittaLand, HenrikMagnuson, AnnBerggren, Gustav

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