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Interactions between the [FeFe]-hydrogenase maturation enzymes from Thermoanaerobacter italicus

Interactions between the [FeFe]-hydrogenase maturation enzymes from Thermoanaerobacter italicus
Interactions between the [FeFe]-hydrogenase maturation enzymes from Thermoanaerobacter italicus
The [FeFe]-hydrogenase enzyme is highly efficient at producing hydrogen under anaerobic conditions, hence a promising target to study in respect to scarce fossil energies and as an alternative zero-carbon energy source. The biosynthesis of the active [FeFe]-hydrogenase cofactor is a result of an interplay between three maturation enzymes named HydF, HydG and HydE. The unique diiron core and its surrounding CO and CN ligands, as well as the azadithiolate bridge form the H-cluster cofactor, responsible for hydrogen production. To understand the role of each maturation enzyme in context to the others, each maturation enzyme is produced via heterologous expression in E. coli and chemically reconstituted to ensure full activity. StrepHydF and StrepHydE expression vectors were generated and the pCDuet vector containing the T7 promoter resulted in a higher expression yield than initial expression studies with a pBAD expression vector. However, both HydE and HydF required chemical reconstitution despite several attempts to incorporate iron-sulfur cluster during expression and purification. The level of reconstitution is characterized by spectroscopic methods, including UV-Vis spectroscopy, electron paramagnetic resonance (EPR) and FT-IR (Fourier-transformed InfraRed) spectroscopy. Furthermore, FT-IR spectroscopy provided a tool to monitor the formation of the [Fe(CO)2(CN)]-synthon by HydG after cleavage of L-tyrosine into p-cresol and dehydroglycine, which decomposes to CO and CN- ligands. The presence of the synthon-iron was confirmed by EPR-spectroscopy and showed a characteristic S = 5/2 signal in the low magnetic field, if L-cysteine was added. HydG and HydE are radical SAM enzymes that are able to reductively cleave SAM into 5'-deoxyadenosyl and methionine which was confirmed by HPLC-based activity assays. Since the substrate of HydE is unknown the reductive SAM cleavage activity is significantly lower compared to HydG in the presence of its substrate L-tyrosine. Screening of compounds that might influence the activity of either HydG or HydE in the presence of all [FeFe]-hydrogenase maturation partner enzymes revealed that GTP has an enhancing effect on HydGs activity. However, alongside activity tests of the GTPase HydF, which has shown to hydrolyze GTP into GDP, HydG was found not to hydrolyze GTP. Suggesting HydG is not using GTP for energetic reasons. ATP and Pyro diphosphate also had a stimulating effect on HydGs activity, supporting the important role of the phosphates which might coordinate the synthon and initiate transport onto HydF. Attempts to study the interaction between the [FeFe]-hydrogenase maturation proteins confirmed the dimeric state of HydF and monomeric state of HydG in solution by gel-filtration. Moreover, binding studies using the pull-down assay method with HydF and HydG, revealed a crucial role of the pH and the presence of L-tyrosine for the complex formation between HydF and HydG.
University of Southampton
Monfort, Beata Marta
a8ab31d2-68c4-41c8-b666-8edbe3a805e4
Monfort, Beata Marta
a8ab31d2-68c4-41c8-b666-8edbe3a805e4
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9

Monfort, Beata Marta (2018) Interactions between the [FeFe]-hydrogenase maturation enzymes from Thermoanaerobacter italicus. University of Southampton, Doctoral Thesis, 401pp.

Record type: Thesis (Doctoral)

Abstract

The [FeFe]-hydrogenase enzyme is highly efficient at producing hydrogen under anaerobic conditions, hence a promising target to study in respect to scarce fossil energies and as an alternative zero-carbon energy source. The biosynthesis of the active [FeFe]-hydrogenase cofactor is a result of an interplay between three maturation enzymes named HydF, HydG and HydE. The unique diiron core and its surrounding CO and CN ligands, as well as the azadithiolate bridge form the H-cluster cofactor, responsible for hydrogen production. To understand the role of each maturation enzyme in context to the others, each maturation enzyme is produced via heterologous expression in E. coli and chemically reconstituted to ensure full activity. StrepHydF and StrepHydE expression vectors were generated and the pCDuet vector containing the T7 promoter resulted in a higher expression yield than initial expression studies with a pBAD expression vector. However, both HydE and HydF required chemical reconstitution despite several attempts to incorporate iron-sulfur cluster during expression and purification. The level of reconstitution is characterized by spectroscopic methods, including UV-Vis spectroscopy, electron paramagnetic resonance (EPR) and FT-IR (Fourier-transformed InfraRed) spectroscopy. Furthermore, FT-IR spectroscopy provided a tool to monitor the formation of the [Fe(CO)2(CN)]-synthon by HydG after cleavage of L-tyrosine into p-cresol and dehydroglycine, which decomposes to CO and CN- ligands. The presence of the synthon-iron was confirmed by EPR-spectroscopy and showed a characteristic S = 5/2 signal in the low magnetic field, if L-cysteine was added. HydG and HydE are radical SAM enzymes that are able to reductively cleave SAM into 5'-deoxyadenosyl and methionine which was confirmed by HPLC-based activity assays. Since the substrate of HydE is unknown the reductive SAM cleavage activity is significantly lower compared to HydG in the presence of its substrate L-tyrosine. Screening of compounds that might influence the activity of either HydG or HydE in the presence of all [FeFe]-hydrogenase maturation partner enzymes revealed that GTP has an enhancing effect on HydGs activity. However, alongside activity tests of the GTPase HydF, which has shown to hydrolyze GTP into GDP, HydG was found not to hydrolyze GTP. Suggesting HydG is not using GTP for energetic reasons. ATP and Pyro diphosphate also had a stimulating effect on HydGs activity, supporting the important role of the phosphates which might coordinate the synthon and initiate transport onto HydF. Attempts to study the interaction between the [FeFe]-hydrogenase maturation proteins confirmed the dimeric state of HydF and monomeric state of HydG in solution by gel-filtration. Moreover, binding studies using the pull-down assay method with HydF and HydG, revealed a crucial role of the pH and the presence of L-tyrosine for the complex formation between HydF and HydG.

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E-Thesis Beata Monfort Dec 2018 - Version of Record
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Published date: December 2018

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Local EPrints ID: 428048
URI: http://eprints.soton.ac.uk/id/eprint/428048
PURE UUID: 421925bd-e814-435c-aad4-177fedab1f47
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 07 Feb 2019 17:30
Last modified: 30 Jan 2020 01:30

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Contributors

Author: Beata Marta Monfort
Thesis advisor: Peter L. Roach ORCID iD

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