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- Németh, Brigitta, 1990-
(författare)
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The birth of the hydrogenase : Studying the mechanism of [FeFe] hydrogenase maturation
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Redman, Holly J., 1995-
(författare)
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Studies of second coordination sphere effects and metal variations on [FeFe]-hydrogenase mimics
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitigation of climate change motivates researchers to explore hydrogen as a potential energy carrier. Unfortunately, widespread use of hydrogen as an energy carrier is limited by numerous challenges in its production, including high energy consumption; high economic cost; current reliance on rare metals such as platinum. Diiron hydrogenases could provide a solution to the above-mentioned challenges because they are able to turnover hydrogen at very high frequencies, and utilise earth abundant iron as the redox active centre. However, diiron hydrogenases are not currently a scalable technology, and more research is needed to fully understand their reaction mechanism, and to allow engineering of optimal proton reduction catalysts. The H-cluster is an hexanuclear iron cluster in the active site, which consists of a [Fe4S4]-cluster and a [Fe2S2] cofactor. The [Fe4S4]-cluster behaves as a redox active ligand for the [Fe2S2] cofactor. The [Fe2S2] cofactor is a diiron complex in which the irons share a bridging azadithiolato ligand, and one bridging carbonyl ligand. Each iron has one terminal carbonyl ligand and one cyanide ligand. The [Fe4S4]-cluster and [Fe2S2] cofactor are coupled via a bridging cysteine thiol. Several mechanisms for the diiron hydrogenase enzyme have been put forth, and are still debated. In parallel synthetic chemists continue to develop a diverse array of structural and functional mimics of the diiron hydrogenase active site. This thesis aims to examine several aspects of the H-cluster from a molecular design perspective, utilising FTIR, electrochemistry, EPR, XAS, among other spectroscopic techniques, to do so. Paper I studies the effects of the bridgehead ligand in the outer coordination sphere, and what effect this has on the oxygen tolerance and reduction chemistry. Papers II and III investigate the effect of tuning the electron density on the iron centres through the incorporation of a redox active ligand (paper II) or through introduction of capping Lewis acidic moieties on the cyanide ligands of the diiron cofactor (paper III). Paper III also reports on the potential role played by the Lewis acid in protecting the cyanide ligands from proton attack during catalysis. In paper IV a new synthetic mimic is synthesised in which the iron is replaced with manganese while the coordination environment remains intact. The results from these studies inform on future design perspectives for diiron hydrogenase active site mimics.
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