| 1. |
- Hola, Katerina, et al.
(författare)
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Carbon Dots and [FeFe] Hydrogenase Biohybrid Assemblies for Efficient Light-Driven Hydrogen Evolution
- 2020
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 10:17, s. 9943-9952
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Tidskriftsartikel (refereegranskat)abstract
- Artificial photosynthesis is seen as a path to convert and store solar energy into chemical energy for our society. In this work, highly fluorescent aspartic acid-based carbon dots (CDs) are synthesized and employed as a photosensitizer to drive photocatalytic hydrogen evolution with an [FeFe] hydrogenase (CrHydA1). The direct interaction in CDs from L-aspartic acid (AspCDs)/CrHydA1 self-assembly systems, which is visualized from native gel electrophoresis, has been systematically investigated to understand the electron-transfer dynamics and its impact on photocatalytic efficiency. The study discloses the significant influence of the electrostatic surrounding generated by sacrificial electron donors on the intimate interplay within the oppositely charged subunits of the biohybrid assembly as well as the overall photocatalytic performance. The system reaches an external quantum efficiency of 1.7% at 420 nm and an initial activity of 1.73 mu mol(H-2) mg(-1) (hydrogenase) min(-1) under favorable electrostatic conditions. Owing to the ability of the synthesized AspCDs to operate efficiently under visible light, in contrast to other materials that require UV illumination, the stability of the biohybrid assembly in the presence of a redox mediator extends beyond 1 week.
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| 2. |
- Land, Henrik, et al.
(författare)
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Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture
- 2020
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Ingår i: Chemical Science. - : The Royal Society of Chemistry. - 2041-6539. ; 11:47, s. 12789-12801
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Tidskriftsartikel (refereegranskat)abstract
- [FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown. It features significant differences in domain- and active site architecture, and is most closely related to the putative sensory [FeFe]-hydrogenases. Here we report the first comprehensive biochemical and spectroscopical characterization of an M2e enzyme, derived from Thermoanaerobacter mathranii. As compared to other [FeFe]-hydrogenases characterized to-date, this enzyme displays an increased H2 affinity, higher activation enthalpies for H+/H2 interconversion, and unusual reactivity towards known hydrogenase inhibitors. These properties are related to differences in active site architecture between the M2e [FeFe]-hydrogenase and “prototypical†[FeFe]-hydrogenases. Thus, this study provides new insight into the role of this subclass in hydrogen metabolism and the influence of the active site pocket on the chemistry of the H-cluster.
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| 3. |
- Meszaros, Livia S., et al.
(författare)
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InVivo EPR Characterization of Semi-Synthetic [FeFe] Hydrogenases
- 2018
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Ingår i: Angewandte Chemie International Edition. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 57:10, s. 2596-2599
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Németh, Brigitta, et al.
(författare)
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[FeFe]-hydrogenase maturation : H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy
- 2020
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Nature. - 0949-8257 .- 1432-1327. ; 25:5, s. 777-788
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Tidskriftsartikel (refereegranskat)abstract
- [FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt =NH(CH2S)(2)).[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe](adt)). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe](adt). The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred'-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster. [GRAPHICS] .
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| 5. |
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| 6. |
- Nemeth, Brigitta, et al.
(författare)
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Monitoring H-cluster assembly using a semi-synthetic HydF protein
- 2019
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Ingår i: Dalton Transactions. - : ROYAL SOC CHEMISTRY. - 1477-9226 .- 1477-9234. ; 48:18, s. 5978-5986
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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| 8. |
- 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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| 9. |
- Németh, Brigitta, et al.
(författare)
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The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions
- 2020
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 295:33, s. 11891-11901
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Tidskriftsartikel (refereegranskat)abstract
- [FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H(2)gas production. H(2)formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase inin vitroassays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly,i.e.in binding and delivering the precatalyst.
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| 10. |
- Tian, Haining, 1983-, et al.
(författare)
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Hydrogen evolution by a photoelectrochemical cell based on a Cu2O-ZnO-[FeFe] hydrogenase electrode
- 2018
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Ingår i: Journal of Photochemistry and Photobiology A. - : Elsevier BV. - 1010-6030 .- 1873-2666. ; 366, s. 27-33
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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