| 1. |
- Esmieu, Charlene, et al.
(författare)
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Synthesis of a miniaturized [FeFe] hydrogenase model system
- 2019
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Ingår i: Dalton Transactions. - : ROYAL SOC CHEMISTRY. - 1477-9226 .- 1477-9234. ; 48:7, s. 2280-2284
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Tidskriftsartikel (refereegranskat)abstract
- The reaction occurring during artificial maturation of [FeFe] hydrogenase has been recreated using molecular systems. The formation of a miniaturized [FeFe] hydrogenase model system, generated through the combination of a [4Fe4S] cluster binding oligopeptide and an organometallic Fe complex, has been monitored by a range of spectroscopic techniques. A structure of the final assembly is suggested based on EPR and FTIR spectroscopy in combination with DFT calculations. The capacity of this novel H-cluster model to catalyze H-2 production in aqueous media at mild potentials is verified in chemical assays.
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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. |
- Land, Henrik, et al.
(författare)
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Discovery of novel [FeFe]-hydrogenases for biocatalytic H-2-production
- 2019
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 10:43, s. 9941-9948
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Tidskriftsartikel (refereegranskat)abstract
- A new screening method for [FeFe]-hydrogenases is described, circumventing the need for specialized expression conditions as well as protein purification for initial characterization. [FeFe]-hydrogenases catalyze the formation and oxidation of molecular hydrogen at rates exceeding 10(3) s(-1), making them highly promising for biotechnological applications. However, the discovery of novel [FeFe]-hydrogenases is slow due to their oxygen sensitivity and dependency on a structurally unique cofactor, complicating protein expression and purification. Consequently, only a very limited number have been characterized, hampering their implementation. With the purpose of increasing the throughput of [FeFe]-hydrogenase discovery, we have developed a screening method that allows for rapid identification of novel [FeFe]-hydrogenases as well as their characterization with regards to activity (activity assays and protein film electrochemistry) and spectroscopic properties (electron paramagnetic resonance and Fourier transform infrared spectroscopy). The method is based on in vivo artificial maturation of [FeFe]-hydrogenases in Escherichia coli and all procedures are performed on either whole cells or non-purified cell lysates, thereby circumventing extensive protein purification. The screening was applied on eight putative [FeFe]-hydrogenases originating from different structural sub-classes and resulted in the discovery of two new active [FeFe]-hydrogenases. The [FeFe]-hydrogenase from Solobacterium moorei shows high H-2-gas production activity, while the enzyme from Thermoanaerobacter mathranii represents a hitherto uncharacterized [FeFe]-hydrogenase sub-class. This latter enzyme is a putative sensory hydrogenase and our in vivo spectroscopy study reveals distinct differences compared to the well established H-2 producing HydA1 hydrogenase from Chlamydomonas reinhardtii.
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| 4. |
- Lorenzi, Marco, et al.
(författare)
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Light-Driven [FeFe] Hydrogenase Based H-2 Production in E. coli : A Model Reaction for Exploring E. coli Based Semiartificial Photosynthetic Systems
- 2022
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 10:33, s. 10760-10767
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Tidskriftsartikel (refereegranskat)abstract
- Biohybrid technologies like semiartificial photosynthesis are attracting increased attention, as they enable the combination of highly efficient synthetic light-harvesters with the self-healing and outstanding performance of biocatalysis. However, such systems are intrinsically complex, with multiple interacting components. Herein, we explore a whole-cell photocatalytic system for hydrogen (H2) gas production as a model system for semiartificial photosynthesis. The employed whole-cell photo catalytic system is based on Escherichia coli cells heterologously expressing a highly efficient, but oxygen-sensitive, [FeFe] hydrogenase. The system is driven by the organic photosensitizer eosin Y under broad-spectrum white light illumination. The direct involvement of the [FeFe] hydrogenase in the catalytic reaction is verified spectroscopically. We also observe that E. coli provides protection against O-2 damage, underscoring the suitability of this host organism for oxygen-sensitive enzymes in the development of (photo) catalytic biohybrid systems. Moreover, the study shows how factorial experimental design combined with analysis of variance (ANOVA) can be employed to identify relevant variables, as well as their interconnectivity, on both overall catalytic performance and O-2 tolerance.
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| 5. |
- Meszaros, Livia S., et al.
(författare)
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Semi-synthetic hydrogenases—in vitro and in vivo applications
- 2021
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Ingår i: Current Opinion in Green and Sustainable Chemistry. - : Elsevier. - 2452-2236. ; 32
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are gas processing redox enzymes central in hydrogen metabolism. The interdisciplinary nature of hydrogenase research is underscored by the development of “artificial maturation”, enabling the preparation of semi-synthetic hydrogenases through the incorporation of synthetic cofactors into a range of apo-hydrogenase hosts under in vitro and in vivo conditions. Herein, we discuss how the preparation of such semi-synthetic [FeFe]-hydrogenases has elucidated structural elements of the cofactor critical for catalysis and reactivity towards known inhibitors. It has also provided a convenient method for exploring the biodiversity of this enzyme family and thereby facilitated investigation of the role of the outer-coordination sphere in tuning the reactivity of the Hcluster. In parallel, hijacking the assembly line of the [FeFe]-hydrogenase through incorporation of synthetic precursors has provided detailed insight into the biosynthesis of the H-cluster. Moreover, it has allowed the preparation of Mn analogs of [Fe] hydrogenase.
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| 6. |
- Meszaros, Livia S., et al.
(författare)
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Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase
- 2020
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Ingår i: Chemical Science. - : ROYAL SOC CHEMISTRY. - 2041-6520 .- 2041-6539. ; 11:18, s. 4608-4617
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are among the fastest H-2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.
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| 7. |
- 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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| 8. |
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| 9. |
- 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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| 10. |
- Redman, Holly J., et al.
(författare)
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Exploring Dimanganese dithiolato carbonyl complexes as models of [FeFe] hydrogenase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- [FeFe]-hydrogenase utilize an organometallic dinuclear iron carbonyl complex to catalyze the formation and oxidation of H2. Herein we explore the related dimanganese complex, Mn2(µ-S2C2H2)(CO)6, and three phosphine subsituted derivates, as structural and functional mimic of this cofactor. The structure and protonation chemistry of the complexes was characterised by a combination of X-ray crystallography, spectroscopy and electrochemical methods. The hexacarbonyl complex was found to be an active catalyst for proton reduction.
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