| 1. |
- Cabotaje, Princess R., et al.
(författare)
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Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis : An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase
- 2023
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:15, s. 10435-10446
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Tidskriftsartikel (refereegranskat)abstract
- [FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogeninterconversion, have attracted intense attention due to their remarkablecatalytic properties and (bio-)technological potential for a futurehydrogen economy. In order to unravel the factors enabling their efficientcatalysis, both their unique organometallic cofactors and proteinstructural features, i.e., "outer-coordination sphere"effects have been intensively studied. These structurally diverseenzymes are divided into distinct phylogenetic groups, denoted asGroup A-D. Prototypical Group A hydrogenases display high turnoverrates (10(4)-10(5) s(-1)).Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity10(-1) & mu;mol H-2 mg(-1) min(-1)) and has been proposed to serve a H-2-sensory function. The various groups of [FeFe] hydrogenaseshare the same catalytic cofactor, the H-cluster, and the structuralfactors causing the diverging reactivities of Group A and D remainto be elucidated. In the case of the highly active Group A enzymes,a well-defined proton transfer pathway (PTP) has been identified,which shuttles H+ between the enzyme surface and the activesite. In Group D hydrogenases, this conserved pathway is absent. Here,we report on the identification of highly conserved amino acid residuesin Group D hydrogenases that constitute a possible alternative PTP.We varied two proposed key amino acid residues of this pathway (E252and E289, TamHydS numbering) via site-directed mutagenesisand analyzed the resulting variants via biochemical and spectroscopicmethods. All variants displayed significantly decreased H-2-evolution and -oxidation activities. Additionally, the variantsshowed two redox states that were not characterized previously. Thesefindings provide initial evidence that these amino acid residues arecentral to the putative PTP of Group D [FeFe] hydrogenase. Since theidentified residues are highly conserved in Group D exclusively, ourresults support the notion that the PTP is not universal for differentphylogenetic groups in [FeFe] hydrogenases.
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| 2. |
- Fasano, Andrea, et al.
(författare)
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Reversible or Irreversible Catalysis of H+/H2 Conversion by FeFe Hydrogenases
- 2021
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:48, s. 20320-20325
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Tidskriftsartikel (refereegranskat)abstract
- Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. “Reversible” catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, “irreversible” catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al. Nat. Rev. Chem.2021, 5, 348–360]. Numerous mechanistic rationales for these contrasting behaviors have been proposed. To understand the determinants of catalytic (ir)reversibility, we examined the steady-state, direct electron transfer voltammetry of a particular FeFe hydrogenase, from Thermoanaerobacter mathranii, which is very unusual in that it irreversibly catalyzes H2 oxidation and production: a large overpotential is required for the reaction to proceed in either direction [Land et al. Chem. Sci.2020, 11, 12789–12801]. In contrast to previous hypotheses, we demonstrate that in this particular enzyme catalytic irreversibility can be explained without invoking slow interfacial electron transfer or variations in the mechanism: the observed kinetics is fully consistent with the same catalytic pathway being used in both directions of the reaction.
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| 3. |
- 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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| 4. |
- Kätterer, Thomas, et al.
(författare)
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Växtföljders påverkan på inlagring av organiskt kol i jordbruksmark : en systematisk översikt och samhällsekonomisk analys
- 2021
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Rapport (populärvet., debatt m.m.)abstract
- Ändrade växtföljder kan göra jordbruket mer klimatsmart Genom väl avvägda växtföljder inom jordbruket kan förlusten av organiskt kol i marken minskas. Det är bra både för klimatet och markens bördighet. Vi har undersökt vad vetenskapen säger om vilka grödor och växtföljder som minskar kolförlusterna mest. I den här rapporten redovisar vi en systematisk översikt (del 1) och en samhällsekonomisk analys (del 2). Den systematiska översikten reder ut hur olika växtföljder påverkar inlagring av kol i jordbruksmark. Syftet med en systematisk översikt är att identifiera all tillgänglig och relevant kunskap, kvalitetsgranska studierna och undersöka hur olika faktorer påverkar studieresultaten. I den här systematiska översikten har vi granskat över 7500 unika sökträffar, varav 125 vetenskapliga artiklar uppfyllde de på förhand uppställda urvalskriterierna och är inkluderade i översikten. Utifrån resultaten från den systematiska översikten har vi därefter genomfört en kostnadsnyttoanalys och beräknat den samhällsekonomiska lönsamheten av olika typer av växtföljder. Vi har också genomfört en styrmedelsanalys där vi diskuterar vad som kan vara ett lämpligt styrmedel för att få till stånd en ändring av växtföljder som leder till en ökad inlagring av kol på jordbruksmark.Del 1: Övergripande evidensgraderade slutsatser av den systematiska översiktenVi är säkra på att flerårsväxter har en positiv effekt på halten av organiskt kol i jorden inom varierade växtföljderVi är säkra på att baljväxter har en positiv effekt på halten av organiskt kol i jorden inom varierade växtföljderVi kan visa att det finns en positiv effekt på halten av organiskt kol i jorden av vissa varierade växtföljder och brukningsmetoder jämfört med en upprepad monokultur.Fler och mer detaljerade slutsatser presenteras i rapporten. De skillnader i halt av organiskt kol i jorden mellan olika typer av växtföljder vi rapporterar om här framkommer tydligast när många studier vägs samman, snarare än i enskilda studier, och det statistiska underlaget därmed blir större. I den systematiska översikten har vi även identifierat några typer av växtföljder som det kan behövas mer forskning om, samt brister i hur metoder och resultat ofta rapporteras och som författare av framtida vetenskapliga artiklar bör beakta.Del 2: Resultat av den samhällsekonomiska analysenModellberäkningar visar att det är samhällsekonomiskt lönsamt att inom jordbruket praktisera växtföljder som främjar inlagring av organiskt kol i jordbruksmark även om de på kort sikt ger en lägre avkastning för den enskilde jordbrukaren.Om styrmedel i form av ekonomiska ersättningar skulle bli aktuella för att öka intresset för växtföljder som främjar inlagring av organiskt kol i jordbruksmark kan omvänd auktionering vara ett fungerande alternativ. Införande och utformning av ett eventuellt styrmedel måste dock utredas djupare och förankras brett genom sedvanligt remissförfarande.
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| 5. |
- 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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| 6. |
- Land, Henrik, et al.
(författare)
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Current State of [FeFe]-Hydrogenase Research : Biodiversity and Spectroscopic Investigations
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:13, s. 7069-7086
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are redox enzymes that catalyze the conversion of protons and molecular hydrogen (H-2). Based on the composition of the active site cofactor, the monometallic [Fe]-hydrogenase is distinguished from the bimetallic [NiFe]- or [FeFe]-hydrogenase. The latter has been reported with particularly high turnover activities for both H-2 release and H-2 oxidation, notably at neutral pH, ambient temperatures, and negligible electric overpotential. Due to these properties, [FeFe]-hydrogenase represents the "gold standard" in enzymatic hydrogen turnover. Understanding hydrogenase chemistry is crucial for the design of transition metal complexes that serve as potentially sustainable proton reduction or H-2 oxidation catalysts, e.g., in electrolytic devices or fuel cells. However, even 20 years after the crystal structures of [FeFe]-hydrogenase have been published, several aspects of biological hydrogen turnover are heatedly discussed. In this perspective, we give an overview on how the diversity of naturally occurring and artificially prepared, semisynthetic [FeFe]-hydrogenases deepens our understanding of hydrogenase chemistry. In parallel, we cover recent results from biophysical techniques that go beyond the scope of conventional X-ray diffraction, EPR, and FTIR spectroscopy. Taking into account both proton transfer and electron transfer as well as the notorious sensitivity of [FeFe]-hydrogenase toward carbon monoxide, the discussion further touches upon the molecular proceedings of biological hydrogen turnover.
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| 7. |
- Land, Henrik, et al.
(författare)
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Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines
- 2020
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Ingår i: Advanced Synthesis and Catalysis. - : Wiley-VCH Verlagsgesellschaft. - 1615-4150 .- 1615-4169. ; 362:4, s. 812-821
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Tidskriftsartikel (refereegranskat)abstract
- A protein engineering approach for expanding the substrate scope of the (S)-selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl- and benzyl-substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2-diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)-1,2-diphenylethylamine with ee >99 % under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20 % (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzyl-substituted primary amines.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- Moloney, Kristen, et al.
(författare)
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Development of a surgical competency assessment tool for sentinel lymph node dissection by minimally invasive surgery for endometrial cancer
- 2021
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Ingår i: International Journal of Gynecological Cancer. - : BMJ. - 1048-891X .- 1525-1438. ; 31:5, s. 647-655
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Tidskriftsartikel (refereegranskat)abstract
- Sentinel lymph node dissection is widely used in the staging of endometrial cancer. Variation in surgical techniques potentially impacts diagnostic accuracy and oncologic outcomes, and poses barriers to the comparison of outcomes across institutions or clinical trial sites. Standardization of surgical technique and surgical quality assessment tools are critical to the conduct of clinical trials. By identifying mandatory and prohibited steps of sentinel lymph node (SLN) dissection in endometrial cancer, the purpose of this study was to develop and validate a competency assessment tool for use in surgical quality assurance. A Delphi methodology was applied, included 35 expert gynecological oncology surgeons from 16 countries. Interviews identified key steps and tasks which were rated mandatory, optional, or prohibited using questionnaires. Using the surgical steps for which consensus was achieved, a competency assessment tool was developed and subjected to assessments of validity and reliability. Seventy percent consensus agreement standardized the specific mandatory, optional, and prohibited steps of SLN dissection for endometrial cancer and informed the development of a competency assessment tool. Consensus agreement identified 21 mandatory and three prohibited steps to complete a SLN dissection. The competency assessment tool was used to rate surgical quality in three preselected videos, demonstrating clear separation in the rating of the skill level displayed with mean skills summary scores differing significantly between the three videos (F score=89.4; P<0.001). Internal consistency of the items was high (Cronbach α=0.88). Specific mandatory and prohibited steps of SLN dissection in endometrial cancer have been identified and validated based on consensus among a large number of international experts. A competency assessment tool is now available and can be used for surgeon selection in clinical trials and for ongoing, prospective quality assurance in routine clinical care.
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