| 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. |
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| 3. |
- Gamache, Mira T., et al.
(författare)
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Elucidating Electron Transfer Kinetics and Optimizing System Performance for Escherichia coli-Based Semi-Artificial H-2 Production
- 2023
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:14, s. 9476-9486
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Tidskriftsartikel (refereegranskat)abstract
- Both photo- and biocatalysis are well-established andintensivelystudied. The combination of these two approaches is also an emergingresearch field, commonly referred to as semi-artificial photosynthesis.Semi-artificial photosynthesis aims at combining highly efficientsynthetic light harvesters with the self-healing and potent catalyticproperties of biocatalysis. In this study, a semi-artificial photocatalyticsystem featuring Escherichia coli bacteria,which heterologously express the [FeFe] hydrogenase enzyme HydA1 fromgreen algae, is employed as a hydrogen gas production catalyst. Toprobe the influence of photochemistry on overall system performance,the E. coli whole-cell catalyst iscombined with two different photosensitizers and redox mediators.The addition of a redox mediator greatly improves the rates and longevityof the photocatalytic system, as reflected in increases of both theturn-over number (0.777 vs 10.9 & mu;mol H-2 mL(-1) OD600 (-1)) and the turn-over frequency(175 vs 334 & mu;mol H-2 mL(-1) h(-1) OD600 (-1)). The redoxmediator is found to both protect from photobleaching and enable electrontransport to the hydrogenase from an extracellular photosensitizer.However, E. coli cells are stronglyaffected by the photocatalytic system, leading to a decrease in cellintegrity and cell viability, possibly due to toxic decompositionproducts formed during the photocatalytic process. We furthermoreemployed steady-state and transient absorption spectroscopy to investigatesolution potentials and the kinetics of electron transfer processesbetween the sacrificial electron donor, photosensitizer, redox mediator,and the [FeFe] hydrogenase as the final electron acceptor. These resultsallowed us to rationalize the different activities observed in photocatalyticassays and offer a better understanding of the factors that influencethe photocatalytic performance of E. coli-based whole-cell systems.
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| 4. |
- Greening, Chris, et al.
(författare)
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Minimal and hybrid hydrogenases are active from archaea
- 2024
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Ingår i: Cell. - : Elsevier. - 0092-8674 .- 1097-4172. ; 187:13
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Tidskriftsartikel (refereegranskat)abstract
- Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.
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| 5. |
- Grinter, Rhys, et al.
(författare)
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Structural basis for bacterial energy extraction from atmospheric hydrogen
- 2023
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Ingår i: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 615:7952, s. 541-547
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Tidskriftsartikel (refereegranskat)abstract
- Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.
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| 6. |
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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- Laun, Konstantin, et al.
(författare)
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Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase
- 2021
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Ingår i: Dalton Transactions. - : Royal Society of Chemistry. - 1477-9226 .- 1477-9234. ; 50:10, s. 3641-3650
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H-2 oxidation activity, [FeFe]-hydrogenases are excellent H-2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H-2 oxidation or H-2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pK(a) differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.
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