105 results on '"Frielingsdorf, Stefan"'
Search Results
2. Stepwise assembly of the active site of [NiFe]-hydrogenase
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Caserta, Giorgio, Hartmann, Sven, Van Stappen, Casey, Karafoulidi-Retsou, Chara, Lorent, Christian, Yelin, Stefan, Keck, Matthias, Schoknecht, Janna, Sergueev, Ilya, Yoda, Yoshitaka, Hildebrandt, Peter, Limberg, Christian, DeBeer, Serena, Zebger, Ingo, Frielingsdorf, Stefan, and Lenz, Oliver
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- 2023
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3. Biotechnological perspective for wireless energy: H2-based power extraction from air
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Frielingsdorf, Stefan
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- 2023
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4. Ultrathin Film Antimony-Doped Tin Oxide Prevents [NiFe] Hydrogenase Inactivation at High Electrode Potentials.
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Davis, Victoria, Frielingsdorf, Stefan, Hu, Qiwei, Elsäßer, Patrick, Balzer, Bizan N., Lenz, Oliver, Zebger, Ingo, and Fischer, Anna
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- 2024
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5. Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle
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Claassens, Nico J., Scarinci, Giovanni, Fischer, Axel, Flamholz, Avi I., Newell, William, Frielingsdorf, Stefan, Lenz, Oliver, and Bar-Even, Arren
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- 2020
6. Tracking the route of molecular oxygen in O₂-tolerant membrane-bound [NiFe] hydrogenase
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Kalms, Jacqueline, Schmidt, Andrea, Frielingsdorf, Stefan, Utesch, Tillmann, Gotthard, Guillaume, von Stetten, David, van der Linden, Peter, Royant, Antoine, Mroginski, Maria Andrea, Carpentier, Philippe, Lenz, Oliver, and Scheerer, Patrick
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- 2018
7. CO synthesized from the central one-carbon pool as source for the iron carbonyl in O₂-tolerant [NiFe]-hydrogenase
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Bürstel, Ingmar, Siebert, Elisabeth, Frielingsdorf, Stefan, Zebger, Ingo, Friedrich, Bärbel, and Lenz, Oliver
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- 2016
8. Stepwise conversion of the Cys6[4Fe–3S] to a Cys4[4Fe–4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase.
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Schmidt, Andrea, Kalms, Jacqueline, Lorent, Christian, Katz, Sagie, Frielingsdorf, Stefan, Evans, Rhiannon M., Fritsch, Johannes, Siebert, Elisabeth, Teutloff, Christian, Armstrong, Fraser A., Zebger, Ingo, Lenz, Oliver, and Scheerer, Patrick
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- 2023
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9. Editorial: Hydrogenase: structure, function, maturation, and application.
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Frielingsdorf, Stefan, Pinske, Constanze, Valetti, Francesca, and Greening, Chris
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HYDROGENASE ,BIOTECHNOLOGY ,METALS - Published
- 2023
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10. Immobilization of O2‑tolerant [NiFe] hydrogenase from Cupriavidus necator on Tin-rich Indium Oxide Alters the Catalytic Bias from H2 Oxidation to Proton Reduction.
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Davis, Victoria, Heidary, Nina, Guiet, Amandine, Ly, Khoa Hoang, Zerball, Maximilian, Schulz, Claudia, Michael, Norbert, von Klitzing, Regine, Hildebrandt, Peter, Frielingsdorf, Stefan, Lenz, Oliver, Zebger, Ingo, and Fischer, Anna
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- 2023
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11. Active site assembly of [NiFe]-hydrogenase scrutinized on the basis of purified maturation intermediates
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Caserta, Giorgio, Hartmann, Sven, van Stappen, Casey, Karafoulidi Retsou, Chara, Lorent, Christian, Yelin, Stefan, Keck, Matthias, Schoknecht, Janna, Sergueev, Ilya, Yoda, Yoshitaka, Hildebrandt, Peter, Limberg, Christian, DeBeer, Serena, Zebger, Ingo, Frielingsdorf, Stefan, and Lenz, Oliver
- Abstract
doi:10.26434/chemrxiv-2022-jvtgw, [NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H$_2$ into 2 e$^- $ and 2 H$^+$ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)$_2$(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN$^–$ molecules. Herein, we investigated the sequential assembly of the [NiFe]-cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O$_2$-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)$_2$(CO) fragment, a precursor that contained all cofactor components but remained redox-inactive, and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption, and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.
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- 2022
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12. A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation
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Hartmann, Sven, Frielingsdorf, Stefan, Caserta, Giorgio, and Lenz, Oliver
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Carbon Monoxide ,Cyanides ,metalloenzyme ,Iron ,lcsh:QR1-502 ,Membrane Proteins ,Original Articles ,cofactor assembly ,lcsh:Microbiology ,Protein Subunits ,nickel ,Hydrogenase ,Catalytic Domain ,hydrogen ,Escherichia coli ,541 Physikalische Chemie ,Original Article ,Cupriavidus necator ,ddc:541 ,Tat transport ,Genetic Engineering ,Plasmids ,chemolithotrophy - Abstract
[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator., The apo‐forms of [NiFe]‐hydrogenase large subunits are usually synthesized with a C‐terminal peptide extension that is proteolytically cleaved off upon incorporation of the catalytic metal center. Although to a limited amount, an artificially precleaved large subunit, which was deleted for the C‐terminal extension by genetic engineering, still received the active site components delivered by the dedicated maturation machinery. This suggests that the C‐terminal extension optimizes metal center incorporation, but is not essential for the formation of catalytically active [NiFe]‐hydrogenase.
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- 2020
13. Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes
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Lorent, Christian, Pelmenschikov, Vladimir, Frielingsdorf, Stefan, Schoknecht, Janna, Caserta, Giorgio, Yoda, Yoshitaka, Wang, Hongxin, Tamasaku, Kenji, Lenz, Oliver, Cramer, Stephen P., Horch, Marius, Lauterbach, Lars, and Zebger, Ingo
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metalloenzymes ,biocatalysis ,541 Physikalische Chemie ,in situ spectroscopy ,vibrational spectroscopy ,[NiFe]-hydrogenase - Abstract
To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2-tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.
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- 2021
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14. Electrografted Interfaces on Metal Oxide Electrodes for Enzyme Immobilization and Bioelectrocatalysis
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Harris, Tomos G. A. A., Heidary, Nina, Frielingsdorf, Stefan, Rauwerdink, Sander, Tahraoui, Abbes, Lenz, Oliver, Zebger, Ingo, and Fischer, Anna
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ddc:540 - Abstract
In this work, we demonstrate that diazonium electrografting of biocompatible interfaces on transparent conducting oxide indium tin oxide (ITO) can be controlled and optimized to achieve low charge transfer resistance, allowing highly efficient electron transfer to an immobilized model enzyme, the oxygen‐tolerant [NiFe]‐hydrogenase from Ralstonia eutropha. The use of a radical scavenger enables control of the interface thickness, and thus facilitates maximization of direct electron transfer processes between the enzyme's active center and the electrode. Using this approach, amine and carboxylic acid functionalities were grafted on ITO, allowing enzyme immobilization both under moderate electrostatic control and covalently via amide bond formation. Despite an initial decrease in catalytic activity, covalent immobilization led to an improvement in current stability compared to just electrostatically immobilized enzyme. Given the superior stability of electrografted interfaces in comparison to adsorbed or self‐assembled interfaces, we propose electrografting as an alternative approach for the functional immobilization of redox‐active enzymes on transparent conducting oxide (TCO) electrodes in bioelectronic devices.
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- 2021
15. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre
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Fritsch, Johannes, Scheerer, Patrick, Frielingsdorf, Stefan, Kroschinsky, Sebastian, Friedrich, Barbel, Lenz, Oliver, and Spahn, Christian M.T.
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Crystals -- Structure ,Iron -- Chemical properties ,Enzymes -- Chemical properties -- Structure ,Sulfur -- Chemical properties ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Hydrogenases are abundant enzymes that catalyse the reversible interconversion of [H.sub.2] into protons and electrons at high rates (1). Those hydrogenases maintaining their activity in the presence of [O.sub.2] are considered to be central to [H.sub.2]-based technologies, such as enzymatic fuel cells and for light-driven [H.sub.2] production (2). Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations (3-8), the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by [O.sub.2] has remained unclear. Here we present the crystal structure of an [O.sub.2]-tolerant [NiFe]-hydrogenase from the aerobic [H.sub.2] oxidizer Ralstonia eutropha H16 at 1.5 A resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the [H.sub.2]-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of [H.sub.2] oxidation and as an electron-delivering device upon [O.sub.2] attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials (7-9). The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical [H.sub.2]-converting catalysts that are capable of cycling [H.sub.2] in air., More than two billion years ago, ancient microbes exploited the reducing power of [H.sub.2] for their metabolism; until today [H.sub.2] provides a valuable energy source which is used by [H.sub.2]-oxidizing [...]
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- 2011
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16. Resonance Raman spectroscopic analysis of the iron–sulfur cluster redox chain of the Ralstonia eutropha membrane‐bound [NiFe]‐hydrogenase.
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Siebert, Elisabeth, Schmidt, Andrea, Frielingsdorf, Stefan, Kalms, Jacqueline, Kuhlmann, Uwe, Lenz, Oliver, Scheerer, Patrick, Zebger, Ingo, and Hildebrandt, Peter
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RALSTONIA eutropha ,CHARGE exchange ,CLUSTER analysis (Statistics) ,CRYSTALLOIDS (Botany) ,PROTEIN engineering - Abstract
Iron–sulfur (Fe–S) centers are versatile building blocks in biological electron transfer chains because their redox potentials may cover a wide potential range depending on the type of the cluster and the specific protein environment. Resonance Raman (RR) spectroscopy is widely used to analyze structural properties of such cofactors, but it remains still a challenge to disentangle the overlapping signals of metalloproteins carrying several Fe–S centers. In this work, we combined RR spectroscopy with protein engineering and X‐ray crystallography to address this issue on the basis of the oxygen‐tolerant membrane‐bound hydrogenase from Ralstonia eutropha that catalyzes the reversible conversion of hydrogen into protons and electrons. Besides the NiFe‐active site, this enzyme harbors three different Fe–S clusters constituting an electron relay with a distal [4Fe–4S], a medial [3Fe–4S], and an unusual proximal [4Fe–3S] cluster that may carry a hydroxyl ligand in the superoxidized state. RR spectra were measured from protein crystals by varying the crystal orientation with respect to the electric field vector of the incident laser to achieve a preferential RR enhancement for individual Fe–S clusters. In addition to spectral discrimination by selective reduction of the proximal cluster, protein engineering allowed for transforming the proximal and medial cluster into standard cubane‐type [4Fe–4S] centers in the C19G/C120G and P242C variants, respectively. The latter variant was structurally characterized for the first time in this work. Altogether, the entirety of the RR data provided the basis for identifying the vibrational modes characteristic of the various cluster states in this "model" enzyme as a prerequisite for future studies of complex (FeS)‐based electron transfer chains. [ABSTRACT FROM AUTHOR]
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- 2021
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17. Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen – veranschaulicht anhand von [NiFe]‐Hydrogenasen.
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Lorent, Christian, Pelmenschikov, Vladimir, Frielingsdorf, Stefan, Schoknecht, Janna, Caserta, Giorgio, Yoda, Yoshitaka, Wang, Hongxin, Tamasaku, Kenji, Lenz, Oliver, Cramer, Stephen P., Horch, Marius, Lauterbach, Lars, and Zebger, Ingo
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HYDROGENASE - Abstract
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- 2021
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18. Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research‐Based Learning of Xenobiology.
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Schmitt, Franz‐Josef, Frielingsdorf, Stefan, Friedrich, Thomas, and Budisa, Nediljko
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- 2021
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19. Double-flow focused liquid injector for efficient serial femtosecond crystallography
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Oberthuer, Dominik, Knoška, Juraj, Wiedorn, Max O., Beyerlein, Kenneth, Bushnell, David A., Kovaleva, Elena G., Heymann, Michael, Gumprecht, Lars, Kirian, Richard A., Barty, Anton, Mariani, Valerio, Tolstikova, Aleksandra, Adriano, Luigi, Awel, Salah, Barthelmess, Miriam, Doerner, Katerina, Xavier, P. Lourdu, Yefanov, Oleksandr, James, Daniel R., Nelson, Garrett, Wang, Dingjie, Calvey, George, Chen, Yujie, Schmidt, Andrea, Szczepek, Michael, Frielingsdorf, Stefan, Lenz, Oliver, Snell, Edward, Robinson, Philip J., Šarler, Božidar, Belšak, Grega, Maček, Marjan, Wilde, Fabian, Aquila, Andrew, Boutet, Sébastien, Liang, Mengning, Hunter, Mark S., Scheerer, Patrick, Lipscomb, John D., Weierstall, Uwe, Kornberg, Roger D., Spence, John C. H., Pollack, Lois, Chapman, Henry N., and Bajt, Saša
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Crystallography ,Time Factors ,X-Ray Diffraction ,ddc:000 ,Temperature ,Computer Simulation ,RNA Polymerase II ,Saccharomyces cerevisiae ,Rheology ,Corrigenda ,Article - Abstract
Scientific reports 7, 44628 (2017). doi:10.1038/srep44628, Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices., Published by Springer Nature, London
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- 2017
20. Corrigendum: Double-flow focused liquid injector for efficient serial femtosecond crystallography
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Oberthuer, Dominik, Knoška, Juraj, Wiedorn, Max O., Beyerlein, Kenneth R., Bushnell, David A., Kovaleva, Elena G., Heymann, Michael, Gumprecht, Lars, Kirian, Richard A., Barty, Anton, Mariani, Valerio, Tolstikova, Aleksandra, Adriano, Luigi, Awel, Salah, Barthelmess, Miriam, Dörner, Katerina, Xavier, P. Lourdu, Yefanov, Oleksandr, James, Daniel R., Nelson, Garrett, Wang, Dingjie, Calvey, George, Chen, Yujie, Schmidt, Andrea, Szczepek, Michael, Frielingsdorf, Stefan, Lenz, Oliver, Snell, Edward, Robinson, Philip J., Šarler, Božidar, Belšak, Grega, Maček, Marjan, Wilde, Fabian, Aquila, Andrew, Boutet, Sébastien, Liang, Mengning, Hunter, Mark S., Scheerer, Patrick, Lipscomb, John D., Weierstall, Uwe, Kornberg, Roger D., Spence, John C. H., Pollack, Lois, Chapman, Henry N., and Bajt, Saša
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ddc:000 - Abstract
Scientific reports 7, 46846 (2017). doi:10.1038/srep46846, Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices., Published by Nature Publishing Group, London
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- 2017
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21. O2‑Tolerant H2 Activation by an Isolated Large Subunit of a [NiFe] Hydrogenase.
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Hartmann, Sven, Frielingsdorf, Stefan, Ciaccafava, Alexandre, Lorent, Christian, Fritsch, Johannes, Siebert, Elisabeth, Priebe, Jacqueline, Haumann, Michael, Zebger, Ingo, and Lenz, Oliver
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- 2018
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22. Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.
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Kalms, Jacqueline, Schmidt, Andrea, Frielingsdorf, Stefan, Utesch, Tillmann, Gotthard, Guillaume, von Stetten, David, van der Linden, Peter, Royant, Antoine, Mroginski, Maria Andrea, Carpentier, Philippe, Lenz, Oliver, and Scheerer, Patrick
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HYDROGENASE ,RALSTONIA eutropha ,CATALYTIC reduction ,METALLOPROTEINS ,X-ray crystallography - Abstract
[NiFe] hydrogenases catalyze the reversible splitting of H
2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2 , a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2 -derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme. [ABSTRACT FROM AUTHOR]- Published
- 2018
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23. Multilayered Lipid Membrane Stacks for Biocatalysis Using Membrane Enzymes.
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Heath, George R., Li, Mengqiu, Rong, Honling, Radu, Valentin, Frielingsdorf, Stefan, Lenz, Oliver, Butt, Julea N., and Jeuken, Lars J. C.
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ENZYMES ,BILAYER lipid membranes ,BIOLOGICAL membranes ,RADIOENZYMATIC assays ,BIOCATALYSIS - Abstract
Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single-lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules, and greatly increase surface area and hence membrane protein concentration. Here, a supramolecular assembly of a multilayered lipid membrane system is reported in which poly- l-lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo
3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane-bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. Using atomic force microscopy, CV, and fluorescence microscopy it is deduced that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and has significant potential for mimicry in biotechnology applications such as energy production or biosensing. [ABSTRACT FROM AUTHOR]- Published
- 2017
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24. CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase.
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Bürstel, Ingmar, Siebert, Elisabeth, Frielingsdorf, Stefan, Zebger, Ingo, Friedrich, Bärbel, and Lenz, Oliver
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HYDROGENASE ,IRON carbonyls ,METALLOENZYMES ,CARBON monoxide ,MICROBIAL metabolism - Abstract
Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H
2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H2 , the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry--that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H2 oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O2 , employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N10 -formyl-tetrahydrofolate (N10 -CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N10 -CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates. [ABSTRACT FROM AUTHOR]- Published
- 2016
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25. Impact of Carbon Nanotube Surface Chemistry on Hydrogen Oxidation by Membrane-Bound Oxygen-Tolerant Hydrogenases.
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Monsalve, Karen, Mazurenko, Ievgen, Gutierrez‐Sanchez, Cristina, Ilbert, Marianne, Infossi, Pascale, Frielingsdorf, Stefan, Giudici‐Orticoni, Marie Thérèse, Lenz, Oliver, and Lojou, Elisabeth
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CARBON nanotubes ,SURFACE chemistry ,HYDROGEN oxidation ,HYDROGENASE ,FUEL cell design & construction ,AQUIFEX aeolicus - Abstract
Oxygen-tolerant [NiFe] hydrogenases are attractive biocatalysts for utilization in H
2 /O2 fuel cells, which thereby reduces the amount of platinum-based catalysts. The O2 -tolerant membrane-bound hydrogenases isolated from Ralstonia eutropha and Aquifex aeolicus were previously studied at planar electrodes. The design of a powerful enzymatic fuel cell, however, requires a considerable increase in enzyme loading. Herein, we immobilized the two hydrogenases on carbon nanotubes, and we demonstrated that the enzyme binding and electron-transfer properties on the 3D networks relied on the same surface chemistry as that of the planar electrodes. We evaluated how the intrinsic properties of each hydrogenase, that is, temperature and O2 tolerance, were affected by immobilization on different electrode surfaces. The role of the detergent used for protein purification was especially emphasized. We also demonstrated that O2 reduction products affected more seriously the enzyme activity than molecular O2 . If immobilized on pyrene-modified carbon nanotubes, both enzymes were used for the first time in a mild-temperature, membraneless H2 /O2 enzymatic fuel cell, fed with O2 -rich gas mixture, opening new avenues toward the development of alternative energy supplies. [ABSTRACT FROM AUTHOR]- Published
- 2016
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26. Krypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.
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Kalms, Jacqueline, Schmidt, Andrea, Frielingsdorf, Stefan, van der Linden, Peter, von Stetten, David, Lenz, Oliver, Carpentier, Philippe, and Scheerer, Patrick
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METALLOENZYMES ,HYDROGENASE ,RENEWABLE energy sources ,KRYPTON ,NOBLE gases - Abstract
[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O
2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. [ABSTRACT FROM AUTHOR]- Published
- 2016
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27. Reactivation from the Ni–B state in [NiFe] hydrogenase of Ralstonia eutropha is controlled by reduction of the superoxidised proximal cluster.
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Radu, Valentin, Frielingsdorf, Stefan, Lenz, Oliver, and Jeuken, Lars J. C.
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HYDROGENASE , *RALSTONIA eutropha , *SUPEROXIDES , *CHARGE exchange , *METALLOENZYMES , *FUEL cells , *ELECTROCHEMISTRY - Abstract
The tolerance towards oxic conditions of O2-tolerant [NiFe] hydrogenases has been attributed to an unusual [4Fe–3S] cluster that lies proximal to the [NiFe] active site. Upon exposure to oxygen, this cluster converts to a superoxidised (5+) state, which is believed to secure the formation of the so-called Ni–B state that is rapidly reactivated under reducing conditions. Here, the reductive reactivation of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha in a native-like lipid membrane was characterised and compared to a variant that instead carries a typical [4Fe–4S] proximal cluster. Reactivation from the Ni–B state was faster in the [4Fe–4S] variant, suggesting that the reactivation rate in MBH is limited by the reduction of the superoxidised [4Fe–3S] cluster. We propose that the [4Fe–3S] cluster plays a major role in protecting MBH by blocking the reversal of electron transfer to the [NiFe] active site, which would produce damaging radical oxygen species. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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28. Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha.
- Author
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Radu,, Valentin, Frielingsdorf, Stefan, Evans, Stephen D., Lenz, Oliver, and Jeuken, Lars J. C.
- Subjects
- *
HYDROGENASE , *OXIDOREDUCTASES , *ENZYMES , *RALSTONIA eutropha , *PSEUDOMONADACEAE , *BILAYER lipid membranes , *PHOTOSYNTHETIC oxygen evolution , *CHEMICAL inhibitors - Abstract
Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membranebound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome h subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
29. Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase.
- Author
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Frielingsdorf, Stefan, Fritsch, Johannes, Schmidt, Andrea, Hammer, Mathias, Löwenstein, Julia, Siebert, Elisabeth, Pelmenschikov, Vladimir, Jaenicke, Tina, Kalms, Jacqueline, Rippers, Yvonne, Lendzian, Friedhelm, Zebger, Ingo, Teutloff, Christian, Kaupp, Martin, Bittl, Robert, Hildebrandt, Peter, Friedrich, Bärbel, Lenz, Oliver, and Scheerer, Patrick
- Subjects
- *
HYDROGENASE , *RALSTONIA , *HISTIDINE kinases , *NITROGENASES , *OXIDATION-reduction reaction , *LIGAND analysis - Abstract
Hydrogenases catalyze the reversible oxidation of H2 into protons and electrons and are usually readily inactivated by O2. However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O2 that enables their host organisms to utilize H2 as an energy source at high O2. This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H2 oxidation at high O2. We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
30. Resonance Raman Spectroscopy as a Tool to Monitor the Active Site of Hydrogenases.
- Author
-
Siebert, Elisabeth, Horch, Marius, Rippers, Yvonne, Fritsch, Johannes, Frielingsdorf, Stefan, Lenz, Oliver, Velazquez Escobar, Francisco, Siebert, Friedrich, Paasche, Lars, Kuhlmann, Uwe, Lendzian, Friedhelm, Mroginski, Maria‐Andrea, Zebger, Ingo, and Hildebrandt, Peter
- Abstract
Insights in active sites: Hydrogen‐conversion by hydrogenase is mediated by a sophisticated, metal‐containing catalytic center. Resonance Raman spectroscopy is used for the first time in the characterization of the active site of these biocatalysts. An integrated spectroscopic and computational approach gives insights into structural and photochemical properties of the active site of an oxygen‐tolerant [NiFe] hydrogenase. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
31. Resonanz-Raman-Spektroskopie als Methode zur Untersuchung des aktiven Zentrums von Hydrogenasen.
- Author
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Siebert, Elisabeth, Horch, Marius, Rippers, Yvonne, Fritsch, Johannes, Frielingsdorf, Stefan, Lenz, Oliver, Velazquez Escobar, Francisco, Siebert, Friedrich, Paasche, Lars, Kuhlmann, Uwe, Lendzian, Friedhelm, Mroginski, Maria ‐ Andrea, Zebger, Ingo, and Hildebrandt, Peter
- Abstract
Mithilfe eines optimierten Metallzentrums ermöglichen Hydrogenasen die Umsetzung von Wasserstoff. Resonanz ‐ Raman ‐ Spektroskopie wird als neue Methode zur Charakterisierung des aktiven Zentrums dieser Biokatalysatoren vorgestellt. Ein kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe] ‐ Zentrums einer sauerstofftoleranten Hydrogenase. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
32. Essential Amino Acid Residues of BioY Reveal That Dimers Are the Functional S Unit of the Rhodobacter capsulatus Biotin Transporter.
- Author
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Kirsch, Franziska, Frielingsdorf, Stefan, Pohlmann, Anne, Ziomkowska, Joanna, Herrmann, Andreas, and Eitinger, Thomas
- Subjects
- *
ESSENTIAL amino acids , *RHODOBACTER capsulatus , *BIOTIN , *PROKARYOTES , *ESCHERICHIA coli - Abstract
Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coil Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp 164 (by Asn) and Lys167 (by Arg or Gin) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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33. A Trimeric Supercomplex of the Oxygen-Tolerant Membrane-Bound [NiFe]-Hydrogenase from Ralstonia eutropha H16.
- Author
-
Frielingsdorf, Stefan, Schubert, Torsten, Pohlmann, Anne, Lenz, Oliver, and Friedrich, Bärbel
- Subjects
- *
RALSTONIA , *MEMBRANE proteins , *MEMBRANE oxygenators , *HYDROGENASE , *CARRIER proteins , *BINDING sites - Abstract
The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H2-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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- View/download PDF
34. Role of the HoxZ Subunit in the Electron Transfer Pathway of the Membrane-Bound [NiFe]-Hydrogenase from Ralstonia eutropha Immobilized on Electrodes.
- Author
-
Sezer, Murat, Frielingsdorf, Stefan, Millo, Diego, Heidary, Nina, Utesch, Tillman, Mroginski, Maria-Andrea, Friedrich, Bärbel, Hildebrandt, Peter, Zebger, Ingo, and Weidinger, Inez M.
- Subjects
- *
CYTOCHROMES , *CHARGE exchange , *ELECTRODES , *SPECTRUM analysis , *RAMAN spectroscopy - Abstract
The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofoctors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H2 addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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- View/download PDF
35. A Stromal Pool of TatA Promotes Tat-dependent Protein Transport across the Thylakoid Membrane.
- Author
-
Frielingsdorf, Stefan, Jakob, Mario, and Klösgen, Ralf Bernd
- Subjects
- *
THYLAKOIDS , *CHLOROPLASTS , *CHROMOSOMAL translocation , *CELL membranes , *MEMBRANE proteins , *BIOLOGICAL transport - Abstract
In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transportin-competent due to extraction with solutions of chaotropic salts. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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- View/download PDF
36. Prerequisites for Terminal Processing of Thylakoidal Tat Substrates.
- Author
-
Frielingsdorf, Stefan and Bernd Klosgen, Ralf
- Subjects
- *
PROTEOLYTIC enzymes , *BIOLOGICAL transport , *PEPTIDES , *ARGININE , *CHLOROPLASTS , *AMINO acids - Abstract
In bacteria and chloroplasts, the Tat (twin arginine translocation) system is capable of translocating folded passenger proteins across the cytoplasmic and thylakoidal membranes, respectively. Transport depends on signal peptides that are characterized by a twin pair of arginine residues. The signal peptides are generally removed after transport by specific processing peptidases, namely the leader peptidase and the thylakoidal processing peptidase. To gain insight into the prerequisites for such signal peptide removal, we mutagenized the vicinity of thylakoidal processing peptidase cleavage sites in several thylakoidal Tat substrates. Analysis of these mutants in thylakoid transport experiments showed that the amino acid composition of both the C-terminal segment of the signal peptide and the N-terminal part of the mature protein plays an important role in the maturation process. Efficient removal of the signal peptide requires the presence of charged or polar residues within at least one of those regions, whereas increased hydrophobicity impairs the process. The relative extent of this effect varies to some degree depending on the nature of the precursor protein. Unprocessed transport intermediates with fully translocated passenger proteins are found in membrane complexes of high molecular mass, which presumably represent Tat complexes, as well as free in the lipid bilayer. This seems to indicate that the Tat substrates can be laterally released from the complexes prior to processing and that membrane transport and terminal processing of Tat substrates are independent processes. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
37. Unassisted Membrane Insertion as the Initial Step in ΔpH/Tat-dependent Protein Transport
- Author
-
Hou, Bo, Frielingsdorf, Stefan, and Klösgen, Ralf Bernd
- Subjects
- *
DNA insertion elements , *CHLOROPLASTS , *CELL membranes , *FUNGUS-bacterium relationships - Abstract
In the thylakoid membrane of chloroplasts as well as in the cytoplasmic membrane of bacteria, the ΔpH/Tat-dependent protein transport pathway is responsible for the translocation of folded proteins. Using the chimeric 16/23 protein as model substrate in thylakoid transport experiments, we dissected the transport process into several distinct steps that are characterized by specific integral translocation intermediates. Formation of the early translocation intermediate Ti-1, which still exposes the N and the C terminus to the stroma, is observed with thylakoids pretreated with (i) solutions of chaotropic salts or alkaline pH, (ii) protease, or (iii) antibodies raised against TatA, TatB, or TatC. Membrane insertion takes place even into liposomes, demonstrating that proteinaceous components are not required. This suggests that Tat-dependent transport may be initiated by the unassisted insertion of the substrate into the lipid bilayer, and that interaction with the Tat translocase takes place only in later stages of the process. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
- View/download PDF
38. Frontispiz: Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen – veranschaulicht anhand von [NiFe]‐Hydrogenasen.
- Author
-
Lorent, Christian, Pelmenschikov, Vladimir, Frielingsdorf, Stefan, Schoknecht, Janna, Caserta, Giorgio, Yoda, Yoshitaka, Wang, Hongxin, Tamasaku, Kenji, Lenz, Oliver, Cramer, Stephen P., Horch, Marius, Lauterbach, Lars, and Zebger, Ingo
- Subjects
CHRISTIANS - Abstract
Redox-Intermediate von [NiFe]-Hydrogenasen durch einen experimentellen Ansatz für solvatisierte, lyophilisierte und kristallisierte Metalloenzyme. Frontispiz: Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen - veranschaulicht anhand von [NiFe]-Hydrogenasen Keywords: [NiFe]-Hydrogenasen; Biokatalyse; In-situ-Spektroskopie; Metalloenzyme; Schwingungsspektroskopie DE [NiFe]-Hydrogenasen Biokatalyse In-situ-Spektroskopie Metalloenzyme Schwingungsspektroskopie 1 1 1 07/10/21 20210712 NES 210712 B Metalloenzyme b Im Forschungsartikel auf S. 15988 erforschen Christian Lorent, Marius Horch, Lars Lauterbach, Ingo Zebger et al. [Extracted from the article]
- Published
- 2021
- Full Text
- View/download PDF
39. Frontispiece: Exploring Structure and Function of Redox Intermediates in [NiFe]‐Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.
- Author
-
Lorent, Christian, Pelmenschikov, Vladimir, Frielingsdorf, Stefan, Schoknecht, Janna, Caserta, Giorgio, Yoda, Yoshitaka, Wang, Hongxin, Tamasaku, Kenji, Lenz, Oliver, Cramer, Stephen P., Horch, Marius, Lauterbach, Lars, and Zebger, Ingo
- Subjects
METALLOENZYMES ,OXIDATION-reduction reaction ,BIOCATALYSIS ,SOLVATION - Abstract
Keywords: [NiFe]-hydrogenase; biocatalysis; in situ spectroscopy; metalloenzymes; vibrational spectroscopy EN [NiFe]-hydrogenase biocatalysis in situ spectroscopy metalloenzymes vibrational spectroscopy 1 1 1 07/10/21 20210712 NES 210712 B Metalloenzymes b In their Research Article on page 15854, Christian Lorent, Marius Horch, Lars Lauterbach, Ingo Zebger et al. explore redox intermediates of [NiFe]-hydrogenases by an advanced experimental approach for solvated, lyophilized, and crystallized metalloenzymes. Frontispiece: Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes [NiFe]-hydrogenase, biocatalysis, in situ spectroscopy, metalloenzymes, vibrational spectroscopy. [Extracted from the article]
- Published
- 2021
- Full Text
- View/download PDF
40. Biomimetics: Multilayered Lipid Membrane Stacks for Biocatalysis Using Membrane Enzymes (Adv. Funct. Mater. 17/2017).
- Author
-
Heath, George R., Li, Mengqiu, Rong, Honling, Radu, Valentin, Frielingsdorf, Stefan, Lenz, Oliver, Butt, Julea N., and Jeuken, Lars J. C.
- Subjects
BILAYER lipid membranes ,PROTEINS ,BIOMIMETIC materials - Abstract
In article number 1606265 Lars J. C. Jeuken and co‐workers use a layer‐by‐layer assembly of lipid bilayers to multiply the surface concentration of electroactive membrane enzymes at electrodes. The interconnected membrane multilayers, akin to those of thylakoid membranes, create a material that exhibits a linear increase in bioelectrocatalytic activity with each additional enzyme‐containing membrane layer (containing either ubiquinol oxidase or an oxygen‐tolerant hydrogenase). [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
41. Back Cover: Resonance Raman Spectroscopy as a Tool to Monitor the Active Site of Hydrogenases (Angew. Chem. Int. Ed. 19/2013).
- Author
-
Siebert, Elisabeth, Horch, Marius, Rippers, Yvonne, Fritsch, Johannes, Frielingsdorf, Stefan, Lenz, Oliver, Velazquez Escobar, Francisco, Siebert, Friedrich, Paasche, Lars, Kuhlmann, Uwe, Lendzian, Friedhelm, Mroginski, Maria‐Andrea, Zebger, Ingo, and Hildebrandt, Peter
- Abstract
The biological conversion of hydrogen is catalyzed by [NiFe] hydrogenases utilizing a tailored bimetallic center. In their Communication on page 5162 ff. P. Hildebrandt, I. Zebger, M. Horch, and co‐workers use resonance Raman spectroscopy for the first time to characterize this active site by directly probing Fe–CO/CN vibrational modes. Applying an integrated spectroscopic and computational approach, this method provides new insights into structural and photochemical aspects of the [NiFe] site. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
42. Rücktitelbild: Resonanz-Raman-Spektroskopie als Methode zur Untersuchung des aktiven Zentrums von Hydrogenasen (Angew. Chem. 19/2013).
- Author
-
Siebert, Elisabeth, Horch, Marius, Rippers, Yvonne, Fritsch, Johannes, Frielingsdorf, Stefan, Lenz, Oliver, Velazquez Escobar, Francisco, Siebert, Friedrich, Paasche, Lars, Kuhlmann, Uwe, Lendzian, Friedhelm, Mroginski, Maria ‐ Andrea, Zebger, Ingo, and Hildebrandt, Peter
- Abstract
[NiFe] ‐ Hydrogenasen katalysieren die biologische Umwandlung von Wasserstoff mithilfe eines maßgeschneiderten bimetallischen Zentrums. In ihrer Zuschrift auf S. 5267 ff. nutzen P. Hildebrandt, I. Zebger, M. Horch et al. die Resonanz ‐ Raman ‐ Spektroskopie als neue Charakterisierungstechnik für dieses aktive Zentrum, die eine Möglichkeit zur direkten Beobachtung der Fe ‐ CO/CN ‐ Schwingungsmoden bietet. Ihr kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe] ‐ Zentrums. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
43. Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts
- Author
-
Gutensohn, Michael, Fan, Enguo, Frielingsdorf, Stefan, Hanner, Peter, Hou, Bo, Hust, Bianca, and Klösgen, Ralf Bernd
- Subjects
- *
CHLOROPLASTS , *THYLAKOIDS , *PROTEINS , *ORGANELLES - Abstract
Summary: The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the ‘export’ of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
- View/download PDF
44. A Strep-tag Imprinted Polymer Platform for Heterogenous Bio(electro)catalysis.
- Author
-
Yarman A, Waffo AFT, Katz S, Bernitzky CCM, Kovács N, Borrero P, Frielingsdorf S, Supala E, Dragelj J, Kurbanoglu S, Neumann B, Lenz O, Gyurcsányi RE, Mroginski MA, Wollenberger U, Scheller FW, Caserta G, and Zebger I
- Abstract
Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising-strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via the widely used Strep-tag II affinity peptide. Electrochemistry, surface-sensitive spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tag labeled enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology., (© 2024 Wiley‐VCH GmbH.)
- Published
- 2024
- Full Text
- View/download PDF
45. Stepwise conversion of the Cys 6 [4Fe-3S] to a Cys 4 [4Fe-4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase.
- Author
-
Schmidt A, Kalms J, Lorent C, Katz S, Frielingsdorf S, Evans RM, Fritsch J, Siebert E, Teutloff C, Armstrong FA, Zebger I, Lenz O, and Scheerer P
- Abstract
The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O
2 -tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e- and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6 [4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5 [4Fe-4S] or Cys4 [4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2 O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions., Competing Interests: The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P. S. (patrick.scheerer@charite.de), O. L. (oliver.lenz@tu-berlin.de), and I. Z. (ingo.zebger@tu-berlin.de)., (This journal is © The Royal Society of Chemistry.)- Published
- 2023
- Full Text
- View/download PDF
46. Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.
- Author
-
Lorent C, Pelmenschikov V, Frielingsdorf S, Schoknecht J, Caserta G, Yoda Y, Wang H, Tamasaku K, Lenz O, Cramer SP, Horch M, Lauterbach L, and Zebger I
- Subjects
- Freeze Drying, Crystallography, X-Ray, Density Functional Theory, Spectrum Analysis, Raman, Models, Molecular, Hydrogenase chemistry, Hydrogenase metabolism, Oxidation-Reduction
- Abstract
To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O
2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)- Published
- 2021
- Full Text
- View/download PDF
47. Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O 2 -Tolerant [NiFe] Hydrogenase.
- Author
-
Schulz AC, Frielingsdorf S, Pommerening P, Lauterbach L, Bistoni G, Neese F, Oestreich M, and Lenz O
- Subjects
- Ligands, Carbon Monoxide chemistry, Enzymes chemistry, Formyltetrahydrofolates chemistry, Hydrogenase chemistry, Oxygen chemistry
- Abstract
[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN
- ) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN)2 (CO) cofactor requires the activity of at least six maturation proteins, designated HypA-F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N10 -formyltetrahydrofolate ( N10 -formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N10 -formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro evidence that purified HypX first transfers the formyl group of N10 -formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.- Published
- 2020
- Full Text
- View/download PDF
48. O 2 -Tolerant H 2 Activation by an Isolated Large Subunit of a [NiFe] Hydrogenase.
- Author
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Hartmann S, Frielingsdorf S, Ciaccafava A, Lorent C, Fritsch J, Siebert E, Priebe J, Haumann M, Zebger I, and Lenz O
- Subjects
- Catalysis, Catalytic Domain, Oxidation-Reduction, Protein Subunits, Bacterial Proteins metabolism, Cupriavidus necator enzymology, Hydrogen metabolism, Hydrogenase metabolism, Oxygen metabolism
- Abstract
The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H
2 ⇌ 2H+ + 2e- . Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H2 -activating [NiFe] site and the small subunit contains iron-sulfur clusters mediating e- transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O2 . Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O2 -tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H2 as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H2 activation was performed by preHoxG even in the presence of O2 , although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O2 was absent. These findings challenge the current understanding of O2 tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.- Published
- 2018
- Full Text
- View/download PDF
49. In Situ Spectroelectrochemical Studies into the Formation and Stability of Robust Diazonium-Derived Interfaces on Gold Electrodes for the Immobilization of an Oxygen-Tolerant Hydrogenase.
- Author
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Harris TGAA, Heidary N, Kozuch J, Frielingsdorf S, Lenz O, Mroginski MA, Hildebrandt P, Zebger I, and Fischer A
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins metabolism, Cupriavidus necator enzymology, Diazonium Compounds chemistry, Electrodes, Enzymes, Immobilized metabolism, Hydrogenase metabolism, Surface Properties, Electrochemical Techniques methods, Enzymes, Immobilized chemistry, Gold chemistry, Hydrogenase chemistry, Spectrum Analysis methods
- Abstract
Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the "Knallgas" bacterium Ralstonia eutropha by relating its electrochemical activity for hydrogen oxidation to the interface properties.
- Published
- 2018
- Full Text
- View/download PDF
50. Tracking the route of molecular oxygen in O 2 -tolerant membrane-bound [NiFe] hydrogenase.
- Author
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Kalms J, Schmidt A, Frielingsdorf S, Utesch T, Gotthard G, von Stetten D, van der Linden P, Royant A, Mroginski MA, Carpentier P, Lenz O, and Scheerer P
- Subjects
- Bacterial Proteins genetics, Binding Sites, Catalytic Domain, Cell Membrane chemistry, Cell Membrane genetics, Crystallography, X-Ray, Cupriavidus necator chemistry, Cupriavidus necator genetics, Hydrogenase genetics, Hydrophobic and Hydrophilic Interactions, Oxygen chemistry, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Cell Membrane enzymology, Cupriavidus necator enzymology, Hydrogenase chemistry, Hydrogenase metabolism, Oxygen metabolism
- Abstract
[NiFe] hydrogenases catalyze the reversible splitting of H
2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2 , a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha , is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2 -derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)- Published
- 2018
- Full Text
- View/download PDF
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