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2. Stepwise assembly of the active site of [NiFe]-hydrogenase

Author

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

Published
2023
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11. Stepwise conversion of the Cys6[4Fe-3S] to a Cys4[4Fe-4S] cluster and its impact on oxygen tolerance of [NiFe]-hydrogenase

Author

Schmidt, Andrea, primary, Kalms, Jaqueline, additional, Lorent, Christian, additional, Katz, Sagie, additional, Frielingsdorf, Stefan, additional, Evans, Rhiannon Mari, additional, Fritsch, Johannes, additional, Siebert, Elisabeth, additional, Teutloff, Christian, additional, Armstrong, Fraser A, additional, Zebger, Ingo, additional, Lenz, Oliver, additional, and Scheerer, Patrick, additional

Published
2023
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14. Active site assembly of [NiFe]-hydrogenase scrutinized on the basis of purified maturation intermediates

Author

Caserta, Giorgio, primary, Hartmann, Sven, additional, van Stappen, Casey, additional, Karafoulidi Retsou, Chara, additional, Lorent, Christian, additional, Yelin, Stefan, additional, Keck, Matthias, additional, Schoknecht, Janna, additional, Sergueev, Ilya, additional, Yoda, Yoshitaka, additional, Hildebrandt, Peter, additional, Limberg, Christian, additional, DeBeer, Serena, additional, Zebger, Ingo, additional, Frielingsdorf, Stefan, additional, and Lenz, Oliver, additional

Published
2022
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15. Active site assembly of [NiFe]-hydrogenase scrutinized on the basis of purified maturation intermediates

Author

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.

Published
2022
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16. A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

Author

Hartmann, Sven, Frielingsdorf, Stefan, Caserta, Giorgio, and Lenz, Oliver

Subjects
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.

Published
2020

18. 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, 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.

Published
2021
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19. 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, primary, Pelmenschikov, Vladimir, additional, Frielingsdorf, Stefan, additional, Schoknecht, Janna, additional, Caserta, Giorgio, additional, Yoda, Yoshitaka, additional, Wang, Hongxin, additional, Tamasaku, Kenji, additional, Lenz, Oliver, additional, Cramer, Stephen P., additional, Horch, Marius, additional, Lauterbach, Lars, additional, and Zebger, Ingo, additional

Published
2021
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20. Frontispiz: Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen – veranschaulicht anhand von [NiFe]‐Hydrogenasen

Author

Lorent, Christian, primary, Pelmenschikov, Vladimir, additional, Frielingsdorf, Stefan, additional, Schoknecht, Janna, additional, Caserta, Giorgio, additional, Yoda, Yoshitaka, additional, Wang, Hongxin, additional, Tamasaku, Kenji, additional, Lenz, Oliver, additional, Cramer, Stephen P., additional, Horch, Marius, additional, Lauterbach, Lars, additional, and Zebger, Ingo, additional

Published
2021
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21. Electrografted Interfaces on Metal Oxide Electrodes for Enzyme Immobilization and Bioelectrocatalysis

Author

Harris, Tomos G. A. A., Heidary, Nina, Frielingsdorf, Stefan, Rauwerdink, Sander, Tahraoui, Abbes, Lenz, Oliver, Zebger, Ingo, and Fischer, Anna

Subjects
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.

Published
2021

23. Exploring Structure and Function of Redox Intermediates in [NiFe]‐Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes

Author

Lorent, Christian, primary, Pelmenschikov, Vladimir, additional, Frielingsdorf, Stefan, additional, Schoknecht, Janna, additional, Caserta, Giorgio, additional, Yoda, Yoshitaka, additional, Wang, Hongxin, additional, Tamasaku, Kenji, additional, Lenz, Oliver, additional, Cramer, Stephen P., additional, Horch, Marius, additional, Lauterbach, Lars, additional, and Zebger, Ingo, additional

Published
2021
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24. Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen – veranschaulicht anhand von [NiFe]‐Hydrogenasen

Author

Lorent, Christian, primary, Pelmenschikov, Vladimir, additional, Frielingsdorf, Stefan, additional, Schoknecht, Janna, additional, Caserta, Giorgio, additional, Yoda, Yoshitaka, additional, Wang, Hongxin, additional, Tamasaku, Kenji, additional, Lenz, Oliver, additional, Cramer, Stephen P., additional, Horch, Marius, additional, Lauterbach, Lars, additional, and Zebger, Ingo, additional

Published
2021
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25. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre

Author

Fritsch, Johannes, Scheerer, Patrick, Frielingsdorf, Stefan, Kroschinsky, Sebastian, Friedrich, Barbel, Lenz, Oliver, and Spahn, Christian M.T.

Subjects
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 [...]

Published
2011
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29. Photorespiration pathways in a chemolithoautotroph

Author

Claassens, Nico J., primary, Scarinci, Giovanni, additional, Fischer, Axel, additional, Flamholz, Avi I., additional, Newell, William, additional, Frielingsdorf, Stefan, additional, Lenz, Oliver, additional, and Bar-Even, Arren, additional

Published
2020
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32. Double-flow focused liquid injector for efficient serial femtosecond crystallography

Author

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

Subjects
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

Published
2017

33. Corrigendum: Double-flow focused liquid injector for efficient serial femtosecond crystallography

Author

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

Subjects
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

Published
2017
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35. 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

Harris, Tomos G. A. A., primary, Heidary, Nina, additional, Kozuch, Jacek, additional, Frielingsdorf, Stefan, additional, Lenz, Oliver, additional, Mroginski, Maria-Andrea, additional, Hildebrandt, Peter, additional, Zebger, Ingo, additional, and Fischer, Anna, additional

Published
2018
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37. Tracking the route of molecular oxygen in O 2 -tolerant membrane-bound [NiFe] hydrogenase

Author

Kalms, Jacqueline, primary, Schmidt, Andrea, additional, Frielingsdorf, Stefan, additional, Utesch, Tillmann, additional, Gotthard, Guillaume, additional, von Stetten, David, additional, van der Linden, Peter, additional, Royant, Antoine, additional, Mroginski, Maria Andrea, additional, Carpentier, Philippe, additional, Lenz, Oliver, additional, and Scheerer, Patrick, additional

Published
2018
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38. Correction: Corrigendum: Double-flow focused liquid injector for efficient serial femtosecond crystallography

Author

Oberthuer, Dominik, primary, Knoška, Juraj, additional, Wiedorn, Max O., additional, Beyerlein, Kenneth R., additional, Bushnell, David A., additional, Kovaleva, Elena G., additional, Heymann, Michael, additional, Gumprecht, Lars, additional, Kirian, Richard A., additional, Barty, Anton, additional, Mariani, Valerio, additional, Tolstikova, Aleksandra, additional, Adriano, Luigi, additional, Awel, Salah, additional, Barthelmess, Miriam, additional, Dörner, Katerina, additional, Xavier, P. Lourdu, additional, Yefanov, Oleksandr, additional, James, Daniel R., additional, Nelson, Garrett, additional, Wang, Dingjie, additional, Calvey, George, additional, Chen, Yujie, additional, Schmidt, Andrea, additional, Szczepek, Michael, additional, Frielingsdorf, Stefan, additional, Lenz, Oliver, additional, Snell, Edward, additional, Robinson, Philip J., additional, Šarler, Božidar, additional, Belšak, Grega, additional, Maček, Marjan, additional, Wilde, Fabian, additional, Aquila, Andrew, additional, Boutet, Sébastien, additional, Liang, Mengning, additional, Hunter, Mark S., additional, Scheerer, Patrick, additional, Lipscomb, John D., additional, Weierstall, Uwe, additional, Kornberg, Roger D., additional, Spence, John C. H., additional, Pollack, Lois, additional, Chapman, Henry N., additional, and Bajt, Saša, additional

Published
2017
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42. Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O2-Tolerant [NiFe] Hydrogenase

Author

Schulz, Anne-Christine, Frielingsdorf, Stefan, Pommerening, Phillip, Lauterbach, Lars, Bistoni, Giovanni, Neese, Frank, Oestreich, Martin, and Lenz, Oliver

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 vivodata 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 vitroevidence 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
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44. Ein Netzwerk aus hydrophoben Tunneln zum Transport gasförmiger Reaktanten in einer O2‐toleranten, membrangebundenen [NiFe]‐ Hydrogenase, aufgedeckt durch Derivatisierung mit Krypton

Author

Kalms, Jacqueline, primary, Schmidt, Andrea, additional, Frielingsdorf, Stefan, additional, van der Linden, Peter, additional, von Stetten, David, additional, Lenz, Oliver, additional, Carpentier, Philippe, additional, and Scheerer, Patrick, additional

Published
2016
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48. Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.

Author

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

Subjects
HYDROGENASE, RALSTONIA eutropha, CATALYTIC reduction, METALLOPROTEINS, X-ray crystallography
Abstract

[NiFe] hydrogenases catalyze the reversible splitting of H2 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 O2tolerance 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 O2molecules 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 O2tolerance of the enzyme. [ABSTRACT FROM AUTHOR]

Published
2018
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49. Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State

Author

Siebert, Elisabeth, primary, Rippers, Yvonne, additional, Frielingsdorf, Stefan, additional, Fritsch, Johannes, additional, Schmidt, Andrea, additional, Kalms, Jacqueline, additional, Katz, Sagie, additional, Lenz, Oliver, additional, Scheerer, Patrick, additional, Paasche, Lars, additional, Pelmenschikov, Vladimir, additional, Kuhlmann, Uwe, additional, Mroginski, Maria Andrea, additional, Zebger, Ingo, additional, and Hildebrandt, Peter, additional

Published
2015
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50. CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase.

Author

Bürstel, Ingmar, Siebert, Elisabeth, Frielingsdorf, Stefan, Zebger, Ingo, Friedrich, Bärbel, and Lenz, Oliver

Subjects
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. H2 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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