27 results on '"Frielingsdorf, Stefan"'
Search Results
2. Stepwise assembly of the active site of [NiFe]-hydrogenase.
- Author
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Caserta G, Hartmann S, Van Stappen C, Karafoulidi-Retsou C, Lorent C, Yelin S, Keck M, Schoknecht J, Sergueev I, Yoda Y, Hildebrandt P, Limberg C, DeBeer S, Zebger I, Frielingsdorf S, and Lenz O
- Subjects
- Catalytic Domain, Oxidation-Reduction, Nickel, Hydrogenase chemistry, Hydrogenase metabolism, Cupriavidus necator chemistry, Cupriavidus necator metabolism
- Abstract
[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H
2 into 2e- and 2H+ 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. Here, 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 O2 -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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)- Published
- 2023
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3. Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.
- Author
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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
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4. A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation.
- Author
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Hartmann S, Frielingsdorf S, Caserta G, and Lenz O
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- Carbon Monoxide chemistry, Catalytic Domain genetics, Cyanides chemistry, Escherichia coli genetics, Hydrogen chemistry, Iron chemistry, Membrane Proteins genetics, Membrane Proteins metabolism, Nickel chemistry, Plasmids genetics, Protein Subunits genetics, Protein Subunits metabolism, Cupriavidus necator genetics, Cupriavidus necator metabolism, Genetic Engineering methods, Hydrogenase genetics, Hydrogenase metabolism
- 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., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)- Published
- 2020
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5. Formyltetrahydrofolate Decarbonylase Synthesizes the Active Site CO Ligand of O 2 -Tolerant [NiFe] Hydrogenase.
- Author
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Schulz AC, Frielingsdorf S, Pommerening P, Lauterbach L, Bistoni G, Neese F, Oestreich M, and Lenz O
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- 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
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6. 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
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7. 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
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- 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
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8. Tracking the route of molecular oxygen in O 2 -tolerant membrane-bound [NiFe] hydrogenase.
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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
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9. O 2 -tolerant [NiFe]-hydrogenases of Ralstonia eutropha H16: Physiology, molecular biology, purification, and biochemical analysis.
- Author
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Lenz O, Lauterbach L, and Frielingsdorf S
- Subjects
- Catalysis, Chromatography, Gas, Hydrogen metabolism, Oxidation-Reduction, Plasmids genetics, Cupriavidus necator enzymology, Cupriavidus necator metabolism, Hydrogenase metabolism, Oxygen metabolism
- Abstract
Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H
2 ⇌2H+ +2e- even in the presence of O2 . Catalytic and probably also noncatalytic mechanisms protect their active sites from being inactivated by reactive oxygen species, which makes them attractive subjects of investigation from both fundamental and applied perspectives. Prominent representatives of the O2 -tolerant [NiFe]-hydrogenases have been isolated from the chemolithoautotrophic model organism Ralstonia eutropha H16, which can thrive in a simple mineral medium supplemented with the gases H2 , O2 , and CO2 . In this chapter, we describe methods for cultivation and genetic manipulation of R. eutropha, both of which are prerequisites for the reproducible manufacturing of high-quality hydrogenase preparations. The purification procedures for two different O2 -tolerant [NiFe]-hydrogenases from R. eutropha are described in detail, as well as the corresponding biochemical procedures used for the determination of the catalytic properties of these sophisticated enzymes., (© 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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10. CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase.
- Author
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Bürstel I, Siebert E, Frielingsdorf S, Zebger I, Friedrich B, and Lenz O
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- Adenosine Diphosphate chemistry, Carbon metabolism, Catalysis, Catalytic Domain, Cupriavidus necator, DNA Primers, Gene Deletion, Glycine chemistry, Hydrogen metabolism, Iron metabolism, Ligands, Mutagenesis, Site-Directed, Mutation, Time Factors, Carbon chemistry, Carbon Monoxide chemistry, Hydrogenase 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., Competing Interests: The authors declare no conflict of interest.- Published
- 2016
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11. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.
- Author
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Kalms J, Schmidt A, Frielingsdorf S, van der Linden P, von Stetten D, Lenz O, Carpentier P, and Scheerer P
- Subjects
- Catalytic Domain, Crystallography, X-Ray, Cupriavidus necator chemistry, Cupriavidus necator metabolism, Hydrogenase metabolism, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Oxidation-Reduction, Oxygen metabolism, Protein Conformation, Cupriavidus necator enzymology, Hydrogenase chemistry
- 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 O2 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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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12. 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
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Siebert E, Rippers Y, Frielingsdorf S, Fritsch J, Schmidt A, Kalms J, Katz S, Lenz O, Scheerer P, Paasche L, Pelmenschikov V, Kuhlmann U, Mroginski MA, Zebger I, and Hildebrandt P
- Subjects
- Crystallization, Cupriavidus necator enzymology, Membrane Proteins chemistry, Models, Molecular, Oxygen chemistry, Quantum Theory, Spectrum Analysis, Raman, Catalytic Domain, Hydrogenase chemistry, Hydrogenase metabolism, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins metabolism, Membrane Proteins metabolism, Oxygen metabolism
- Abstract
We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.
- Published
- 2015
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13. Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha.
- Author
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Radu V, Frielingsdorf S, Evans SD, Lenz O, and Jeuken LJ
- Subjects
- Electrodes, Lipid Bilayers chemistry, Molecular Structure, Oxygen chemistry, Cupriavidus necator enzymology, Hydrogenase metabolism, Lipid Bilayers metabolism, Oxygen metabolism
- Abstract
Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [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 b 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.
- Published
- 2014
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14. Reversible [4Fe-3S] cluster morphing in an O(2)-tolerant [NiFe] hydrogenase.
- Author
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Frielingsdorf S, Fritsch J, Schmidt A, Hammer M, Löwenstein J, Siebert E, Pelmenschikov V, Jaenicke T, Kalms J, Rippers Y, Lendzian F, Zebger I, Teutloff C, Kaupp M, Bittl R, Hildebrandt P, Friedrich B, Lenz O, and Scheerer P
- Subjects
- Catalysis, Hydrogen metabolism, Ligands, Models, Molecular, Oxidation-Reduction, Hydrogenase metabolism, Iron-Sulfur Proteins metabolism, Oxygen metabolism
- Abstract
Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). 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 H(2) oxidation at high O(2). 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.
- Published
- 2014
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15. Resonance Raman spectroscopy as a tool to monitor the active site of hydrogenases.
- Author
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Siebert E, Horch M, Rippers Y, Fritsch J, Frielingsdorf S, Lenz O, Velazquez Escobar F, Siebert F, Paasche L, Kuhlmann U, Lendzian F, Mroginski MA, Zebger I, and Hildebrandt P
- Subjects
- Catalysis, Catalytic Domain, Hydrogen metabolism, Hydrogenase metabolism, Models, Chemical, Cupriavidus necator enzymology, Hydrogen chemistry, Hydrogenase chemistry, Photochemistry, Spectrum Analysis, Raman
- Published
- 2013
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16. A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16.
- Author
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Frielingsdorf S, Schubert T, Pohlmann A, Lenz O, and Friedrich B
- Subjects
- Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Cardiolipins metabolism, Cupriavidus necator metabolism, Cytochrome b Group chemistry, Cytochrome b Group genetics, Cytochrome b Group metabolism, Digitonin chemistry, Enzyme Stability, Hydrogenase chemistry, Hydrogenase genetics, Hydrogenase metabolism, Models, Molecular, Molecular Weight, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Multiprotein Complexes isolation & purification, Multiprotein Complexes metabolism, Oxidation-Reduction, Phosphatidylethanolamines metabolism, Phosphatidylglycerols metabolism, Protein Multimerization, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Surface-Active Agents chemistry, Bacterial Outer Membrane Proteins isolation & purification, Cupriavidus necator enzymology, Cytochrome b Group isolation & purification, Hydrogenase isolation & purification, Protein Subunits isolation & purification
- 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. H(2)-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.
- Published
- 2011
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17. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre.
- Author
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Fritsch J, Scheerer P, Frielingsdorf S, Kroschinsky S, Friedrich B, Lenz O, and Spahn CM
- Subjects
- Catalytic Domain, Cell Membrane metabolism, Crystallography, X-Ray, Cysteine metabolism, Hydrogenase metabolism, Iron analysis, Iron-Sulfur Proteins metabolism, Models, Molecular, Oxidation-Reduction, Protein Multimerization, Protein Structure, Quaternary, Protein Subunits chemistry, Protein Subunits metabolism, Protons, Sulfur analysis, Water chemistry, Water metabolism, Cupriavidus necator enzymology, Hydrogenase chemistry, Iron chemistry, Iron-Sulfur Proteins chemistry, Oxygen metabolism, Sulfur chemistry
- Abstract
Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(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(2) oxidation and as an electron-delivering device upon O(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. 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(2)-converting catalysts that are capable of cycling H(2) in air.
- Published
- 2011
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18. Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.
- Author
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Sezer M, Frielingsdorf S, Millo D, Heidary N, Utesch T, Mroginski MA, Friedrich B, Hildebrandt P, Zebger I, and Weidinger IM
- Subjects
- Biocatalysis, Cytochromes b chemistry, Electrodes, Electron Transport, Enzymes, Immobilized chemistry, Hydrogenase chemistry, Models, Molecular, Protein Multimerization, Protein Structure, Quaternary, Protein Subunits chemistry, Spectrum Analysis, Raman, Surface Properties, Cell Membrane metabolism, Cupriavidus necator enzymology, Cytochromes b metabolism, Enzymes, Immobilized metabolism, Hydrogenase metabolism, Protein Subunits metabolism
- 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 cofactors 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 H(2) 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., (© 2011 American Chemical Society)
- Published
- 2011
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19. Editorial: Hydrogenase: structure, function, maturation, and application.
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Frielingsdorf, Stefan, Pinske, Constanze, Valetti, Francesca, and Greening, Chris
- Subjects
HYDROGENASE ,BIOTECHNOLOGY ,METALS - Published
- 2023
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20. Resonance Raman spectroscopic analysis of the iron–sulfur cluster redox chain of the Ralstonia eutropha membrane‐bound [NiFe]‐hydrogenase.
- Author
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Siebert, Elisabeth, Schmidt, Andrea, Frielingsdorf, Stefan, Kalms, Jacqueline, Kuhlmann, Uwe, Lenz, Oliver, Scheerer, Patrick, Zebger, Ingo, and Hildebrandt, Peter
- Subjects
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]
- Published
- 2021
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21. 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
- Subjects
HYDROGENASE - Abstract
Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Published
- 2021
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22. Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.
- Author
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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
- Subjects
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. CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase.
- Author
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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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24. 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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25. Krypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.
- Author
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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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26. Reactivation from the Ni–B state in [NiFe] hydrogenase of Ralstonia eutropha is controlled by reduction of the superoxidised proximal cluster.
- Author
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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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27. Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase.
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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
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