91 results on '"Horch, Marius"'
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2. Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values
- Author
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Wilkening, Svea, Schmitt, Franz-Josef, Horch, Marius, Zebger, Ingo, Lenz, Oliver, and Friedrich, Thomas
- Published
- 2017
- Full Text
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3. Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment.
- Author
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Wrathall, Solomon L. D., Procacci, Barbara, Horch, Marius, Saxton, Emily, Furlan, Chris, Walton, Julia, Rippers, Yvonne, Blaza, James N., Greetham, Gregory M., Towrie, Michael, Parker, Anthony W., Lynam, Jason, Parkin, Alison, and Hunt, Neil T.
- Abstract
Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)
2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir -SI/II , that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2 ] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation. [ABSTRACT FROM AUTHOR]- Published
- 2022
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4. 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
- 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.
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- 2021
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5. Understanding 2D-IR Spectra of Hydrogenases: A Descriptive and Predictive Computational Study.
- Author
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Rippers, Yvonne, Procacci, Barbara, Hunt, Neil T., and Horch, Marius
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HYDROGENASE ,POTENTIAL energy surfaces ,METALLOENZYMES ,ENZYMES ,ENERGY transfer ,DIATOMIC molecules - Abstract
[NiFe] hydrogenases are metalloenzymes that catalyze the reversible cleavage of dihydrogen ( H 2 ), a clean future fuel. Understanding the mechanism of these biocatalysts requires spectroscopic techniques that yield insights into the structure and dynamics of the [NiFe] active site. Due to the presence of CO and CN − ligands at this cofactor, infrared (IR) spectroscopy represents an ideal technique for studying these aspects, but molecular information from linear IR absorption experiments is limited. More detailed insights can be obtained from ultrafast nonlinear IR techniques like IR
pump -IRprobe and two-dimensional (2D-)IR spectroscopy. However, fully exploiting these advanced techniques requires an in-depth understanding of experimental observables and the encoded molecular information. To address this challenge, we present a descriptive and predictive computational approach for the simulation and analysis of static 2D-IR spectra of [NiFe] hydrogenases and similar organometallic systems. Accurate reproduction of experimental spectra from a first-coordination-sphere model suggests a decisive role of the [NiFe] core in shaping the enzymatic potential energy surface. We also reveal spectrally encoded molecular information that is not accessible by experiments, thereby helping to understand the catalytic role of the diatomic ligands, structural differences between [NiFe] intermediates, and possible energy transfer mechanisms. Our studies demonstrate the feasibility and benefits of computational spectroscopy in the 2D-IR investigation of hydrogenases, thereby further strengthening the potential of this nonlinear IR technique as a powerful research tool for the investigation of complex bioinorganic molecules. [ABSTRACT FROM AUTHOR]- Published
- 2022
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6. Shedding Light on Proton and Electron Dynamics in [FeFe] Hydrogenases
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Lorent, Christian, Katz, Sagie, Duan, Jifu, Kulka, Catharina Julia, Caserta, Giorgio, Teutloff, Christian, Yadav, Shanika, Apfel, Ulf-Peter, Winkler, Martin, Happe, Thomas, Horch, Marius, Zebger, Ingo, and Publica
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reaction mechanisms ,ligands ,infrared light ,541 Physikalische Chemie ,electron paramagnetic resonance spectroscopy ,anions - Abstract
[FeFe] hydrogenases are highly efficient catalysts for reversible dihydrogen evolution. H2 turnover involves different catalytic intermediates including a recently characterized hydride state of the active site (H-cluster). Applying cryogenic infrared and electron paramagnetic resonance spectroscopy to an [FeFe] model hydrogenase from Chlamydomonas reinhardtii (CrHydA1), we have discovered two new hydride intermediates and spectroscopic evidence for a bridging CO ligand in two reduced H-cluster states. Our study provides novel insights into these key intermediates, their relevance for the catalytic cycle of [FeFe] hydrogenase, and novel strategies for exploring these aspects in detail.
- Published
- 2020
7. Understanding the Structure and Dynamics of Hydrogenases by Ultrafast and Two-Dimensional Infrared Spectroscopy
- Author
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Hunt, Neil Terrence and Horch, Marius
- Abstract
Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H2, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN− ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail. Using an O2-tolerant [NiFe] hydrogenase as a model system, we show that IR pump-probe spectroscopy can explore catalytically relevant ligand bonding by accessing high-lying vibrational states. This ultrafast technique also shows that the protein matrix is influential in vibrational relaxation, which may be relevant for energy dissipation from the active site during fast reaction steps. Further insights into the relevance of the active site environment are provided by 2D-IR spectroscopy, which reveals equilibrium dynamics and structural constraints imposed on the H2-accepting intermediate of [NiFe] hydrogenases. Both techniques offer new strategies for uniquely identifying redox-structural states in complex catalytic mixtures via vibrational quantum beats and 2D-IR off-diagonal peaks. Together, these findings considerably expand the scope of IR spectroscopy in hydrogenase research, and new perspectives for the characterization of these enzymes and other (bio‑)organometallic targets are presented.
- Published
- 2019
8. Rational redox tuning of transition metal sites : Learning from superoxide reductase
- Author
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Horch, Marius
- Abstract
Using superoxide reductase as a model system, a computational approach reveals how histidine tautomerism tunes the redox properties of metalloenzymes to enable their catalytic function. Inspired by these experimentally inaccessible insights, non-canonical histidine congeners are introduced as new versatile tools for the rational engineering of biological transition metal sites.
- Published
- 2019
9. 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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10. X‐ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases.
- Author
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Ilina, Yulia, Lorent, Christian, Katz, Sagie, Jeoung, Jae‐Hun, Shima, Seigo, Horch, Marius, Zebger, Ingo, and Dobbek, Holger
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HYDROGENASE ,EXTRACELLULAR matrix proteins ,ELECTRON distribution ,SPECTRUM analysis ,MULTIENZYME complexes ,X-ray crystallography ,NICKEL catalysts - Abstract
[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational‐spectroscopic insights into the unexplored structure of the H2‐binding [NiFe] intermediate. Using an F420‐reducing [NiFe]‐hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit‐bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio‐inspired H2‐conversion catalysts. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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11. Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.
- Author
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Horch, Marius, Schoknecht, Janna, Wrathall, Solomon L. D., Greetham, Gregory M., Lenz, Oliver, and Hunt, Neil T.
- Published
- 2019
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12. Struktur-Funktionsbeziehungen von Metalloenzymen
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Horch, Marius, Hildebrandt, Peter, Technische Universität Berlin, Fakultät II - Mathematik und Naturwissenschaften, Teixeira, Miguel, and Zebger, Ingo
- Subjects
ddc:572 ,ddc:541 - Abstract
Anhand der Modellsysteme [NiFe] Hydrogenase und Superoxid Reduktase wurden Struktur-Funktionsbeziehungen von Metalloenzymen, die die Umwandlung kleiner Moleküle katalysieren, mittels theoretischer und spektroskopischer Methoden analysiert. Der erste Teil dieser Arbeit ist der Charakterisierung O2-toleranter [NiFe] Hydrogenasen gewidmet, die die reversible Spaltung von H2 in Anwesenheit von O2 katalysieren. Die strukturellen Aspekte aerober H2-Umwandlung wurden hierbei detailliert für die lösliche NAD+-reduzierende [NiFe] Hydrogenase (SH) aus Ralstonia eutropha untersucht. Aufgrund zusätzlicher CN-Streckschwingungsbanden im Infrarot(IR)-Spektrum der isolierten SH wurde in früheren Arbeiten die Sauerstofftoleranz auf eine sterische Abschirmung des scheinbar redoxinaktiven katalytischen Zentrums durch zusätzlichen Cyanidliganden zurückgeführt. Mittels Dichtefunktionaltheorie (DFT) konnte nun gezeigt werden, dass dieses Modell nicht mit den experimentellen Befunden kompatibel ist. In Übereinstimmung mit früheren in vivo spektroskopischen Untersuchungen des Autors können überzählige Cyanidliganden als Erklärung für zusätzliche CN-Streckschwingungsbanden ausgeschlossen werden. Um die eigentliche Ursache dieser ungewöhnlichen IR-Signale aufzuklären, wurde die isolierte SH unter verschiedenen Redoxbedingungen untersucht. Mittels DFT Rechnungen konnten so sämtliche IR-spektroskopischen Eigenschaften der SH auf der Grundlage eines Standard [NiFe] Zentrums sowie dessen reversible Sulfoxigenierung im voll oxidierten Zustand erklärt werden. Auf dieser Grundlage wurde die katalytische Detoxifizierung von Sauerstoff über eine NADH-abhängige, Schwefel-basierte Peroxidasereaktion als neues Modell für die Sauerstofftoleranz der SH vorgeschlagen. Dieses Prinzip bietet einen wertvollen Ansatzpunkt für die Konzipierung entsprechender biomimetischer Katalysatoren, da H2-Konversion und O2-Detoxifizierung hierbei vom selben Cofaktor bewerkstelligt werden. Neben ihrer Sauerstofftoleranz unterscheidet sich die SH von anderen Hydrogenasen auch durch die Kopplung der reversiblen H2-Spaltung an die Redoxumwandlung von NAD(H). Die Gesamtheit der mutmaßlich an dieser Kopplung beteiligten FeS Cluster konnte nun erstmal experimentell mittels Kernresonanzschwingungsspektroskopie (NRVS) nachgewiesen werden. Das Fehlen entsprechender Elektronenspinresonanz (ESR) Signale ist hierbei auf eine nur partielle Reduktion der Cluster durch den nativen Elektronendonor der SH, NADH, zurückzuführen, so dass der langjährige Widerspruch zwischen vorhergesagten und ESR-spektroskopisch detektierten Clustern aufgelöst werden konnte. Der zweite Teil dieser Arbeit führt die Resonanz Raman (RR) Spektroskopie als neue Methode zur strukturellen und funktionalen Charakterisierung des aktiven [NiFe] Zentrums von Hydrogenasen ein. Mit Hilfe theoretischer Methoden konnten Fe-CO/CN und Ni-S Moden eindeutig über charakteristische Isotopenverschiebungen identifiziert werden. Durch die Nutzung dieser Moden als strukturelle Marker für die zugrunde liegenden molekularen Koordinaten gewährte die Theorie-gestützte RR Spektroskopie wertvolle Einblicke in katalytische Intermediate von [NiFe] Hydrogenasen. Es wurde gezeigt, dass die hohen Photonendichten während des RR Experiments die photochemische Bildung von Ni-L aus dem vollständig reduzierten Nia-SR Zustand ermöglichen. Über das experimentelle Spektrum konnte dieses photoinduzierbare Intermediat als Ni(I) Spezies mit einem protonierten terminalen Cystein sowie einer freien Koordinationstelle zwischen den beiden Metallen identifiziert und damit ein Vorschlag aus der Literatur bestätigt werden. In Übereinstimmung mit DFT Rechnungen wurde ein ähnliches Spektrum auch für das H2-bindende Intermediat Nia-S beobachtet, so dass auch für diese Spezies eine freie Koordinationsstelle angenommen werden kann. Experimentelle und theoretische Daten belegen weiterhin, dass Nia-S eine wippenförmige Ni-Geometrie sowie einen elektronischen Ni(II), S = 0 Grundzustand aufweist. Somit konnten über die fundamentalen strukturellen Eigenschaften von Ni-L und Nia-S essentielle funktionelle Determinanten der biologischen H2-Umwandlung erschlossen werden. Superoxid Reduktasen (SOR) sind Nichthäm-Eisenenzyme, die die Reduktion von Superoxid katalysieren und somit ein weiteres wertvolles Modellsystem für die Untersuchung reversibler Interaktionen von Metalloenzymen mit Sauerstoff-Spezies darstellen. Mit Hilfe der IR-Differenzspektroskopie und theoretischer Methoden wurden redoxabhängige strukturelle Änderungen einer SOR detailliert untersucht. Auf diesem Wege wurden die reduktive Dissoziation des Glutamatliganden vom aktiven Zentrum sowie dadurch hervorgerufene Konformationsänderungen einer nahegelegen beta-Schleife, angrenzender Helices sowie entfernter beta-Faltblätter nachgewiesen. Mittels Normalmodenanalyse auf Grundlage eines elastischen Netzwerk-Modells (ENM-NMA) konnten diese Beobachtungen auf eine niederfrequente thermische Mode des gesamten Proteins zurückgeführt werden. Diese ist vermutlich für den definierten strukturellen Übergang zwischen der oxidierten und reduzierten SOR Form und somit für die enzymatische Funktion, z.B. im Rahmen kooperativer Effekte, relevant. Über IR-differenzspektroskopische Untersuchungen wurden auch H/D-insensitive Imidazol-Moden beobachtet, was auf eine metallinduzierte Deprotonierung von Histidinliganden hindeuten könnte. Dies wäre von katalytischer Relevanz, da hierdurch die Redoxeigenschaften des aktiven Zentrums sowie das umgebende Wasserstoffbrückenbindungsnetzwerk beeinflusst würden. Durch experimentelle und quantenmechanische Daten konnte dies jedoch ausgeschlossen werden, d.h. alle Histidinliganden des aktiven Zentrums im Neutralzustand vorliegen. Deren Deprotonierung würde vielmehr die Geometrie des aktiven Zentrums verzerren, so dass der fehlende H/D-Austausch durch eine hohe Reorganisationsenergie erklärt wurde. Durch die Einbeziehung statischer wie dynamischer Aspekte gewährten diese Untersuchungen wertvolle Einblicke in die lokale und globale SOR-Struktur sowie Säure/Base-Eigenschaften koordinierter Histidine und dibasischer Liganden im Allgemeinen. Using [NiFe] hydrogenase and superoxide reductase as model systems, a combined approach of spectroscopic and theoretical methods was applied to reveal structure-function relationships of metalloenzymes that catalyze the transformation of small molecules. The first part of this thesis is dedicated to the characterization of oxygen-tolerant [NiFe] hydrogenases, which catalyze the reversible cleavage of dihydrogen in the presence of molecular oxygen. The structural aspects of aerobic hydrogen cycling have been investigated in detail for the soluble NAD+-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha. Based on the presence of additional CN stretching bands in the infrared (IR) spectrum of isolated SH, a previous model proposed the presence of additional cyanide ligands at the apparently redox-inactive catalytic center, and one of these ligands was claimed to sterically prevent oxygen attack. Using density functional theory (DFT), these proposals were revisited in this thesis and shown to be incompatible with the experimental data. In line with the author's previous results from in vivo spectroscopic studies, the computational data show that additional CN stretching bands in the IR spectrum do not reflect extra cyanide ligands. To elucidate the actual origin of these unique features, isolated purified SH was characterized under different redox conditions by IR spectroscopy. Supported by DFT calculations, these studies were able to consistently explain all IR spectroscopic properties of the SH by a standard-like [NiFe] active site that exhibits reversible cysteine sulfoxygenation in the fully oxidized state. Based on this finding, a new model for the oxygen-tolerance of the SH is proposed, where oxygen is detoxified catalytically through a NADH-dependent sulfur-centered peroxidase reaction. Combining hydrogen cycling and oxygen detoxification in a single cofactor, this scheme represents a valuable inspiration for the design of biomimetic catalysts for aerobic hydrogen conversion. Besides its oxygen tolerance, the SH differs from other hydrogenases by coupling the reversible cleavage of hydrogen to the redox conversion of NAD(H), presumably via a chain of FeS clusters. Using nuclear resonance vibrational spectroscopy (NRVS), the full set of clusters was experimentally confirmed for the first time, and the lack of corresponding electron paramagnetic resonance (EPR) signals could be explained by the fact that most clusters remained in their oxidized state upon incubation of SH with its native electron donor NADH. In this way, the long-lasting discrepancy between sequence-predicted and EPR spectroscopically detected clusters was resolved. The second part of this thesis introduces resonance Raman (RR) spectroscopy as a technique for the characterization of the [NiFe] active site of hydrogenase, thereby establishing a novel tool to elucidate structural and functional aspects of these enzymes. Supported by theoretical methods, Fe-CO/CN and Ni-S modes of the catalytic center could be unambiguously assigned on the basis of characteristic isotopic shifts. Using these normal modes as structural markers for the underlying molecular coordinates, valuable information on catalytic intermediates of [NiFe] hydrogenase was obtained by computationally assisted RR spectroscopy. A novel photochemical reaction path for the formation of Ni-L from the fully reduced Nia-SR state was shown to be feasible under high photon densities as available during the RR experiment. The proposed structure of this photo-inducible intermediate was confirmed by showing that the experimental spectra are only consistent with a Ni(I) species exhibiting a protonated terminal cysteine and a vacant coordination site between both metals. In line with DFT data, RR spectra of Ni-L and the hydrogen-binding intermediate Nia-S were found to be very similar, suggesting that the latter species provides a vacant coordination site as well, as generally anticipated. Experimental and computational data also support the suggestion that Nia-S exhibits a seesaw-shaped Ni coordination geometry and a Ni(II), S = 0 electronic ground state. Essentially, these studies revealed the fundamental structural aspects of Ni-L and Nia-S, which represent important functional determinants in biological hydrogen cycling. Superoxide reductase (SOR) is a non-heme iron enzyme that catalyzes the reductive detoxification of superoxide and, thus, represents another valuable model system to study the reversible interaction of metalloenzymes with dioxygen derivatives. Using potential-dependent IR difference spectroscopy and a set of computational methods, redox-related structural changes of SOR were explored in detail. These data revealed the reductive dissociation of an iron-bound glutamate ligand from the active site, which triggered conformational changes in nearby loop and helical regions as well as more remote beta-sheets of the protein. According to normal mode analysis based on an elastic network model (ENM-NMA), these structural changes could be associated with a low-frequency thermal mode of the entire protein, which is proposed to guide the structural transition between the ferric and ferrous state. This type of motion may facilitate the enzymatic function, possibly in a cooperative manner. IR difference spectroscopic studies on SOR also revealed H/D exchange-insensitive imidazole modes, which could indicate the metal-induced deprotonation of histidine ligands. Notably, the protonation states of coordinated histidines may considerably affect the catalytic mechanism of SOR by tuning active site redox properties and the surrounding H-bonding network. Based on experimental and quantum mechanical data, metal-induced deprotonation could be excluded, showing that all active site histidines reside in their neutral state at physiological pH. Instead, the deprotonation of these ligands was found to distort the active site, which is proposed to prevent H/D exchange by a high reorganization energy. Covering both static and dynamic aspects, these findings provide important insights into the local and global structure of SOR as well as acid-base properties of coordinated imidazole and dibasic ligands in general.
- Published
- 2015
13. 2nd coordination sphere controlled electron transfer of iron hangman complexes on electrodes probed by surface enhanced vibrational spectroscopy
- Author
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Ly, Hoang Khoa, Wrzolek, Pierre, Heidary, Nina, Götz, R., Horch, Marius, Kozuch, Jacek, Schwalbe, Matthias, and Weidinger, Inez M.
- Subjects
ddc:540 - Abstract
Iron hangman complexes exhibit improved catalytic properties regarding O-2 and H2O2 reduction, which are attributed to the presence of a proton donating group in defined vicinity of the catalytic metal centre. Surface enhanced resonance Raman (SERR) and IR (SEIRA) spectro-electrochemistry has been applied concomitantly for the first time to analyse such iron hangman porphyrin complexes attached to electrodes in aqueous solution. While the SERR spectra yield information about the redox state of the central iron, the SEIRA spectra show protonation and deprotonation events of the 2nd coordination sphere. To investigate the influence of a proton active hanging group on the heterogeneous electron transfer between the iron porphyrin and the electrode, two hangman complexes with either an acid or ester functional group were compared. Using time resolved SERR spectroscopy the electron transfer rates of both complexes were determined. Complexes with an acid group showed a slow electron transfer rate at neutral pH that increased significantly at pH 4, while complexes with an ester group exhibited a much faster, but pH independent rate. SEIRA measurements were able to determine directly for the first time a pK(a) value of 3.4 of a carboxylic hanging group in the immobilized state that shifted to 5.2 in D2O buffer solution. The kinetic data showed an increase of the heterogeneous electron transfer rate with the protonation degree of the acid groups. From these results, we propose a PCET which is strongly modulated by the protonation state of the acid hanging group via hydrogen bond interactions.
- Published
- 2015
14. Microporous polymer network films covalently bound to gold electrodes
- Author
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Becker, Daniel, Heidary, Nina, Horch, Marius, Gernert, Ulrich, Zebger, Ingo, Schmidt, Johannes, Fischer, Anna, and Thomas, Arne
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ddc:540 - Abstract
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. Covalent attachment of a microporous polymer network (MPN) on a gold surface is presented. A functional bromophenyl-based self-assembled monolayer (SAM) formed on the gold surface acts as co-monomer in the polymerisation of the MPN yielding homogeneous and robust coatings. Covalent binding of the films to the electrode is confirmed by SEIRAS measurements.
- Published
- 2015
15. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O-2-tolerant NAD(+)-reducing [NiFe] hydrogenase
- Author
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Lauterbach, Lars, Wang, Hongxin, Horch, Marius, Gee, Leland B., Yoda, Yoshitaka, Tanaka, Yoshihito, Zebger, Ingo, Lenz, Oliver, and Cramer, Stephen P.
- Subjects
ddc:540 - Abstract
Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD(+)-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H-2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by Fe-57 specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD(+)-reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective C-13 labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.
- Published
- 2015
16. Hydrogen evolution by cobalt hangman porphyrins under operating conditions studied by vibrational spectro-electrochemistry.
- Author
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Kielb, Patrycja, Horch, Marius, Wrzolek, Pierre, Goetz, Robert, Ly, Khoa H., Kozuch, Jacek, Schwalbe, Matthias, and Weidinger, Inez M.
- Published
- 2018
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17. An S-Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O2-Tolerant Hydrogenase.
- Author
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Lindenmaier, Nils J., Wahlefeld, Stefan, Bill, Eckhard, Szilvási, Tibor, Eberle, Christopher, Yao, Shenglai, Hildebrandt, Peter, Horch, Marius, Zebger, Ingo, and Driess, Matthias
- Subjects
HYDROGENASE ,INTERMEDIATES (Chemistry) ,OXYGENATION (Chemistry) ,NAD (Coenzyme) ,MOLECULAR structure ,HYDROGEN - Abstract
To understand the molecular details of O
2 -tolerant hydrogen cycling by a soluble NAD+ -reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S-oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen-inhibited [NiFe] active site. This compound and its non-S-oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S-oxygenated intermediates in hydrogenases and similar enzymes. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
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18. Domain motions and electron transfer dynamics in 2Fe-superoxide reductase.
- Author
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Horch, Marius, Utesch, Tillmann, Hildebrandt, Peter, Mroginski, Maria Andrea, and Zebger, Ingo
- Abstract
Superoxide reductases are non-heme iron enzymes that represent valuable model systems for the reductive detoxification of reactive oxygen species. In the present study, we applied different theoretical methods to study the structural dynamics of a prototypical 2Fe-superoxide reductase and its influence on electron transfer towards the active site. Using normal mode and essential dynamics analyses, we could show that enzymes of this type are capable of well-defined, electrostatically triggered domain movements, which may allow conformational proofreading for cellular redox partners involved in intermolecular electron transfer. Moreover, these global modes of motion were found to enable access to molecular configurations with decreased tunnelling distances between the active site and the enzyme's second iron centre. Using all-atom classical molecular dynamics simulations and the tunnelling pathway model, however, we found that electron transfer between the two metal sites is not accelerated under these conditions. This unexpected finding suggests that the unperturbed enzymatic structure is optimized for intramolecular electron transfer, which provides an indirect indication of the biological relevance of such a mechanism. Consistently, efficient electron transfer was found to depend on a distinct route, which is accessible via the equilibrium geometry and characterized by a quasi conserved tyrosine that could enable multistep-tunnelling (hopping). Besides these explicit findings, the present study demonstrates the importance of considering both global and local protein dynamics, and a generalized approach for the functional analysis of these aspects is provided. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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19. Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase.
- Author
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Heidary, Nina, Utesch, Tillmann, Zerball, Maximilian, Horch, Marius, Millo, Diego, Fritsch, Johannes, Lenz, Oliver, von Klitzing, Regine, Hildebrandt, Peter, Fischer, Anna, Mroginski, Maria Andrea, and Zebger, Ingo
- Subjects
ELECTROCATALYSIS ,HYDROGENASE ,PHYSIOLOGICAL effects of oxygen ,VOLTAMMETRY ,BIOCOMPATIBILITY - Abstract
Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H
2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme. [ABSTRACT FROM AUTHOR]- Published
- 2015
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20. Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD+-Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties.
- Author
-
Horch, Marius, Lauterbach, Lars, Mroginski, Maria Andrea, Hildebrandt, Peter, Lenz, Oliver, and Zebger, Ingo
- Subjects
- *
PHOTOSYNTHETIC oxygen evolution , *OXYGEN compounds , *NONMETALS , *HYDROGENASE , *REFRIGERANTS - Abstract
Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD+-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD+ and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
21. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase.
- Author
-
Lauterbach, Lars, Wang, Hongxin, Horch, Marius, Gee, Leland B., Yoda, Yoshitaka, Tanaka, Yoshihito, Zebger, Ingo, Lenz, Oliver, and Cramer, Stephen P.
- Published
- 2015
- Full Text
- View/download PDF
22. Impact of the Iron–Sulfur Cluster Proximal to the Active Site on the Catalytic Function of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase.
- Author
-
Karstens, Katja, Wahlefeld, Stefan, Horch, Marius, Grunzel, Miriam, Lauterbach, Lars, Lendzian, Friedhelm, Zebger, Ingo, and Lenz, Oliver
- Published
- 2015
- Full Text
- View/download PDF
23. Metal-induced histidine deprotonation in biocatalysis? Experimental and theoretical insights into superoxide reductase.
- Author
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Horch, Marius, Pinto, Ana Filipa, Mroginski, Maria Andrea, Teixeira, Miguel, Hildebrandt, Peter, and Zebger, Ingo
- Published
- 2014
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24. Resonance Raman Spectroscopy on [NiFe] Hydrogenase Provides Structural Insights into Catalytic Intermediates and Reactions.
- Author
-
Horch, Marius, Schoknecht, Janna, Mroginski, Maria Andrea, Lenz, Oliver, Hildebrandt, Peter, and Zebger, Ingo
- Subjects
- *
RESONANCE Raman spectroscopy , *HYDROGENASE , *CATALYSIS research , *OXIDOREDUCTASES , *RAMAN spectroscopy - Abstract
[NiFe] hydrogenases catalyze the reversible cleavage of hydrogen and, thus, represent model systems for the investigation and exploitation of emission-free energy conversion processes. Valuable information on the underlying molecular mechanisms can be obtained by spectroscopic techniques that monitor individual catalytic intermediates. Here, we employed resonance Raman spectroscopy and extended it to the entire binuclear active site of an oxygen-tolerant [NiFe] hydrogenase by probing the metal–ligand modes of both the Fe and, for the first time, the Ni ion. Supported by theoretical methods, this approach allowed for monitoring H-transfer from the active site and revealed novel insights into the so far unknown structure and electronic configuration of the hydrogen-binding intermediate of the catalytic cycle, thereby providing key information about catalytic intermediates and reactions of biological hydrogen activation. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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- View/download PDF
25. 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
26. Resonanz-Raman-Spektroskopie als Methode zur Untersuchung des aktiven Zentrums von Hydrogenasen.
- 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
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
27. Combining Spectroscopy and Theory to Evaluate Structural Models of Metalloenzymes: A Case Study on the Soluble [NiFe] Hydrogenase from Ralstonia eutropha.
- Author
-
Horch, Marius, Rippers, Yvonne, Mroginski, Maria A., Hildebrandt, Peter, and Zebger, Ingo
- Published
- 2013
- Full Text
- View/download PDF
28. Revealing the Absolute Configuration of the CO and CN− Ligands at the Active Site of a [NiFe] Hydrogenase.
- Author
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Rippers, Yvonne, Horch, Marius, Hildebrandt, Peter, Zebger, Ingo, and Mroginski, Maria Andrea
- Published
- 2012
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29. The Hydrogenase Subcomplex of the NAD.
- Author
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Lauterbach, Lars, Liu, Juan, Horch, Marius, Hummel, Phillip, Schwarze, Alexander, Haumann, Michael, Vincent, Kylie A., Lenz, Oliver, and Zebger, Ingo
- Published
- 2011
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30. Untersuchung des katalytischen Zentrums der O2-toleranten NAD+-reduzierenden [NiFe]-Hydrogenase von Ralstonia eutropha H16 mit In-situ-EPR- und -FTIR-Spektroskopie.
- Author
-
Horch, Marius, Lauterbach, Lars, Saggu, Miguel, Hildebrandt, Peter, Lendzian, Friedhelm, Bittl, Robert, Lenz, Oliver, and Zebger, Ingo
- Published
- 2010
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31. Probing the Active Site of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 by In Situ EPR and FTIR Spectroscopy.
- Author
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Horch, Marius, Lauterbach, Lars, Saggu, Miguel, Hildebrandt, Peter, Lendzian, Friedhelm, Bittl, Robert, Lenz, Oliver, and Zebger, Ingo
- Published
- 2010
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- View/download PDF
32. 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
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- View/download PDF
33. 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
34. A Beginner's Guide to Thermodynamic Modelling of [FeFe] Hydrogenase.
- Author
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Birrell, James A., Rodríguez-Maciá, Patricia, Hery-Barranco, Adrian, and Horch, Marius
- Subjects
HYDROGENASE ,METALLOENZYMES ,INFRARED spectroscopy ,HYDROGEN oxidation ,REDUCTION potential - Abstract
[FeFe] hydrogenases, which are considered the most active naturally occurring catalysts for hydrogen oxidation and proton reduction, are extensively studied as models to learn the important features for efficient H
2 conversion catalysis. Using infrared spectroscopy as a selective probe, the redox behaviour of the active site H-cluster is routinely modelled with thermodynamic schemes based on the Nernst equation for determining thermodynamic parameters, such as redox midpoint potentials and pKa values. Here, the thermodynamic models usually applied to [FeFe] hydrogenases are introduced and discussed in a pedagogic fashion and their applicability to additional metalloenzymes and molecular catalysts is also addressed. [ABSTRACT FROM AUTHOR]- Published
- 2021
- Full Text
- View/download PDF
35. Electrocatalysis by Heme Enzymes—Applications in Biosensing.
- Author
-
Zuccarello, Lidia, Barbosa, Catarina, Todorovic, Smilja, Silveira, Célia M., and Horch, Marius
- Subjects
HEMOPROTEINS ,IMMOBILIZED proteins ,CYTOCHROME oxidase ,HEME ,ENZYMES ,MYOGLOBIN ,CATALASE - Abstract
Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
36. 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
37. 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
38. Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2.
- Author
-
Preissler, Janina, Wahlefeld, Stefan, Lorent, Christian, Teutloff, Christian, Horch, Marius, Lauterbach, Lars, Cramer, Stephen P., Zebger, Ingo, and Lenz, Oliver
- Subjects
- *
ENZYMES , *PYRIDINE nucleotides , *HYDROGENASE , *IRON-sulfur compounds , *NAD (Coenzyme) - Abstract
Biocatalysts that mediate the H 2 -dependent reduction of NAD + to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD + -reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O 2 , which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH , encoding the soluble NAD + -reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 T ( Ht ). The Ht SH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H 2 -oxidizing betaproteobacterium Ralstonia eutropha H16 ( Re ). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H 2 -mediated NAD + reduction activity was observed at 80 °C and pH 6.5, and catalytic activity was found to be sustained at low O 2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated Ht SH that is remarkably different from those of the closely related Re SH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in Ht SH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD + -reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the Ht SH as well as application of this enzyme for H 2 -driven cofactor recycling under oxic conditions at elevated temperatures. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
39. Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.
- Author
-
Tejwani, Vijay, Schmitt, Franz-Josef, Wilkening, Svea, Zebger, Ingo, Horch, Marius, Lenz, Oliver, and Friedrich, Thomas
- Subjects
- *
RALSTONIA eutropha , *FLUORESCENCE , *HYDROGENASE , *AEROBIC metabolism , *HYDROGEN production - Abstract
Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H 2 -driven production of biodegradable polymers and hydrocarbons. H 2 oxidation by R. eutropha takes place in the presence of O 2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H 2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H 2 oxidation with the reduction of NAD + to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD + pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD + ] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD + ] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD + ] ratios represents a novel and sensitive tool to determine the redox state of cells. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
40. Light-Induced Electron Transfer in a [NiFe] Hydrogenase Opens a Photochemical Shortcut for Catalytic Dihydrogen Cleavage.
- Author
-
Karafoulidi-Retsou C, Lorent C, Katz S, Rippers Y, Matsuura H, Higuchi Y, Zebger I, and Horch M
- Abstract
[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen-tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Nia-C and Nia-SR'', were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron-sulfur clusters of the enzyme. In addition, IR and EPR experiments performed under illumination revealed an unusual photochemical response of the enzyme. Nia-SR'', a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Nia-L. In contrast to the well-known photolysis of the more oxidized hydride intermediate Nia-C, photoconversion of Nia-SR'' into Nia-L is an active-site redox reaction that involves light-driven electron transfer towards the enzyme's iron-sulfur clusters. Omitting the ground-state intermediate Nia-C, this direct interconversion of these two states represents a potential photochemical shortcut of the catalytic cycle that integrates multiple redox sites of the enzyme. In total, our findings reveal the non-local redistribution of electrons via thermal and photochemical reaction channels and the potential of accelerating or controlling [NiFe] hydrogenases by light., (© 2024 Wiley‐VCH GmbH.)
- Published
- 2024
- Full Text
- View/download PDF
41. Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN) 2 ] in Polar and Protic Solvents.
- Author
-
Procacci B, Wrathall SLD, Farmer AL, Shaw DJ, Greetham GM, Parker AW, Rippers Y, Horch M, Lynam JM, and Hunt NT
- Abstract
The [CpFe(CO)(CN)
2 ]- unit is an excellent structural model for the Fe(CO)(CN)2 moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)2 ] ( M1 ) in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of M1 in H2 O, D2 O, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN)2 unit. Comparisons of the spectroscopic and dynamic data obtained for M1 in solution and in a dry film with those obtained for the enzyme led to the conclusion that the protein backbone forms an important part of the bimetallic active site environment via secondary coordination sphere interactions.- Published
- 2024
- Full Text
- View/download PDF
42. Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.
- Author
-
Kulka-Peschke CJ, Schulz AC, Lorent C, Rippers Y, Wahlefeld S, Preissler J, Schulz C, Wiemann C, Bernitzky CCM, Karafoulidi-Retsou C, Wrathall SLD, Procacci B, Matsuura H, Greetham GM, Teutloff C, Lauterbach L, Higuchi Y, Ishii M, Hunt NT, Lenz O, Zebger I, and Horch M
- Subjects
- Alanine metabolism, Aspartic Acid metabolism, Catalytic Domain, Glutamic Acid metabolism, Glutamine metabolism, Hydrogenophilaceae, Iron chemistry, Ligands, NAD metabolism, Nickel chemistry, Oxidation-Reduction, Oxygen chemistry, Hydrogenase chemistry
- Abstract
NAD
+ -reducing [NiFe] hydrogenases are valuable biocatalysts for H2 -based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+ -reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus ( Ht SH) is O2 -tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native Ht SH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized Ht SH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.- Published
- 2022
- Full Text
- View/download PDF
43. 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
44. X-ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases.
- Author
-
Ilina Y, Lorent C, Katz S, Jeoung JH, Shima S, Horch M, Zebger I, and Dobbek H
- Subjects
- Catalysis, Humans, Crystallography, X-Ray methods, Electron Spin Resonance Spectroscopy methods, Hydrogenase metabolism
- Abstract
[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H
2 ). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H2 -binding [NiFe] intermediate. Using an F420 -reducing [NiFe]-hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit-bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio-inspired H2 -conversion catalysts., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)- Published
- 2019
- Full Text
- View/download PDF
45. An S-Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O 2 -Tolerant Hydrogenase.
- Author
-
Lindenmaier NJ, Wahlefeld S, Bill E, Szilvási T, Eberle C, Yao S, Hildebrandt P, Horch M, Zebger I, and Driess M
- Subjects
- Catalytic Domain, Crystallography, X-Ray, Cupriavidus necator chemistry, Models, Molecular, Spectrophotometry, Infrared, Spectrum Analysis, Raman, Cupriavidus necator enzymology, Hydrogenase chemistry, Oxygen chemistry
- Abstract
To understand the molecular details of O
2 -tolerant hydrogen cycling by a soluble NAD+ -reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S-oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen-inhibited [NiFe] active site. This compound and its non-S-oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S-oxygenated intermediates in hydrogenases and similar enzymes., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2017
- Full Text
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46. Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO.
- Author
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Ash PA, Liu J, Coutard N, Heidary N, Horch M, Gudim I, Simler T, Zebger I, Lenz O, and Vincent KA
- Subjects
- Carbon Monoxide chemistry, Hydrogen chemistry, Hydrogen metabolism, Hydrogenase chemistry, Oxidation-Reduction, Oxygen chemistry, Spectrophotometry, Infrared, Carbon Monoxide metabolism, Cupriavidus necator enzymology, Electrochemical Techniques, Hydrogenase metabolism, Oxygen metabolism
- Abstract
The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.
- Published
- 2015
- Full Text
- View/download PDF
47. Microporous polymer network films covalently bound to gold electrodes.
- Author
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Becker D, Heidary N, Horch M, Gernert U, Zebger I, Schmidt J, Fischer A, and Thomas A
- Abstract
Covalent attachment of a microporous polymer network (MPN) on a gold surface is presented. A functional bromophenyl-based self-assembled monolayer (SAM) formed on the gold surface acts as co-monomer in the polymerisation of the MPN yielding homogeneous and robust coatings. Covalent binding of the films to the electrode is confirmed by SEIRAS measurements.
- Published
- 2015
- Full Text
- View/download PDF
48. Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.
- Author
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Karstens K, Wahlefeld S, Horch M, Grunzel M, Lauterbach L, Lendzian F, Zebger I, and Lenz O
- Subjects
- Amino Acid Substitution, Catalytic Domain, Cupriavidus necator chemistry, Cupriavidus necator genetics, Cupriavidus necator metabolism, Electron Spin Resonance Spectroscopy, Hydrogenase genetics, Iron chemistry, Iron metabolism, Models, Molecular, NAD metabolism, Spectroscopy, Fourier Transform Infrared, Sulfur chemistry, Sulfur metabolism, Cupriavidus necator enzymology, Hydrogenase chemistry, Hydrogenase metabolism, Oxygen metabolism
- Abstract
The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.
- Published
- 2015
- Full Text
- View/download PDF
49. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O 2 -tolerant NAD + -reducing [NiFe] hydrogenase.
- Author
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Lauterbach L, Wang H, Horch M, Gee LB, Yoda Y, Tanaka Y, Zebger I, Lenz O, and Cramer SP
- Abstract
Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD
+ -reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by57 Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+ -reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective13 C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.- Published
- 2015
- Full Text
- View/download PDF
50. Revealing the absolute configuration of the CO and CN- ligands at the active site of a [NiFe] hydrogenase.
- Author
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Rippers Y, Horch M, Hildebrandt P, Zebger I, and Mroginski MA
- Subjects
- Catalytic Domain, Crystallography methods, Cupriavidus necator metabolism, Iron chemistry, Iron metabolism, Ligands, Molecular Dynamics Simulation, Nickel chemistry, Nickel metabolism, Quantum Theory, Carbon Monoxide chemistry, Cyanides chemistry, Hydrogenase chemistry, Hydrogenase metabolism
- Abstract
Combined molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the crystal structure of the reduced membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha to determine the absolute configuration of the CO and the two CN(-) ligands bound to the active-site iron of the enzyme. For three models that include the CO ligand at different positions, often indistinguishable on the basis of the crystallographic data, we optimized the structures and calculated the ligand stretching frequencies. Comparison with the experimental IR data reveals that the CO ligand is in trans position to the substrate-binding site of the bimetallic [NiFe] cluster., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
- Full Text
- View/download PDF
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