Insights into the structure of the active site of the O-2-tolerant membrane bound [NiFe] hydrogenase of R. eutropha H16 by molecular modelling

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.Structural models for the Ni-B state of the wild-type and C81S protein variant of the membrane-bound [NiFe] hydrogenase from Ralstonia eutrophaH16 were derived by applying the homology model technique combined with molecular simulations and a hybrid quantum mechanical/molecular mechanical approach. The active site structure was assessed by comparing calculated and experimental IR spectra, confirming the view that the active site structure is very similar to those of anaerobic standard hydrogenases. In addition, the data suggest the presence of a water molecule in the second coordination sphere of the active centre.DFG, EXC 314, Unifying Concepts in Catalysi

Understanding 2D-IR Spectra of Hydrogenases: A Descriptive and Predictive Computational Study

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible cleavage of dihydrogen (H2), 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 IRpump-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

Structural and electronic properties of the active site of [ZnFe] SulE

The function of the recently isolated sulerythrin (SulE) has been investigated using a combination of structural and electronic analyses based on quantum mechanical calculations. In the SulE structure of Fushinobu et al. (2003), isolated from a strictly aerobic archaeon, Sulfolobus tokadaii, a dioxygen-containing species was tentatively included at the active site during crystallographic refinement although the substrate specificity of SulE remains unclear. Studies have suggested that a structurally related enzyme, rubrerythrin, functions as a hydrogen peroxide reductase. Since SulE is a truncated version of rubrerythrin, the enzymes are hypothesized to function similarly. Hence, using available X-ray crystallography data (1.7 Å), we constructed various models of SulE containing a ZnII–Fe active site, differing in the nature of the substrate specificity (O2, H2O2), the oxidation level and the spin state of the iron ion, and the protonation states of the coordinating glutamate residues. Also, the substrate H2O2 is modeled in two possible configurations, differing in the orientation of the hydrogen atoms. Overall, the optimized geometries with an O2 substrate do not show good agreement with the experimentally resolved geometry. In contrast, excellent agreement between crystal structure arrangement and optimized geometries is achieved considering a H2O2 substrate and FeII in both spin states, when Glu92 is protonated. These results suggest that the dioxo species detected at the [ZnFe] active site of sulerythrin is H2O2, rather than an O2 molecule in agreement with experimental data indicating that only the diferrous oxidation state of the dimetal site in rubrerythrin reacts rapidly with H2O2. Based on our computations, we proposed a possible reaction pathway for substrate binding at the ZnFeII site of SulE with a H2O2 substrate. In this reaction pathway, Fe or another electron donor, such as NAD(P)H, catalyzes the reduction of H2O2 to water at the zinc–iron site

Understanding 2D-IR Spectra of Hydrogenases : A Descriptive and Predictive Computational Study

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible cleavage of dihydrogen (H2), 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 IRpump-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

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment

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

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

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 H2O, D2O, 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

QM/MM Berechnungen an der Membran gebundenen Hydrogenase von Ralstonia eutropha

Molekularer Wasserstoff ist ein einfaches Molekül, das in der Knallgasreaktion mit Sauerstoff viel Energie freisetzt (572 kJ/mol). Aus diesem Grund wird es immer häufiger als saubere Energiequelle und als Alternative zu CO2 emittierenden Verbrennungsenergiequellen in Betracht gezogen. Hydrogenasen sind Metallenzyme, die in der Lage sind, Wasserstoff bei milden Reaktionsbedingungen zu aktivieren und in zwei Protonen und zwei Elektronen zu spalten. Da dies ein reversibler Prozess ist, können Hydrogenasen zur effizienten Lagerung und Freisetzung großer Energiemengen eingesetzt werden. Aus diesem Grund gewinnen sie immer mehr Aufmerksamkeit, da sie ein besonderes Potential in bioenergetischen Verwendungen für die alternative Treibstoffproduktion aufweisen. Allerdings sind die meisten Hydrogenasen sehr empfindlich gegenüber Sauerstoff. Die in dieser Arbeit untersuchte membrangebundene Hydrogenase (MBH) von Ralstonia eutropha kann Wasserstoff auch in der Gegenwart von natürlichen Sauerstoffmengen umsetzen. Ein detailliertes Wissen über den Mechanismus der reversiblen Wasserstoffspaltung und der Sauerstofftoleranz würde sehr zur Entwicklung biomimetischer Katalysatoranwendungen beitragen. Diese Mechanismen sind jedoch immer noch nicht im Detail geklärt. Das Ziel dieser Arbeit war es, strukturelle Details des katalytisch aktiven [NiFe] Zentrums, an dem dieWasserstoffreaktion statt findet, und des proximalen Eisen-Schwefel-clusters, der eine wichtige Rolle in der Sauerstofftoleranz der MBH spielt, zu klären. Dazu wurden theoretische Berechnungen der Schwingungseigenschaften mittels des quantenmechanischen/molekularmechanischen (QM/MM) Ansatzes in Kombination mit molek¨uldynamischen (MD) Simulationen durchgeführt. Bevor die röntgenkristallographische Struktur der MBH von Ralstonia eutropha verfügbar war, wurde in Kapitel 5 ein Strukturmodel für das Enzym mit Hilfe der Homologiemodell- Technik erstellt. Die modellierte Struktur wurde durch den Vergleich von QM/MM berechneten Infrarotspektren (IR) mit experimentellen Ergebnissen best¨atigt. Diese modellierte MBH ermöglichte erstmals Einblicke in die Struktur des aktiven Zentrums einer sauerstofftoleranten Hydrogenase. In Kapitel 6 wurde die absolute Konfiguration der an Eisen gebundenen, anorganischen Liganden im aktiven Zentrum aufgeklärt und best¨atigte vorherige Vorschläge für die strukturelle Konfiguration. Diese Arbeit trug außerdem dazu bei, Zuordnungsprobleme der anorganischen Liganden im aktiven Zentrum der röntgenkristallographisch bestimmten Struktur der reduzierten MBH, die 2011 veröffentlicht wurde, zu klären. Nachdem die Kristallstruktur der reduzierten MBH veröffentlicht worden war, wurden schwingungsspektroskopische Berechnungen auf QM/MM Level in Kombination mit MD Simulationen an verschiedenen reduzierten und lichtinduzierten Zuständen des aktiven Zentrums durchgeführt und mit experimentellen Resonanz Raman (RR) Spektren verglichen. Die Ergebnisse demonstrierten das Potential der RR Spektroskopie als Alternative zur IR Technik, Signale des aktiven Zentrums zu messen. In Kombination mit theoretischen Berechnungen konnte ein lichtinduzierter Zustand identifiziert und seine zuvor vorgeschlagene Struktur bestätigt werden. In Kapitel 8 werden die Ergebnisse zu ähnlichen Berechnungen wie in Kapitel 7 an verschiedenen Zuständen der oxidierten MBH vorgestellt. Zusätzlich wurden schwingungsspektroskopische Berechnungen für den proximalen Eisen-Schwefel-Cluster der superoxidierten MBH durchgeführt. Der Vergleich mit experimentellen RR Spektren bestätigte die Anwesenheit eines an ein Eisenatom des proximalen Clusters gebundenen Hydroxylliganden, der auch in der röntgenkristallographischen Struktur des oxidierten Enzyms detektiert werden konnte. Außerdem ermöglichten die QM/MM Rechnungen die Zuordnung aufgezeichneter Signale vom aktiven Zentrum zum aktivierten Zustand der MBH. Schließlich wird in Abschnitt 4.2 die Simulation von resonanzverstärkten Raman-Intensitäten behandelt. Hierzu wurde ein neuer Ansatz entwickelt und angewendet, der Raman-Intensitäten auf der Basis individueller Schwingungsbeiträge zur potentiellen Energie gewichtet.Molecular hydrogen is a simple molecule which releases a large amount of energy when reacting with oxygen in the Knall-Gas-Reaction (572 kJ/mol). For this reason it is more frequently used as a clean energy source and as an alternative to CO2 emitting combustioning energy sources. Hydrogenases are metalloenzymes that are able to activate the hydrogen molecule at mild reaction conditions and cleave it into two protons and two electrons. Since this is a reversible process hydrogenases might be utilized to efficiently store and release high amounts of energy. For this reason gained a lot of attention given its particular potential in bioenergetic applications for alternative fuel productions. However, most hydrogenases are very sensitive towards oxygen. The membrane bound hydrogenase (MBH) from Ralstonia eutropha examined in this work is able to metabolize hydrogen even at ambient oxygen levels. Detailed knowledge of the mechanisms for the reversible hydrogen cleavage and the oxygen tolerance would greatly contribute to biomimetic catalysts applications. However, these mechanisms are still not clarified in all detail. The aim of this work was to elucidate structural details at the [NiFe] active site, where the hydrogen reaction takes place, and at the proximal iron sulfur cluster, which is essential for the oxygen tolerance of the MBH. As a tool, theoretical calculations of vibrational properties using the hybrid quantum mechanical / molecular mechanical (QM/MM) approach in combination with molecular dynamics (MD) simulations was chosen. Before the x-ray crystallographic structure of the MBH from Ralstonia eutropha became available a structural model of the enzyme was constructed with the homology model technique in chapter 5. The modelled structure was confirmed by the comparison of vibrational infrared (IR) spectra computed at QM/MM level with experimental results. The modelled MBH could give first insights into the structure at the active site of an oxygen tolerant hydrogenase. In chapter 6 the absolute active site configuration of the three inorganic ligands bound to the iron could be revealed, confirming former structural configuration suggestions. This work also aimed clarifying assigning problems concerning the inorganic ligands at the active site of the x-ray crystallographic structure of the reduced MBH published in 2011. After publication of the crystall structure of the reduced MBH, vibrational calculations at QM/MM level in combination with MD simulations on different reduced and light induced states of the active site were performed and compared with experimental resonance Raman (RR) spectra. The results in chapter 7 demonstrate the potential of the RR spectroscopical technique to probe active site signals as alternative to IR spectroscopy. In combination with theoretical calculations a light induced state was identified and its structure previously proposed by Brecht et al. was confirmed. In chapter 8 results are presented for similar calculations as in chapter 7 that were repeated for different active site states of the oxidized MBH. In addition vibrational calculations were carried out on the proximal cluster of the superoxidzed MBH. The comparison with RR experimental results confirmed the presence of a hydroxyl ligand at the iron sulfur cluster, which also has been detected in the crystal structure of the oxidized enzyme[9]. Furthermore, the QM/MM calculations enabled the assignment of probed active site signals to the MBH active state. Finally, the simulation of resonance Raman intensities was addressed and a new approach based on potential energy contribution weighting was developed as introduced in section 4.2

The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase