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[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.

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.

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.

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.

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.

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.

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.