Models for the Active Site of the [FeFe]-Hydrogenase

In der Natur spielen Enzyme für viele Prozesse des Lebens eine entscheidende Rolle. Sie bilden die Basis für das Leben auf der Erde. Je nach Wirkungsweise werden Enzyme in unterschiedliche Klassen unterteilt. Eine dieser Klasse bilden die Hydrogenasen, welche das Gleichgewicht zwischen Protonen und Wasserstoff beeinflussen. Besonders in methanogenen (Methan-produzierend), acetogenen (Essigsäure-produzierend), stickstofffixierenden (Umwandlung von N2 in Ammoniak – biologisches Äquivalent zum Haber-Bosch-Verfahren), photosynthetischen und sulfatreduzierenden Bakterien sind diese Enzyme anzufinden und verdeutlichen die Rolle des Wasserstoffes als Reduktionsmittel und Energiespeicher. Die Hydrogenasen lassen sich je nach Aufbau ihres aktiven Zentrums in verschiedene Unterarten einteilen: [FeFe]-, [FeNi]- und [Fe]-Hydrogenasen. Die [FeFe]-Enzyme sind in der Lage das Gleichgewicht (2H+/H2) so zu verschieben, dass Protonen zu Wasserstoff reduziert werden und somit ein effizienter Energieträger zur Verfügung steht. Obwohl das Enzym isoliert werden konnte, war es bisher, bedingt durch analytische Grenzen, nicht möglich die Struktur des aktiven Zentrums vollständig aufzuklären. Aufgrund dieses Tatsache gibt es auch heute noch viele Diskussion über den Mechanismus der Wasserstoffkatalyse in diesen Enzymsystemen. Mit Hilfe verschiedenartiger Modellverbindungen soll diese Frage gelöst und die katalytischen Fähigkeiten zur Wasserstoffsynthese der industriellen Nutzung zugänglich gemacht werden. Besonders Peptid-basierte, Zucker-haltige Komplexe, aber auch Komplexe mit (SCH2)2SiR2 Ligandsystemen standen dabei im Fokus dieser Arbeit. Die elektrochemischen und elektrokatalytischen Eigenschaften dieser Komplexe wurden mit Hilfe der Zyklovoltammetrie untersucht

Insights from 125Te and 57Fe nuclear resonance vibrational spectroscopy: a [4Fe-4Te] cluster from two points of view.

Iron-sulfur clusters are common building blocks for electron transport and active sites of metalloproteins. Their comprehensive investigation is crucial for understanding these enzymes, which play important roles in modern biomimetic catalysis and biotechnology applications. We address this issue by utilizing (Et4N)3[Fe4Te4(SPh)4], a tellurium modified version of a conventional reduced [4Fe-4S]+ cluster, and performed both 57Fe- and 125Te-NRVS to reveal its characteristic vibrational features. Our analysis exposed major differences in the resulting 57Fe spectrum profile as compared to that of the respective [4Fe-4S] cluster, and between the 57Fe and 125Te profiles. DFT calculations are applied to rationalize structural, electronic, vibrational, and redox-dependent properties of the [4Fe-4Te]+ core. We herein highlight the potential of sulfur/tellurium exchange as a method to isolate the iron-only motion in enzymatic systems

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle

The Geometry of the Catalytic Active Site in [FeFe]-hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H2-forming catalysts

A dithiacyclam-coordinated silver(i) polymer with anti-cancer stem cell activity

A cancer stem cell (CSC) active, solution stable, silver(i) polymeric complex bearing a dithiacyclam ligand is reported. The complex displays similar potency towards CSCs to salinomycin in monolayer and three-dimensional cultures. Mechanistic studies suggest CSC death results from cytosol entry, an increase in intracellular reactive oxygen species, and caspase-dependent apoptosis

Bridging hydride at reduced H-cluster species in [FeFe]-hydrogenases revealed by infrared spectroscopy, isotope editing, and quantum chemistry

[FeFe]-Hydrogenases contain a H2-converting cofactor (H-cluster) in which a canonical [4Fe–4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN–) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN– stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during μH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a μCO ligand are likely involved in rapid H2 turnover

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe–4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe–4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe–Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe–4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe–4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed

Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total- reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH

Protonengekoppelte Reduktion des katalytischen [4Fe-4S]-Zentrums in [FeFe]-Hydrogenasen

In der Natur katalysieren [FeFe]-Hydrogenasen die Abgabe und Aufnahme von molekularem Wasserstoff (H2) an einem einzigartigen Eisen-Schwefel-Kofaktor. Das geringe elektrochemische Überpotential in der Wasserstoffabgabe-Reaktion macht die [FeFe]-Hydrogenasen zu einem hervorragenden Beispiel für effiziente Biokatalyse. Gegenwärtig sind die molekularen Details des Wasserstoffumsatzes jedoch noch nicht vollständig verstanden. Daher adressieren wir in dieser Untersuchung die initiale Reduktion des katalytischen Zentrums der [FeFe]-Hydrogenasen mittels Infrarotspektroskopie und Elektrochemie und zeigen, dass der reduzierte Zustand Hred′ durch protonengekoppelten Elektronentransport gebildet wird. Ladungskompensation bindet das überschüssige Elektron am [4Fe-4S]-Zentrum und führt zu einer Stabilisierung der konservativen Konfiguration des [FeFe]-Kofaktors. Die Rolle von Hred′ beim Wasserstoffumsatz und mögliche Auswirkungen auf den katalytischen Mechanismus werden diskutiert. Es liegt nahe, dass die Regulation elektronischer Eigenschaften in der Umgebung von metallischen Kofaktoren die Grundlage für Multielektronenprozesse bildet