Biomimetic mono- and dinuclear Ni(I) and Ni(II) complexes studied by X-ray absorption and emission spectroscopy and quantum chemical calculations

Five biomimetic mono- or dinuclear nickel complexes featuring Ni(I) or Ni(II) sites were studied by X-ray absorption and emission spectroscopy and DFT calculations. Ni K-edge XANES spectra and Kβ main and satellite emission lines were collected on powder samples. The pre-edge absorption transitions (core-to-valence excitation) and Kβ2,5 emission transitions (valence-to-core decay) were calculated using DFT (TPSSh/TZVP) on crystal structures. This yielded theoretical ctv and vtc spectra in near-quantitative agreement with the experiment, showing the adequacy of the DFT approach for electronic structure description, emphasizing the sensitivity of the XAS/XES spectra for ligation/redox changes at nickel, and revealing the configuration of unoccupied and occupied valence levels, as well as the spin-coupling modes in the dinuclear complexes. XAS/XES-DFT is valuable for molecular and electronic structure analysis of synthetic complexes and of nickel centers in H2 or COx converting metalloenzymes.Peer Reviewe

Kombination von fortschrittlicher Röntgenspektroskopie und quantenchemischen Kalkulationen zur Bestimmung von elektronischen Zuständen von metallorganischen Kofaktoren in Proteinen

Proteins with metal cofactors (metalloproteins) are capable to facilitate a large variety of chemical processes and are involved in the most challenging catalytic reactions e.g. activating small and stable molecules like N2, CO2, H2, H2O, CH4 and O2 with superior energetic efficiency and turnover rates. Each of these processes was optimized for millennia in nature under conditions of high interest for industrial application such as atmospheric pressure, room temperature and neutral pH, while utilizing earth abundant elements. Understanding of these enzymatic reactions and metal-ligand bonding thus will be helpful in addressing challenges in synthetic catalysts. Advanced X-ray absorption and emission spectroscopy (XAS/XES) allows to determine the oxidation and spin state of metal cofactors by probing unoccupied and occupied electronic states. However, metal cofactors in reactive high-valent states are prone to radiation damage. In this thesis, XAS/XES data collection was significantly accelerated by development of time-resolved energy-sampling (TRES) detection approaches and efficiency evaluation in comparison with conventional measurement approaches. TRES facilitated monitoring of weak spectral XES features with high signal-to-noise ratio on highly diluted and X-ray sensitive materials. Quantitative correlation of XAS/XES data with quantum chemical (QC) simulations derived from density functional theory (DFT) or the complete active space self-consistent field (CASSCF) approach from molecular orbital theory allows description of key structural and electronic parameters in metalloproteins and synthetic compounds. In this thesis, QC calculation protocols were established in our laboratory and spectral simulations were benchmarked with XAS/XES data from selected metalloproteins and synthetic compounds with a special focus on heme proteins. The heme group is the dominant cofactor in proteins that involve oxygen as reactant. Hemoglobin (HB) and myoglobin (MB) are essential for transport, storage and sensing of molecular oxygen in vertebrates and their physiological function has been investigated for more than a century. However, the nature of the electronic configuration of the Fe – O2 bonding in oxygenated HB/MB (oxy) was not settled so far. Mostly differing in the spin state of Fe and the O2 ligand, apparent contradictory models have dominated the debate. We combined advanced XAS/XES with QC simulations from DFT and CASSCF to determine the electronic configuration of HB/MB in different ligation states and three porphyrin model compounds. Our finding on the spin state and electronic configuration in oxygenated heme in HB and MB provided an adequate description of the metal-ligand interaction, which merged the classical models in a holistic description of the Fe – O2 bonding situation.Proteine mit metallischen Kofaktoren (Metalloproteine) vollführen eine große Bandbreite an chemischen Prozessen und sind involviert in anspruchsvolle katalytische Reaktionen, z. B. die Aktivierung von kleinen und stabilen Molekülen wie N2, H2, H2O, CH4 und O2, mit hoher energetischer Effizienz und Umsetzungsrate. Jeder dieser Prozesse wurde über Jahrmillionen von der Natur optimiert unter Konditionen, die von hohem Interesse für industrielle Anwendung sind, wie Atmosphärendruck, Raumtemperatur und neutralem pH, unter Einsatz von weitverbreiteten Elementen. Das Verständnis dieser enzymatischen Reaktionen und der Metall-Liganden Bindungen wird daher helfen, Herausforderungen in synthetischer Katalyse zu begegnen. Fortschrittliche Röntgenabsorptions- und Röntgenemissionsspektroskopie (RAS/RES) ermöglicht die Bestimmung von Oxidations- und Spinzustand von metallischen Kofaktoren durch Detektieren von unbesetzten und besetzten elektronischen Zuständen. Jedoch sind metallische Kofaktoren in hoch reaktiven hochvalenten Zuständen anfällig für Strahlenschäden. In dieser Arbeit wurde die Messung von RAS/RES Daten durch Entwicklung einer Messmethode mit zeitaufgelösten Fluoreszenzmessungen (ZAFM) und Evaluierung der Messeffizienz im Vergleich zu konventionellen Ansätzen signifikant beschleunigt. ZAFM ermöglichte die Detektion von schwachen spektralen Merkmalen mit hohem Signal-Rausch Verhältnis von dünn konzentrierten und hoch strahlungssensitiven Proben. Quantitative Korrelation von RAS/RES Daten mit quantenchemischen (QC) Kalkulationen aus der Dichtefunktionaltheorie (DFT) oder der complete active space self-consistent field (CASSCF) Methode als Teil der Molekülorbitaltheorie (MO-Theorie) ermöglicht die Beschreibung von wichtigen strukturellen und elektronischen Parametern in Metalloproteinen und synthetischen Verbindungen. QC Kalkulationen wurden während dieser Arbeit in unserem Labor etabliert und die Simulation von spektralen Merkmalen verifiziert mit RAS/RES Daten von ausgewählten Metalloproteinen und synthetischen Verbindungen. Ein Fokus wurde insbesondere auf Proteine der Hämgruppe gelegt, welche der dominante Kofaktor bei Sauerstoffreaktionen ist. Hämoglobin (HB) und Myoglobin (MB) sind essenziell für Transport, Aufbewahrung und Detektion von molekularem Sauerstoff in Wirbeltieren und deren physiologische Funktion seit mehr als einem Jahrhundert untersucht. Nichtsdestotrotz wurde die Natur der elektronischen Konfiguration in mit Sauerstoff angereichertem HB/MB (oxy) noch nicht hinreichend geklärt. Die sich widersprechenden, debattierten Modelle unterscheiden sich hauptsächlich im Spinzustand des Fe Atoms und des O2 Liganden. Wir haben fortschrittliche RAS/RES mit QC Simulationen kombiniert um die elektronische Konfiguration von HB/MB in verschiedenen Präparationen und drei Porphyrin Modelkomplexen, zu untersuchen. Unsere Ergebnisse bezüglich des Spinzustandes und der elektronischen Konfiguration in oxy HB und MB lieferte eine adäquate Beschreibung der Metall-Liganden Interaktion und vereinte die klassischen Modelle in ein holistisches Bild der Fe – O2 Bindung

An ATCUN-like copper site in B2-crystallin plays a protective role in cataract-associated aggregation

Cataracts is the leading cause of blindness worldwide and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce aggregation of human -crystallins. Here we evaluated the impact of divalent metal ions in the aggregation of human B2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of B2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of B2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism (CD) and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in B2-crystallin; one of them with spectroscopic features typical of Cu2+ bound to an amino-terminal copper and nickel binding motif (ATCUN), a motif found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of B2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Removal of the N-terminus yields an N-truncated form of B2-crystallin that is more susceptible to Cu-induced aggregation and loss of thermal stability, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy (XAS) studies reveal the presence of a copper redox active site in B2-crystallin that is associated to metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of cataract-related B2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in B2-crystallin plays a functional/protective role or constitute a vestige from its evolution as a lens structural protein, remains to be elucidated

Electronic and molecular structure relations in diiron compounds mimicking the [FeFe]-hydrogenase active site studied by X-ray spectroscopy and quantum chemistry

International audienceSynthetic diiron compounds of the general formula Fe2(μ-S2R)(CO)n(L)6−n (R = alkyl or aromatic groups; L = CN− or phosphines) are versatile models for the active-site cofactor of hydrogen turnover in [FeFe]-hydrogenases. A series of 18 diiron compounds, containing mostly a dithiolate bridge and terminal ligands of increasing complexity, was characterized by X-ray absorption and emission spectroscopy in combination with density functional theory. Fe K-edge absorption and Kβ main-line emission spectra revealed the varying geometry and the low-spin state of the Fe(I) centers. Good agreement between experimental and calculated core-to-valence-excitation absorption and radiative valence-to-core-decay emission spectra revealed correlations between spectroscopic and structural features and provided access to the electronic configuration. Four main effects on the diiron core were identified, which were preferentially related to variation either of the dithiolate or of the terminal ligands. Alteration of the dithiolate bridge affected mainly the Fe–Fe bond strength, while more potent donor substitution and ligand field asymmetrization changed the metal charge and valence level localization. In contrast, cyanide ligation altered all relevant properties and, in particular, the frontier molecular orbital energies of the diiron core. Mutual benchmarking of experimental and theoretical parameters provides guidelines to verify the electronic properties of related diiron compounds

Copper reductase activity and free radical chemistry by cataract-associated human lens γ-crystallins

Cataracts are caused by high-molecular weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS- crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, SDS-PAGE, circular dichroism and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: i) loss of beta-sheet structure in the N-terminal domain; ii) decreased thermal and kinetic stability; iii) formation of metal-bridged species; and iv) formation of disulfide-bridged dimers. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins’ Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could be acting, not only as structural proteins, but also as key players for metal and redox homeostasis

Kα X‑ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S₃ State

The unique manganese–calcium catalyst in photosystem II (PSII) is the natural paragon for efficient light-driven water oxidation to yield O₂. The oxygen-evolving complex (OEC) in the dark-stable state (S₁) comprises a Mn₄CaO₄ core with five metal-bound water species. Binding and modification of the water molecules that are substrates of the water-oxidation reaction is mechanistically crucial but controversially debated. Two recent crystal structures of the OEC in its highest oxidation state (S₃) show either a vacant Mn coordination site or a bound peroxide species. For purified PSII at room temperature, we collected Mn Kα X-ray emission spectra of the S₀, S₁, S₂, and S₃ intermediates in the OEC cycle, which were analyzed by comparison to synthetic Mn compounds, spectral simulations, and OEC models from density functional theory. Our results contrast both crystallographic structures. They indicate Mn oxidation in three S-transitions and suggest additional water binding at a previously open Mn coordination site. These findings exclude Mn reduction and render peroxide formation in S₃ unlikely

Inhibitory and Non-Inhibitory NH₃ Binding at the Water-Oxidizing Manganese Complex of Photosystem II Suggests Possible Sites and a Rearrangement Mode of Substrate Water Molecules

The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH₄⁺/NH₃ binding. Time-resolved detection of O₂ formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH₄Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O₂ formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn­(IV)₄ (S₃) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH₃ binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the “dangling” Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S₂–S₃ transition as well as their reversal in the O₂ formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) <i>Biochemistry 54</i>, 5783]. (5) We observe the same partial inhibition of PSII by NH₄Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII

Molecular Dynamics and Structural Studies of Zinc Chloroquine Complexes

Chloroquine (CQ) is a first-choice drug against malaria and autoimmune diseases. It may act as a zinc ionophore. In this study, state-of-the-art computations and experiments were leveraged to solve the structure of the Zn chloride-CQ complex in solution and in solid state. The integration of different techniques (NMR, ESI-MS, X-ray absorption and diffraction methods) together with ab initio molecular dynamics simulations, overcomes the issues related to the kinetic lability of zinc complexes. Within the physiological pH range, CQ binds Zn2+ through the quinoline ring nitrogen, forming [Zn(CQH)Clx(H2O)3–x](3+)–x (x = 0, 1, 2, 3) tetrahedral complexes. The Zn(CQH)Cl3 species is stable at neutral pH and at high chloride concentrations typical of the extracellular medium, but metal coordination is lost at moderately low pH, suggesting the release of Zn2+ ions into the lysosomal lumen. [Zn(CQH)(H2O)4]3+ may exist in the absence of chloride. This in vitro / in silico approach can be extended to other metal-targeting drugs and and bioinorganic systems