Philipp Stefan Simon

Chiral topology in semiconductors introduces a structural degree of freedom for tuning the chiroptical, excitonic, and spintronic behavior. In metal–organic chalcogenolates (MOChas) semiconductors, how ligand stereochemistry and supramolecular organization influence chiral topology, and thus optical and excitonic responses, remains poorly understood. Here, we demonstrate intrinsic circularly polarized luminescence (CPL) in sugar-based 1-dimensional MOChas, with structures solved via small-molecule serial femtosecond crystallography. These materials exhibit blue and green CPLs, arising from the chiral inorganic core. Despite the close chemical similarity between the glucose- and galactose-derived ligands, we observed dramatic differences in their inorganic structure. Silver glucosethiolate shows reversible hydration-driven photoluminescence switching with a hydration-linked emission energy. Labile water molecules in the glucose-based system correlate with a blueshift in excitonic emission. Our results suggest a new direction in the search for chirophotonic hybrid semiconductors, exploiting intrinsically chiral topologies and excitonic optical frequencies, which are sensitively modulated by ligand identity and supramolecular interactions.

Biosynthesis of penicillin and cephalosporin antibiotics involves the oxidation of the tripeptide δ-( l -α-aminoadipoyl)- l -cysteinyl- d -valine (ACV) to give the conformationally strained bicyclic penicillin ring system, as catalysed by the Fe(II)-dependent oxidase isopenicillin N synthase (IPNS). The structures of the precursors for the sequential β-lactam and thiazolidine ring forming steps in IPNS have remained elusive. Here we studied the reaction of anaerobic IPNS:Fe(II):ACV microcrystals with O ₂ using time-resolved serial femtosecond crystallography and X-ray emission spectroscopy. High-resolution structures reveal that ACV cysteinyl oxidation produces a thioaldehyde that unexpectedly does not coordinate the iron centre. The use of deuterium-labelled ACV enabled the observation of a monocyclic β-lactam with restoration of the Fe–S bond. Together with mutagenesis, spectroscopic and computational studies, the results support Fe(IV)-mediated penicillin formation involving active-site water molecules. These findings provide structural insight into IPNS catalysis, inform on the wider Fe(II) oxygenase superfamily and highlight the power of X-ray free-electron laser methods for resolving transient enzymatic intermediates.

During photosynthetic water oxidation, the Mn₄Ca cluster in Photosystem II progresses through five intermediate S_i (i = 0–4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S₃ state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S₃ state or in the subsequent S₃ to S₀ transition. Here we report conventional high-resolution data for the S₁, S₂, and S₃ states to a resolution of ~1.9 Å, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, O_X (or O6), in the S₃ state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The O_X-O5 distance is ~2.1 Å, supporting no strong interaction between them in the S₃ state, suggesting that if this is the O-O bond formation site, it is formed during the S₃ to S₀ transition initiated by the final oxidation of the cluster.

In all photosynthetic reaction centers (RC), transmembrane electron-transfer (ET) cofactor pathways are coordinated by homologous peptides and arranged with approximate C ₂ symmetry, providing two possible paths for charge separation (designated A and B). In type II RCs—Photosystem II and the purple bacterial RCs—only the A branch is active for ET. A variant bacterial RC containing nine amino acid substitutions that result in high-yield ET along the normally nonfunctional B branch was designed. Structural and theoretical studies were combined to understand factors that control unique ET reactions in the complex. Serial femtosecond crystallography performed at an X-ray Free Electron Laser afforded the room temperature X-ray-damage-free crystal structure of the variant RC, revealing interactions between the substituted amino acids and the ET cofactors. The positions of the bacteriochlorin cofactors on the symmetry-related pathways are unaffected, underscoring that the substitutions alter the energetics of ET, leading to the functional changes. In the binding pockets for the terminal electron acceptor quinones, alterations in the positions of neighboring amino acids and water molecules are coordinated with the change of the substituted side chains. Modifications deactivated A-branch ET and activated transmembrane charge separation along the B branch. Electrostatic calculations based on the structure reveal how differences in stabilization of charge-separated states by the protein environment underlie the change in direction of electron flow in this variant RC and provide insights into mechanisms by which B-branch charge separation across the photosynthetic membrane can be achieved in high yield.

Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R )-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O ₂ surrogate nitric oxide imply O ₂ binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O ₂ is the sole oxygen source in alcohol products, as shown by ¹⁸ O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.

The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H ₂ O ₂ have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (Fe ^IV -OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (Fe ^III –O ^•– ) character. Such states offer insight into the nature of ferryl heme.

Light-driven water splitting by plants, algae and cyanobacteria is pivotal for global bioenergetics and biomass formation. A manganese cluster bound to the photosystem II proteins catalyzes the complex reaction at high rate, but the rate-determining factors are insufficiently understood. Here we trace the oxygen-evolution transition by time-resolved polarography and infrared spectroscopy for cyanobacterial photosystems genetically modified at two strategic sites, complemented by computational chemistry. Our results highlight three rate-determining roles of the protein environment of the metal cluster: acceleration of proton-coupled electron transfer, acceleration of substrate-water insertion after O ₂ -formation, and balancing of rate-determining enthalpic and entropic contributions. Whereas in general the substrate-water insertion step may be unresolvable in time-resolved experiments, here it likely becomes traceable because of deceleration by genetic modification. Our results may stimulate new time-resolved experiments on substrate-water insertion in photosynthesis, clarification of enthalpy-entropy compensation in enzyme catalysis, and knowledge-guided development of inorganic catalyst materials.

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

Summary: Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the β-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

Accessing the electrode–electrolyte interface under operating conditions and capturing time-resolved kinetics remain challenging in electrochemical studies. Copper's interfacial oxidation dynamics remain unclear despite extensive research. Modulation excitation X-ray absorption spectroscopy (ME-XAS) was used to probe Cu in 100 mM KHCO₃ with sub-second sensitivity, revealing that hydroxide forms 30 ± 10 ms before the appearance of Cu₂O at positive potentials (0 to 0.5 V vs. RHE) near open-circuit conditions. From −0.4 to 0.8 V vs. RHE, hydroxide coverage reaches 49%, accompanied by a balanced presence of Cu(I) and Cu(II) oxides. These insights into Cu interfacial redox behavior under intermittent renewable energy operation—relevant to CO₂ electrolyzer durability—enhance our fundamental understanding of electrochemical interfaces.